Immunofluorescence showing normal ( a , b , e , f , i , l , o , p ) altered ( c , d , g , h , m , n , q , r ) intracellular distribution of SRF ( a b , ; c d , ) , MURF2 ( e f , sarcomeric ; g h , ) , Nbr1 ( i l , sarcomeric ; m n , diffused ) , SQSTM1 / p62 ( o p , sarcomeric ; q r , diffused ) in representative single cardiomyocytes ( left ) . Sarcomeres are marked by α - actinin . On right , of CTR DCM cardiomyocytes showing expression of SRF , accumulation of MURF2 , diffused expression of Nbr1 of SQSTM1 / p62 after infection with control U7snRNA - ScrambleAONs - IRES - GFP ( Scr - AON ) U7snRNA - TTNAONs - IRES - GFP ( TTN - AON ) lentiviruses ( right ) . Data represent mean values ± from two control two DCM clones . Statistical difference was tested using two - sided chi - squared ( CTR Scr - AON : n = 874 , n = 874 , n = 882 n = 890 , CTR TTN - AON : n = 880 , n = 990 , n = 878 n = 890 , DCM Scr - AON : n = 890 , n = 887 , n = 884 n = 886 , DCM TTN - AON : n = 900 , n = 875 , n = 899 n = 891 for SRF , MURF2 , Nbr1 SQSTM1 / p62 , respectively ; * P = 0 . 01 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 04 , DCM Scr - AON versus DCM TTN - AON for SRF ; * P = 0 . 02 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 04 , DCM Scr - AON versus DCM TTN - AON for MURF2 ; * P = 0 . 03 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 03 , DCM Scr - AON versus DCM TTN - AON for Nbr1 ; * * P = 0 . 009 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 01 , DCM Scr - AON versus DCM TTN - AON for SQSTM1 / p62 ) . No significant differences were observed comparing CTR Scr - AON CTR TTN - AON groups . Scale bars , 50 μm .

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Immunofluorescence images showing normal (a, b, e, f, i, l, o, p) and altered (c, d, g, h, m, n, q, r) intracellular distribution of SRF (a and b, nuclear; c and d, cytoplasmic), MURF2 (e and f, sarcomeric; g and h, nuclear), Nbr1 (i and l, sarcomeric; m and n, diffused), and SQSTM1/p62 (o and p, sarcomeric; q and r, diffused) in representative single cardiomyocytes (left). Sarcomeres are marked by α-actinin. On the right, percentage of CTR and DCM cardiomyocytes showing cytoplasmic expression of SRF, nuclear accumulation of MURF2, and diffused expression of Nbr1 and of SQSTM1/p62 after infection with control U7snRNA-ScrambleAONs-IRES-GFP (Scr-AON) and the U7snRNA-TTNAONs-IRES-GFP (TTN-AON) lentiviruses (right). Data represent mean values ± SEM from two control and two DCM clones. Statistical difference was tested using the two-sided chi-squared test (CTR Scr-AON: n = 874, n = 874, n = 882 and n = 890, CTR TTN-AON: n = 880, n = 990, n = 878 and n = 890, DCM Scr-AON: n = 890, n = 887, n = 884 and n = 886, DCM TTN-AON: n = 900, n = 875, n = 899 and n = 891 for SRF, MURF2, Nbr1 and SQSTM1/p62, respectively; *P = 0.01, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for SRF; *P = 0.02, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for MURF2; *P = 0.03, CTR Scr-AON versus DCM Scr-AON; *P = 0.03, DCM Scr-AON versus DCM TTN-AON for Nbr1; **P = 0.009, CTR Scr-AON versus DCM Scr-AON; *P = 0.01, DCM Scr-AON versus DCM TTN-AON for SQSTM1/p62). No significant differences were observed comparing CTR Scr-AON and CTR TTN-AON groups. Scale bars, 50 μm.


B , C Immunofluorescence of sarcomeric α - actinin myosin ( B ) assessment of cardiac function ( C ) in homozygous Ttn knock - in cultured for 24 h after mScrAON mTtnAON transient transfection ( n = 3 per group ) or lentivirus delivery ( n = 7 per group ) . Scale bars , 10 μm .

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B, C Immunofluorescence images of sarcomeric α-actinin and myosin (B) and assessment of cardiac function (C) in homozygous Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection (n = 3 per group) or lentivirus delivery (n = 7 per group). Scale bars, 10 μm.


D Electron microscopy in Ttn knock - in cultured for 24 h after mScrAON mTtnAON transient transfection . mTtnAON treatment of homozygous rescued myofibril formation ( left , black arrows ) , resulting in thicker filaments ( right ) .

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D Electron microscopy analysis in Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection. mTtnAON treatment of homozygous embryos rescued myofibril formation (left, black arrows), resulting in thicker filaments (right).


Echocardiographic of adult knock - in mice . Statistical difference was tested using two - sided Student ' s t - ( WT : n = 6 , HET : n = 6 ; HET + vPMO - mScrAON : n = 10 HET + vPMO - mTtnAON : n = 14 ; * P = 0 . 013 , HET versus HET + vPMO - mTtnAON * P = 0 . 02 , HET + vPMO - mScrAON versus HET + vPMO - mTtnAON for LVEDD differences ; * * P = 0 . 006 , HET versus HET + vPMO - mTtnAON * P = 0 . 02 , HET + vPMO - mScrAON versus HET + vPMO - mTtnAON for LV - EF differences ; * P = 0 . 03 , HET versus HET + vPMO - mTtnAON * P = 0 . 04 , HET + vPMO - mScrAON versus HET + vPMO - mTtnAON for IVSd differences ; * * P = 0 . 001 , HET versus HET + vPMO - mTtnAON * P = 0 . 03 , HET + vPMO - mScrAON versus HET + vPMO - mTtnAON for PWd differences ) . No significant differences were observed among groups at day 0 day 7 . Data represent mean values ± .

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Echocardiographic analysis of adult knock-in mice. Statistical difference was tested using the two-sided Student's t-test (WT: n = 6, HET: n = 6; HET + vPMO-mScrAON: n = 10 and HET + vPMO-mTtnAON: n = 14; *P = 0.013, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LVEDD differences; **P = 0.006, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LV-EF differences; *P = 0.03, HET versus HET + vPMO-mTtnAON and *P = 0.04, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for IVSd differences; **P = 0.001, HET versus HET + vPMO-mTtnAON and *P = 0.03, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON and for PWd differences). No significant differences were observed among groups at day 0 and day 7. Data represent mean values ± SEM.


Masson ' s trichrome of heart sections from HET injected with vPMO - mScrAONs vPMO - mTtnAON . Scale bars , 1 mm .

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Masson's trichrome staining of heart sections from HET animals injected with vPMO-mScrAONs and vPMO-mTtnAON. Scale bars, 1 mm.


in situ hybridization ( FISH ) of heart muscle tissue from adult knock - in mice with a probe complementary to vPMO - mTtnAON . Scale bars , 250 50 μm ( magnification ) .

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Fluorescence in situ hybridization (FISH) of heart muscle tissue from adult knock-in mice with a probe complementary to vPMO-mTtnAON. Scale bars, 250 and 50 μm (magnification).


RT - PCR of Ttn exon 326 transcripts from heart tissue of untreated WT HET vPMO - mScrAON - vPMO - mTtnAON - treated mice ( left ) . Representative direct sequencing of Ttn exon 326 transcripts from vPMO - mTtnAON - treated HET heart tissue ( right ) .

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RT-PCR analysis of Ttn exon 326 transcripts from heart tissue of untreated WT and HET animals and vPMO-mScrAON- and vPMO-mTtnAON-treated mice (left). Representative direct sequencing of Ttn exon 326 transcripts from vPMO-mTtnAON-treated HET heart tissue (right).


Mass spectrometry - of titin peptides in adult knock - in mice injected with vPMO - mScrAONs vPMO - mTtnAONs . Unsupervised hierarchical clustering identified a cluster enriched in exon 326 peptides that was down - regulated in vPMO - mTtnAON - treated compared to vPMO - mScrAON - treated . Another cluster enriched in C - terminal peptides was up - regulated in vPMO - mTtnAON group compared to vPMO - mScrAON group ( n = 3 , P = 0 . 02 , Fisher ' s exact , FDR = 0 . 04 ) .

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Mass spectrometry-based analysis of titin peptides in adult knock-in mice injected with vPMO-mScrAONs and vPMO-mTtnAONs. Unsupervised hierarchical clustering identified a cluster enriched in exon 326 peptides that was down-regulated in vPMO-mTtnAON-treated animals compared to vPMO-mScrAON-treated littermates. Another cluster enriched in C-terminal peptides was up-regulated in the vPMO-mTtnAON group compared to the vPMO-mScrAON group (n = 3, P = 0.02, Fisher's exact test, FDR = 0.04).


A - B ) Western - blot for GAPDH LC3 of lysates obtained from HeLa treated with DMSO or 1 µM rapamycin ( Rapa ) for indicated time periods ( A ) ( n = 7 ) or for 5 h with indicated concentrations ( B ) ( n = 6 ) . One hour before harvesting , 100 nM bafilomycin A1 was added . Upper panels : representative Western blots ; lower panels : of LC3 - II / GAPDH .

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A-B) Western-blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated with DMSO or 1 µM rapamycin (Rapa) for the indicated time periods (A) (n = 7) or for 5 h with the indicated concentrations (B) (n = 6). One hour before harvesting, 100 nM bafilomycin A1 was added. Upper panels: representative Western blots; lower panels: quantification of the LC3-II/GAPDH ratio.


C ) GFP - LC3 - in HeLa treated for 5 h with different concentrations of rapamycin . Left : representative pictures . scale bar represents 10 µm . Concentrations are mentioned in right lower corner . Right : of number of per ( n = 3 ) . * p < 0 . 05 , repeated measurements ANOVA .

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C) GFP-LC3-punctae quantification in HeLa cells treated for 5 h with different concentrations of rapamycin. Left: representative pictures. The scale bar represents 10 µm. Concentrations are mentioned in the right lower corner. Right: Quantification of the number of punctae per cell (n = 3). * p<0.05, repeated measurements ANOVA.


A ) Representative measurements ( n = 4 ) of Ca2 + signals , displayed as Fura2 ( F340 / F380 ) , showing effect of 0 . 3 µM 100 µM ATP , 1 µM thapsigargin ( Tg ) or 10 µM ionomycin ( Iono ) in intact HeLa treated with different concentrations of rapamycin ( Rapa ) for 5 h . 45 s prior to addition of ATP , Tg or Iono , EGTA ( 3 mM ) was given to buffer extracellular Ca2 + as indicated .

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A) Representative measurements (n = 4) of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 0.3 µM and 100 µM ATP, 1 µM thapsigargin (Tg) or 10 µM ionomycin (Iono) in intact HeLa cells treated with different concentrations of rapamycin (Rapa) for 5 h. 45 s prior to the addition of ATP, Tg or Iono, EGTA (3 mM) was given to buffer extracellular Ca2+ as indicated.


B ) of average amplitude of response ( F − F0 ) ( n = 4 ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 , repeated measurements ANOVA .

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B) Quantification of the average amplitude of the response (F−F0) (n = 4). * p<0.05; ** p<0.01; *** p<0.001, repeated measurements ANOVA.


C ) Mean resting [ Ca2 + ] , measured in Fura2 - loaded HeLa treated with indicated concentrations of rapamycin for 5 h , as well as in absence ( Ctrl ) or presence of DMSO ( n = 3 ) . * p < 0 . 05 , repeated measurements ANOVA .

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C) Mean resting cytosolic [Ca2+], measured in Fura2-loaded HeLa cells treated with the indicated concentrations of rapamycin for 5 h, as well as in the absence (Ctrl) or presence of DMSO (n = 3). * p<0.05, repeated measurements ANOVA.


D ) Unidirectional 45Ca2 + - flux experiments in permeabilized pretreated with 1 µM rapamycin for 5 h or with DMSO ( Ctrl ) . Mean fractional 45Ca2 + release ( % / 2 min ) is shown as a function of time with effect of 0 . 7 µM IP3 ( circles ) or no addition ( squares ) . horizontal bar indicates presence of IP3 .

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D) Unidirectional 45Ca2+-flux experiments in permeabilized cells pretreated with 1 µM rapamycin for 5 h or with DMSO (Ctrl). Mean fractional 45Ca2+ release (%/2 min) is shown as a function of time with the effect of 0.7 µM IP3 (circles) or no addition (squares). The horizontal bar indicates the presence of IP3.


E ) Quantitative of IP3 - induced 45Ca2 + release in pretreated for 5 h with 1 µM rapamycin or DMSO ( Ctrl ) ( n = 8 ) . * * * p < 0 . 001 , paired Student ' s t - .

E) Quantitative analysis of the IP3-induced 45Ca2+ release in cells pretreated for 5 h with 1 µM rapamycin or DMSO (Ctrl) (n = 8). *** p<0.001, paired Student's t-test.


A - B ) Western - blot for luminal Ca2 + - binding in HeLa treated with indicated concentrations of rapamycin ( Rapa ) for 5 h : calreticulin ( CRT ) ( A ) BiP / Grp78 ( BiP ) ( B ) . Upper panels : representative Western blots ; lower panels : of / GAPDH ( n = 4 ) .

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A-B) Western-blot analysis for luminal Ca2+-binding proteins in HeLa cells treated with the indicated concentrations of rapamycin (Rapa) for 5 h: calreticulin (CRT) (A) and BiP/Grp78 (BiP) (B). Upper panels: representative Western blots; lower panels: quantification of the protein/GAPDH ratio (n = 4).


C ) Western - blot for SERCA2 in HeLa treated with indicated concentrations of rapamycin for 5 h . Upper panels : representative Western blots ; lower panel : of SERCA2 / GAPDH ( n = 4 ) .

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C) Western-blot analysis for SERCA2 in HeLa cells treated with the indicated concentrations of rapamycin for 5 h. Upper panels: representative Western blots; lower panel: quantification of the SERCA2/GAPDH ratio (n = 4).


D ) Representative plot showing decrease in ER 45Ca2 + content ( logarithmic scale ) in a Ca2 + - free efflux medium without ATP as a function of time in permeabilized HeLa pretreated for 5 h with 1 µM rapamycin or with DMSO . passively bound Ca2 + was determined by loading with 45Ca2 + in presence of 10 µM of Ca2 + ionophore A23187 then subtracted from stored 45Ca2 + . ER Ca2 + - leak rate can be estimated as rate of decline of ER 45Ca2 + - store content as a function of time .

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D) Representative plot showing the decrease in ER 45Ca2+ content (logarithmic scale) in a Ca2+-free efflux medium without ATP as a function of time in permeabilized HeLa cells pretreated for 5 h with 1 µM rapamycin or with DMSO. The passively bound Ca2+ was determined by loading the cells with 45Ca2+ in the presence of 10 µM of the Ca2+ ionophore A23187 and then subtracted from the stored 45Ca2+. The ER Ca2+-leak rate can be estimated as the rate of decline of the ER 45Ca2+-store content as a function of time.


E ) of mean 45Ca2 + - store content at beginning of measurement ( t0 ) ( n = 5 ) .

E) Quantification of the mean 45Ca2+-store content at the beginning of the measurement (t0) (n = 5).


F ) of mean slope of curve in D after transformation to a linear scale , which is a measure of 45Ca2 + - leak rate ( n = 5 ) . * p < 0 . 05 ; * * p < 0 . 01 , paired Student ' s t - .

F) Quantification of the mean slope of the curve in D after transformation to a linear scale, which is a measure of the 45Ca2+-leak rate (n = 5). * p<0.05; ** p<0.01, paired Student's t-test.


A ) Representative Western - blot for Atg12 ( showing autophagic Atg12 - Atg5 ) , GAPDH LC3 of lysates obtained from MEF pretreated with ( + Dox ) or without ( - Dox ) doxycycline treated with DMSO or 0 . 1 , 1 or 5 µM rapamycin ( Rapa ) for 5 h ( n = 3 ) .

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A) Representative Western-blot analysis for Atg12 (showing the autophagic Atg12-Atg5 complex), GAPDH and LC3 of protein lysates obtained from MEF cells pretreated with (+Dox) or without (-Dox) doxycycline and treated with DMSO or 0.1, 1 or 5 µM rapamycin (Rapa) for 5 h (n = 3).


B - C ) Representative measurements of Ca2 + signals , displayed as Fura2 ( F340 / F380 ) , showing effect of 1 mM ATP ( B ) or 10 µM ionomycin ( Iono ) in intact MEF pretreated with or without doxycycline treated with different concentrations of rapamycin for 5 h . Prior to addition of ATP or Iono , EGTA ( 3 mM ) was added to chelate extracellular Ca2 + as indicated .

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B-C) Representative measurements of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 1 mM ATP (B) or 10 µM ionomycin (Iono) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h. Prior to the addition of ATP or Iono, EGTA (3 mM) was added to chelate the extracellular Ca2+ as indicated.


D ) of average amplitude of response ( F − F0 ) ( n = 3 , 4 , 5 6 for ATP - Dox , ATP + Dox , Iono - Dox Iono + Dox , resp . ) * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 , repeated measurements ANOVA .

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D) Quantification of the average amplitude of the response (F−F0) (n = 3, 4, 5 and 6 for ATP-Dox, ATP+Dox, Iono-Dox and Iono+Dox, resp.) * p<0.05; ** p<0.01; *** p<0.001, repeated measurements ANOVA.


2 + - dependent . Western - blot for GAPDH LC3 of lysates obtained from HeLa treated for 5 h with DMSO , 1 µM rapamycin ( Rapa ) , 10 µM BAPTA - AM or both . One hour before harvesting , 100 nM bafilomycin A1 was added . Left : representative Western blots ; right : of LC3 - II / GAPDH ( n = 6 ) . * p < 0 . 05 , repeated measurements ANOVA .

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2+-dependent. Western-blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated for 5 h with DMSO, 1 µM rapamycin (Rapa), 10 µM BAPTA-AM or both. One hour before harvesting, 100 nM bafilomycin A1 was added. Left: representative Western blots; right: quantification of the LC3-II/GAPDH ratio (n = 6). * p<0.05, repeated measurements ANOVA.


( b ) - sensitive growth . Yeast strains ( wild - type or deletion mutants , indicated on left ) were transformed with either an empty vector or a plasmid carrying an artificial guide gene targeting U2 pseudouridylation at position 42 44 ( indicated on top ) , grown at various temperatures ( indicated at bottom ) .

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(b) Temperature-sensitive growth assay. Yeast strains (wild-type or deletion mutants, indicated on the left) were transformed with either an empty vector or a plasmid carrying an artificial guide RNA gene targeting U2 pseudouridylation at position 42 and 44 (indicated on the top), and grown at various temperatures (indicated at the bottom).


( c ) Blockage of pseudouridylation by deletion of specific pseudouridylase genes . RNAs isolated from wild - type pseudouridylase - deletion strains ( indicated on top ) were used for U2 pseudouridylation ( CMC modification followed by primer - extension ) . Primer - extension pauses / stops correspond to ψ sites ( indicated on left right ) . Note : above - background signal of ψ42 in snr81Δ lane ( to some extent , in snr81Δ pus7Δ lane ) is likely caused by presence of strong ψ44 band . This is a primer - extension artifact , which often occurs when there is a strong ψ signal in neighboring position . Alternatively , it is possible that having ψ at position 44 allows for an unidentified enzyme other than snR81 to modify U42 ( albeit inefficiently ) ( Perhaps Pus1 , which is responsible for ψ44 formation , can be a bit " processive " ) . Lanes 1 - 8 lanes 9 - 16 are from two separate gels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Blockage of pseudouridylation by deletion of specific pseudouridylase genes. RNAs isolated from wild-type and pseudouridylase-deletion strains (indicated on the top) were used for U2 pseudouridylation assay (CMC modification followed by primer-extension). Primer-extension pauses/stops correspond to ψ sites (indicated on the left and right). Note: The above-background signal of ψ42 in the snr81Δ lane (and to some extent, in the snr81Δ pus7Δ lane) is likely caused by the presence of strong ψ44 band. This is a primer-extension artifact, which often occurs when there is a strong ψ signal in the neighboring position. Alternatively, it is possible that having ψ at position 44 allows for an unidentified enzyme other than snR81 to modify U42 (albeit inefficiently) (Perhaps Pus1, which is responsible for ψ44 formation, can be a bit "processive"). Lanes 1-8 and lanes 9-16 are from two separate gels.


( d ) Restoration of U2 pseudouridylation by an artificial box H / ACA . RNAs isolated from various yeast strains ( indicated on top ) , including snr81Δ pusΔ that was transformed with an artificial guide targeting positions 42 44 ( lanes 9 10 ) , were assayed for pseudouridylation [ see legend to ( c ) ] .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Restoration of U2 pseudouridylation by an artificial box H/ACA RNA. RNAs isolated from various yeast strains (indicated on the top), including the snr81Δ pusΔ strain that was transformed with an artificial guide RNA targeting positions 42 and 44 (lanes 9 and 10), were assayed for pseudouridylation [see legend to (c)].


( a ) Growth using ACT1 - CUP1 reporter system . Yeast carrying ACT1 - CUP1 reporter were deleted of pseudouridylase genes ( indicated on left ) , were then assayed for growth at 30 ° C on media containing various concentrations of [ Cu2 + ] ( indicated at bottom ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Growth assay using the ACT1-CUP1 reporter system. Yeast cells carrying the ACT1-CUP1 reporter were deleted of pseudouridylase genes (indicated on the left), and were then assayed for growth at 30°C on media containing various concentrations of [Cu2+] (indicated at the bottom).


( b ) Growth rescue by restoration of pseudouridylation . wild - type [ from ( a ) ] or snr81Δ pusΔ [ also from ( a ) ] , which exhibited most severe growth - deficiency phenotype , was transformed with an empty vector or a plasmid carrying a pseudouridylation guide gene targeting either position 42 , position 44 , or both ( indicated ) . resulting were assayed for growth at 30 ° C on media containing various concentrations of [ Cu2 + ] ( indicated at bottom ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) Growth rescue by restoration of pseudouridylation. The wild-type strain [from (a)] or the snr81Δ pusΔ strain [also from (a)], which exhibited the most severe growth-deficiency phenotype, was transformed with an empty vector or a plasmid carrying a pseudouridylation guide RNA gene targeting either position 42, position 44, or both (indicated). The resulting cells were assayed for growth at 30°C on media containing various concentrations of [Cu2+] (indicated at the bottom).


( d ) Splicing using wild - type mutant ACT1 - CUP1 reporters . RNAs isolated from wild - type snr81Δ pusΔ strains ( indicated on top ) , which carry wild - type ACT1 - CUP1 reporter pre - mRNA or any of mutant ACT1 - CUP1 reporter pre - mRNAs [ indicated , also see ( c ) ] , were used for splicing ( primer - extension ) . un - spliced pre - mRNA , lariat intermediate , spliced mRNA are indicated . A primer complementary to U6 was also used ( as an internal control ) in , U6 band is indicated as well . In addition , growth phenotype of each ( in various concentrations of [ Cu2 + ] ) is also shown . Lanes 1 - 12 , lanes 13 14 , lanes 15 16 are from separate gels .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Splicing assay using the wild-type and mutant ACT1-CUP1 reporters. RNAs isolated from the wild-type and the snr81Δ pusΔ strains (indicated on the top), which carry the wild-type ACT1-CUP1 reporter pre-mRNA or any of the mutant ACT1-CUP1 reporter pre-mRNAs [indicated, also see (c)], were used for splicing assay (primer-extension analysis). The un-spliced pre-mRNA, lariat intermediate, and spliced mRNA are indicated. A primer complementary to U6 was also used (as an internal control) in the assay, and the U6 band is indicated as well. In addition, the growth phenotype of each strain (in various concentrations of [Cu2+]) is also shown. Lanes 1-12, lanes 13 and 14, and lanes 15 and 16 are from separate gels.


( e ) of ACT1 - CUP1 mRNA . Spliced ACT1 - CUP1 mRNA were calculated relative to U6 in each lane . was on three independent experiments .

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(e) Quantification of ACT1-CUP1 mRNA levels. Spliced ACT1-CUP1 mRNA levels were calculated relative to U6 in each lane. The quantification was based on three independent experiments.


( f ) Relative improvement / reduction for mutant reporters . Relative improvement or reduction in splicing was calculated by normalizing splicing efficiency ( mRNA / U6 ) of each mutant reporter to that of WT reporter ( set at 1 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

(f) Relative improvement/reduction for the mutant reporters. Relative improvement or reduction in splicing was calculated by normalizing the splicing efficiency (mRNA/U6) of each mutant reporter to that of WT reporter (set at 1).


Figure 3 . Synthetic lethality . Prp5 domain structure , mutations in some of domains ( indicated ) are schematized at top . Each of these PRP5 genes ( wild - type or mutants , plasmid - borne ) was used to replace chromosomal PRP5 . numbers in parentheses are relative ATPase activities of wild - type ( SAT ) Prp5 some of Prp5 mutants tested before ( Xu Query , 2007 ) . U2 status , either containing Ψ42 Ψ44 ( WT - U2 snR81Δ pus1Δ with pΨ42 or Ψ44 ) or lacking Ψ42 Ψ44 ( snR81Δ pus1Δ ) , is indicated ( on right ) . Upon transformation with plasmids carrying PRP5 genes ( wild - type or any of mutants ) , were grown on regular medium ( - FOA ) or on medium containing 5 - FOA ( + FOA ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3. Synthetic lethality assay. The Prp5 domain structure, and the mutations in some of the domains (indicated) are schematized at the top. Each of these PRP5 genes (wild-type or mutants, plasmid-borne) was used to replace the chromosomal PRP5. The numbers in parentheses are the relative ATPase activities of the wild-type (SAT) Prp5 and some of the Prp5 mutants tested before (Xu Query, 2007). The U2 status, either containing Ψ42 and Ψ44 (WT-U2 and snR81Δ pus1Δ with pΨ42 or Ψ44) or lacking Ψ42 and Ψ44 (snR81Δ pus1Δ), is indicated (on the right). Upon transformation with the plasmids carrying PRP5 genes (wild-type or any of the mutants), cells were grown on the regular medium (-FOA) or on medium containing 5-FOA (+FOA).


( a ) Prp5 - U2 co - immunoprecipitation ( IP ) . Wild - type ( lanes 1 2 ) snR81Δ pus1Δ ( lane 3 ) strains , each containing a FLAG - tagged PRP5 gene ( indicated on top ) , were used for anti - FLAG IP in absence ( lane 1 ) or presence ( lanes 2 3 ) of ATP . precipitated Prp5 ( WT Prp5 - FLAG ) as well as co - precipitated U1 U2 , detected by primer - extension , are indicated .

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(a) Prp5-U2 co-immunoprecipitation (IP). Wild-type (lanes 1 and 2) and snR81Δ pus1Δ (lane 3) strains, each containing a FLAG-tagged PRP5 gene (indicated on the top), were used for anti-FLAG IP in the absence (lane 1) or presence (lanes 2 and 3) of ATP. The precipitated Prp5 (WT Prp5-FLAG) as well as co-precipitated U1 and U2, detected by primer-extension, are indicated.


( b ) of co - precipitated U2 snRNA . For each lane [ WT lane snr81Δ pus1Δ lane , corresponding to lane 2 lane 3 of ( a ) , respectively ] , Prp5 - co - precipitated U2 was normalized against co - precipitated U1 [ ( of U2 band ) / ( of U1 band ) ] . snr81Δ pus1Δ lane was then further normalized against WT lane [ ( value of normalized U2 from snr81Δ pus1Δ lane ) / ( value of normalized U2 from WT lane ) ] . was on three independent experiments .

O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O

(b) Quantification of co-precipitated U2 snRNA. For each lane [the WT lane and the snr81Δ pus1Δ lane, corresponding to lane 2 and lane 3 of (a), respectively], Prp5-co-precipitated U2 was normalized against co-precipitated U1 [(intensity of the U2 band) / (intensity of the U1 band)]. The snr81Δ pus1Δ lane was then further normalized against the WT lane [(the value of normalized U2 from the snr81Δ pus1Δ lane) / (the value of normalized U2 from the WT lane)]. Quantification was based on three independent experiments.


( c ) U2 expression level in wild - type snR81Δ pus1Δ strains . Total , isolated from wild - type ( lane 1 ) or snR81Δ pus1Δ ( lane 2 ) strains , was used for primer - extension to measure of U2 U6 . U2 U6 bands are indicated .

O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O

(c) U2 expression level in wild-type and snR81Δ pus1Δ strains. Total RNA, isolated from the wild-type (lane 1) or snR81Δ pus1Δ (lane 2) strains, was used for primer-extension to measure the levels of U2 and U6. The U2 and U6 bands are indicated.


d ) Depletion of Prp5 . Wild - type ( lanes 1 , 3 4 ) snR81Δ pus1Δ ( lanes 2 5 ) containing a FLAG - tagged PRP5 were lysed , anti - FLAG IP was performed at a high of salt . extracts , before ( lanes 1 2 ) , after [ lanes 3 ( mock ) , 4 5 ] anti - FLAG IP , were used for western using anti - FLAG . Prp5 band is indicated .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O

d) Depletion of Prp5. Wild-type (lanes 1, 3 and 4) and snR81Δ pus1Δ (lanes 2 and 5) cells containing a FLAG-tagged PRP5 were lysed, and anti-FLAG IP was performed at a high concentration of salt. Cell extracts, before (lanes 1 and 2), and after [lanes 3 (mock), 4 and 5] anti-FLAG IP, were used for western analysis using anti-FLAG. The Prp5 band is indicated.


( e ) IP of reconstituted Prp5 ( GAR ) - U2 . An equal amount of FLAG - tagged Prp5 ( GAR ) was added to wild - type ( lane 1 ) snR81Δ pus1Δ ( lane 2 ) extracts depleted of endogenous Prp5 [ see ( c ) ] . After salt was brought back to its original level , anti - FLAG IP was carried out . Prp5 ( GAR ) band , detected by western , is indicated . co - precipitated U1 U2 bands , detected by primer - extension , are also indicated .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) IP of reconstituted Prp5 (GAR)-U2. An equal amount of FLAG-tagged Prp5 (GAR) was added to the wild-type (lane 1) and snR81Δ pus1Δ (lane 2) cell extracts depleted of endogenous Prp5 [see (c)]. After the salt concentration was brought back to its original level, anti-FLAG IP was carried out. The Prp5 (GAR) band, detected by western, is indicated. The co-precipitated U1 and U2 bands, detected by primer-extension, are also indicated.


f ) of U2 that is co - precipitated with mutant Prp5 ( GAR ) . For each lane [ including WT lane snr81Δ pus1Δ lane , corresponding to lane 1 lane 2 of ( e ) , respectively ] , U2 co - precipitated with Prp5 ( GAR ) was normalized against co - precipitated U1 [ ( of U2 band ) / ( of U1 band ) ] . snr81Δ pus1Δ lane was then further normalized against WT lane [ ( value of normalized U2 from snr81Δ pus1Δ lane ) / ( value of normalized U2 from WT lane ) ] . was on three independent experiments .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O

f) Quantification of U2 that is co-precipitated with mutant Prp5 (GAR). For each lane [including the WT lane and the snr81Δ pus1Δ lane, corresponding to lane 1 and lane 2 of (e), respectively], U2 co-precipitated with Prp5 (GAR) was normalized against co-precipitated U1 [(intensity of the U2 band) / (intensity of the U1 band)]. The snr81Δ pus1Δ lane was then further normalized against the WT lane [(the value of normalized U2 from the snr81Δ pus1Δ lane) / (the value of normalized U2 from the WT lane)]. Quantification was based on three independent experiments.


( g ) Prp5 - U2 - pre - mRNA co - IP . Reconstitution was carried out as described in ( d ) . Prior to IP , an equal amount of radiolabeled pre - mRNA was also added to each reaction . Anti - FLAG IP was then performed in presence of ATP . precipitated Prp5 ( WT GAR ) band , detected by western , is indicated . co - precipitated pre - mRNA , directly visualized after electrophoresis , is also indicated . pre - mRNA input is also shown . In lanes 1 2 , wild - type snR81Δ pus1Δ extracts , depleted of Prp5 , were reconstituted with wild - type FLAG - Prp5 . In lanes 3 4 , wild - type snR81Δ pus1Δ extracts , depleted of Prp5 , were reconstituted with mutant FLAG - Prp5 ( GAR ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O

(g) Prp5-U2-pre-mRNA co-IP. Reconstitution was carried out as described in (d). Prior to IP, an equal amount of radiolabeled pre-mRNA was also added to each reaction. Anti-FLAG IP was then performed in the presence of ATP. The precipitated Prp5 (WT and GAR) band, detected by western, is indicated. The co-precipitated pre-mRNA, directly visualized after electrophoresis, is also indicated. The pre-mRNA input is also shown. In lanes 1 and 2, wild-type and snR81Δ pus1Δ cell extracts, depleted of Prp5, were reconstituted with the wild-type FLAG-Prp5. In lanes 3 and 4, wild-type and snR81Δ pus1Δ cell extracts, depleted of Prp5, were reconstituted with mutant FLAG-Prp5 (GAR).


( h ) of pre - mRNA co - precipitated with Prp5 U2 . For each lane in ( g ) , pre - mRNA co - precipitated with Prp5 ( Wild - type or GAR ) was normalized against Prp5 ( WT / GAR ) band in same lane ( detected by Anti - FLAG ) [ ( of pre - mRNA ) / ( of WT / GAR Prp5 ) ] . Lanes 2 , 3 , 4 ( see ( g ) ) were then further normalized against lane 1 ( see ( g ) ) [ ( value of normalized pre - mRNA from lane 2 , 3 or 4 in ( g ) ) / ( value of normalized pre - mRNA from lane 1 in ( g ) ) ] . numbers at bottom of ( h ) correspond to lane numbers of ( g ) . was on three independent experiments .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h) Quantification of pre-mRNA co-precipitated with Prp5 and U2. For each lane in (g), pre-mRNA co-precipitated with Prp5 (Wild-type or GAR) was normalized against the Prp5 (WT/GAR) band in the same lane (detected by Anti-FLAG) [(intensity of pre-mRNA) / (intensity of WT/GAR Prp5)]. Lanes 2,3,and 4 (see (g)) were then further normalized against lane 1 (see (g)) [(the value of normalized pre-mRNA from lane 2, 3 or 4 in (g)) / (the value of normalized pre-mRNA from lane 1 in (g))]. The numbers at the bottom of (h) correspond to the lane numbers of (g). Quantification was based on three independent experiments.


( a ) Prp5 ' s ATPase activity using synthetic U2 . 5 ' of S . cerevisiae U2 is shown ( top ) . three ψs ( 35 , 42 44 ) as well as branch site recognition ( underlined ) are indicated . In ATPase ( bottom ) , no ( lane 3 ) , or an equal amount of LiCl - precipitated ( lane 1 ) or tRNA ( lane 2 ) , a short U2 fragment ( nts 1 - 76 ) ( lane 4 ) , a fully pseudouridylated short U2 fragment ( nts 1 - 76 ) ( lane 5 ) , a long U2 fragment ( nts 1 - 120 ) ( lane 6 ) , or a fully pseudouridylated long U2 fragment ( nts 1 - 120 ) ( lane 7 ) , was used . un - hydrolyzed ATP hydrolyzed product ADP are indicated . Lanes 1 2 , lanes 3 - 5 , lanes 6 7 are from separate gels .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Prp5's ATPase activity assay using synthetic U2. The 5' sequence of S. cerevisiae U2 is shown (top). The three ψs (35, 42 and 44) as well as the branch site recognition sequence (underlined) are indicated. In the ATPase assay (bottom), no RNA (lane 3), or an equal amount of LiCl-precipitated RNA (lane 1) or tRNA (lane 2), a short U2 fragment (nts 1-76) (lane 4), a fully pseudouridylated short U2 fragment (nts 1-76) (lane 5), a long U2 fragment (nts 1-120) (lane 6), or a fully pseudouridylated long U2 fragment (nts 1-120) (lane 7), was used. The un-hydrolyzed ATP and hydrolyzed product ADP are indicated. Lanes 1 and 2, lanes 3-5, and lanes 6 and 7 are from separate gels.


( b ) of Prp5 ' s ATPase activity activated by in vitro transcribed U2 . Prp5 ' s ATPase activity shown in ( a ) was calculated using formula ( ADP ) / ( ADT + ATP ) . numbers at bottom of ( b ) correspond to lane numbers of ( a ) . was on three independent experiments .

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(b) Quantification of Prp5's ATPase activity activated by in vitro transcribed U2. Prp5's ATPase activity shown in (a) was calculated using the formula (ADP) / (ADT+ATP). The numbers at the bottom of (b) correspond to the lane numbers of (a). Quantification was based on three independent experiments.


( c ) U2 - Prp5 binding . FLAG - Prp5 a radiolabeled short ( nts 1 - 76 ) ( lanes 1 2 ) or long ( nts 1 - 120 ) ( lanes 5 6 ) U2 fragment , or FLAG - Prp5 a radiolabeled fully pseudouridylated short ( nts 1 - 76 ) ( lanes 3 4 ) or long ( nts 1 - 120 ) ( lanes 7 8 ) U2 fragment , were incubated under conditions used for ATPase activity . Anti - FLAG IP was then carried out . Co - precipitated ( lanes 2 , 4 , 6 8 ) un - precipitated ( lanes 1 , 3 , 5 7 ) U2 RNAs were analyzed by electrophoresis . U2 band is indicated .

O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O

(c) U2-Prp5 binding assay. FLAG-Prp5 and a radiolabeled short (nts 1-76) (lanes 1 and 2) or long (nts 1-120) (lanes 5 and 6) U2 fragment, or FLAG-Prp5 and a radiolabeled fully pseudouridylated short (nts 1-76) (lanes 3 and 4) or long (nts 1-120) (lanes 7 and 8) U2 fragment, were incubated under the conditions used for ATPase activity assay. Anti-FLAG IP was then carried out. Co-precipitated (lanes 2, 4, 6 and 8) and un-precipitated (lanes 1, 3, 5 and 7) U2 RNAs were analyzed by electrophoresis. The U2 band is indicated.


( d ) of Prp5 - U2 binding . Relative intensities of U2 bands shown in ( c ) were calculated [ setting un - precipitated uridine - containing U2 ( lane 1 5 ) to 1 ] . numbers at bottom of ( d ) correspond to lane numbers of ( c ) . was on three independent experiments .

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(d) Quantification of Prp5-U2 binding. Relative intensities of U2 bands shown in (c) were calculated [setting the un-precipitated uridine-containing U2 (lane 1 and 5) to 1]. The numbers at the bottom of (d) correspond to the lane numbers of (c). Quantification was based on three independent experiments.


( e ) Prp5 ' s ATPase activity using U2 . U2 isolated from wild - type ( lanes 2 4 ) or from snR81Δ pus1Δ ( lanes 6 8 ) was used for ATPase activity . In odd - numbered lanes , no was added . Both wild - type Prp5 ( lanes 1 - 4 ) mutant Prp5 ( GAR ) ( lanes 5 - 8 ) were tested .

O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

(e) Prp5's ATPase activity assay using cellular U2. U2 RNA isolated from the wild-type strain (lanes 2 and 4) or from the snR81Δ pus1Δ strain (lanes 6 and 8) was used for the ATPase activity assay. In odd-numbered lanes, no RNA was added. Both the wild-type Prp5 (lanes 1-4) and the mutant Prp5 (GAR) (lanes 5-8) were tested.


( f ) of Prp5 ' s ATPase activity activated by U2 from . Prp5 ' s ATPase activity shown in ( e ) was calculated using formula ( ADP ) / ( ADT + ATP ) . numbers at bottom of ( f ) correspond to lane numbers of ( e ) . was on four independent experiments .

O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) Quantification of Prp5's ATPase activity activated by U2 from cells. Prp5's ATPase activity shown in (e) was calculated using the formula (ADP) / (ADT+ATP). The numbers at the bottom of (f) correspond to the lane numbers of (e). Quantification was based on four independent experiments.


( g ) U2 level in ATPase activity reactions . RNAs from lanes 2 4 in ( c ) were recovered , U2 level was measured using primer - extension . Lanes 1 - 5 , a titration of U2 snRNA . Lane 6 , U2 from wild - type [ lane 2 of ( c ) ] . Lane 7 , U2 from snR81Δ pus1Δ [ lane 4 of ( c ) ] .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O

(g) U2 level in the ATPase activity reactions. RNAs from lanes 2 and 4 in (c) were recovered, and U2 level was measured using primer-extension. Lanes 1-5, a titration of U2 snRNA. Lane 6, U2 from the wild-type strain [lane 2 of (c)]. Lane 7, U2 from the snR81Δ pus1Δ strain [lane 4 of (c)].


( a ) Native gel of pre - splicing complexes . Pre - splicing assembly was carried out in tube with Prp5 - reconstituted extracts labeled pre - mRNA . Lane 1 , Prp5 - depleted wild - type extract . Lane 2 , Prp5 - depleted wild - type extract , plus wild - type Prp5 . Lane 3 , Prp5 - depleted snR81Δ pus1Δ extract , plus wild - type Prp5 . Lane 4 , Prp5 - depleted wild - type extract , plus mutant Prp5 ( GAR ) . Lane 5 , Prp5 - depleted snR81Δ pus1Δ extract , plus mutant Prp5 ( GAR ) . pre - splicing complexes A B are indicated .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

(a) Native gel analysis of pre-splicing complexes. Pre-splicing complex assembly was carried out in the test tube with Prp5-reconstituted cell extracts and labeled pre-mRNA. Lane 1, Prp5-depleted wild-type cell extract. Lane 2, Prp5-depleted wild-type cell extract, plus wild-type Prp5. Lane 3, Prp5-depleted snR81Δ pus1Δ cell extract, plus wild-type Prp5. Lane 4, Prp5-depleted wild-type cell extract, plus mutant Prp5 (GAR). Lane 5, Prp5-depleted snR81Δ pus1Δ cell extract, plus mutant Prp5 (GAR). The pre-splicing complexes A and B are indicated.


( b ) DMS in vivo probing . After being exposed to DMS , yeast were lysed , total collected , primer - extension carried out ( left panel ) . Lane 1 , that were not exposed to DMS . Lane 2 , wild - type exposed to DMS . Lane 3 , snR81Δ pus1Δ exposed to DMS . Lane 4 , snR81Δ pus1Δ , transformed with a plasmid carrying an artificial guide gene targeting positions 42 44 , exposed to DMS . Lane 5 , snR81Δ pus1Δ , transformed with a plasmid carrying a control guide gene with random guide sequences , exposed to DMS . nucleotides in parentheses are DMS - modified C A residues that are one nucleotide after actual primer - extension stops ( indicated by arrows ) . A partial U2 ( along with BSL structure ) is shown ( right panel ) . DMS - modified adenosine cytosine in BSL are also indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

(b) DMS in vivo probing. After being exposed to DMS, yeast cells were lysed, total RNA collected, and primer-extension analysis carried out (left panel). Lane 1, cells that were not exposed to DMS. Lane 2, wild-type cells exposed to DMS. Lane 3, snR81Δ pus1Δ cells exposed to DMS. Lane 4, snR81Δ pus1Δ cells, transformed with a plasmid carrying an artificial guide RNA gene targeting positions 42 and 44, and exposed to DMS. Lane 5, snR81Δ pus1Δ cells, transformed with a plasmid carrying a control guide RNA gene with random guide sequences, and exposed to DMS. The nucleotides in parentheses are DMS-modified C and A residues that are one nucleotide after the actual primer-extension stops (indicated by the arrows). A partial U2 sequence (along with the BSL structure) is shown (right panel). DMS-modified adenosine and cytosine in the BSL are also indicated.


( c ) Residue accessibility . Relative accessibility ( relative band ) was quantified on three independent experiments . of every band in each lane was normalized against C46 band ( least changed band ; compare lanes 2 - 5 in ( b ) ) in its respective lane [ ( of a band ) / ( of C46 in same lane ) ] . Each C46 - normailzed band in lanes 3 - 5 ( refer to ( b ) ) was then further normalized against its respective counterpart in lane 2 [ ( value of a C46 - normalized band in lanes 3 - 5 ) / ( value of its counterpart in lane 2 ) ] . error bars represent standard deviations of measurements .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Residue accessibility quantification. Relative accessibility (relative band intensity) was quantified based on three independent experiments. The intensity of every band in each lane was normalized against the C46 band (the least changed band; compare lanes 2-5 in (b)) in its respective lane [(intensity of a band) / (intensity of C46 in the same lane)]. Each C46-normailzed band in lanes 3-5 (refer to (b)) was then further normalized against its respective counterpart in lane 2 [(the value of a C46-normalized band in lanes 3-5) / (the value of its counterpart in lane 2)]. The error bars represent the standard deviations of the measurements.


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza assessed as follows ( A ) loss Data information : Data are representative of three independent experiments with 3 - 5 mice per group represented as mean ± S . E . M

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (A) Percentage body weight loss Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza assessed as follows ( B ) Relative amount of PR8 viral NP mRNA , by qPCR Data information : Data are representative of three independent experiments with 3 - 5 mice per group represented as mean ± S . E . M

O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (B) Relative amount of PR8 viral NP mRNA, by qPCR Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza assessed as follows ( C ) Histology of lungs by H & E with pathology scores . Scale bars = 200 μm Data information : Data are representative of three independent experiments with 3 - 5 mice per group represented as mean ± S . E . M

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (C) Histology of lungs by H&E staining with pathology scores. Scale bars = 200 μm Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza assessed as follows ( D ) Absolute number of live CD45 + in whole lungs BAL , by flow cytometry Data information : Data are representative of three independent experiments with 3 - 5 mice per group represented as mean ± S . E . M Statistical significanc was determined with unpaired student ' s T -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (D) Absolute number of live CD45+ cells in whole lungs and BAL, by flow cytometry Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M Statistical significanc was determined with unpaired student's T-test


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza assessed as follows ( E ) Level of LDH in BAL , by LDH cytotoxicity Data information : Data are representative of three independent experiments with 3 - 5 mice per group represented as mean ± S . E . M

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (E) Level of LDH in BAL, by LDH cytotoxicity assay Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza assessed as follows ( F ) Expression of PDPN in lungs , by western blot Data information : Data are representative of three independent experiments with 3 - 5 mice per group represented as mean ± S . E . M

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (F) Expression of PDPN in lungs, by western blot Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M


Mice were infected with sub - lethal dose ( 25 pfu ) of influenza assessed as follows ( G ) Total in BAL , by Bradford . ( H ) of albumin in BAL , by ELISA Data information : Data are representative of three independent experiments with 3 - 5 mice per group represented as mean ± S . E . M . Statistical significanc was determined with unpaired student ' s T -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (G) Total protein in BAL, by Bradford assay. (H) Concentration of albumin in BAL, by ELISA Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M. Statistical significanc was determined with unpaired student's T-test


( A ) Absolute from lungs of sub - lethally influenza infected mice following by flow cytometry . After gating out alveolar macrophages by forward side scatter , immune are defined as follows : Neutrophil ( Ly6G + CD11b + SiglecF - Ly6Cint ) , Eosinophil ( SiglecF + CD11c - / + F4 / 80 - CD64 - ) , Monocyte ( Ly6G - CD11b + MHCII - SiglecF - ) , Natural killer ( NK , B220 - / intNK1 . 1 + ) , B ( B220hiNK1 . 1 - ) , CD8 + T ( TCRβ + CD8 + ) , CD4 + T ( TCRβ + CD4 + ) Data information : Data are representative of three independent experiments with 3 - 4 mice per group represented as mean ± S . E . M . Statistical significance in ( A ) was determined with unpaired student ' s T - . n . s . = not significant

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Absolute cell counts from lungs of sub-lethally influenza virus infected mice following analysis by flow cytometry. After gating out alveolar macrophages by forward and side scatter, immune cells are defined as follows: Neutrophil (Ly6G+CD11b+SiglecF-Ly6Cint), Eosinophil (SiglecF+CD11c-/+F4/80-CD64-), Monocyte (Ly6G-CD11b+MHCII-SiglecF-), Natural killer cell (NK, B220-/intNK1.1+), B cell (B220hiNK1.1-), CD8+ T cell (TCRβ+CD8+), CD4+ T cell (TCRβ+CD4+) Data information: Data are representative of three independent experiments with 3-4 mice per group and represented as mean ± S.E.M. Statistical significance in (A) was determined with unpaired student's T-test. n.s.= not significant


( B ) Flow cytometry plots of neutrophils eosinophils in lungs at days 9 15 post sub - lethal influenza infection . Numbers in plots indicate frequency of gated immune in each throughput Data information : Data are representative of three independent experiments with 3 - 4 mice per group represented as mean ± S . E . M

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Flow cytometry plots of neutrophils and eosinophils in lungs at days 9 and 15 post sub-lethal influenza virus infection. Numbers in plots indicate frequency of gated immune cells in each throughput Data information: Data are representative of three independent experiments with 3-4 mice per group and represented as mean ± S.E.M


Mice were superinfected as described in ( A ) ( B ) Survival of mice following superinfection Data information : Data from ( B ) are combined from two independent experiments , n = 13 mice per experimental group data are represented as mean ± S . E . M . Statistical significance was determined by logrank in (

Mice were superinfected as described in (A) (B) Survival of mice following bacterial superinfection Data information: Data from (B) are combined from two independent experiments, n=13 mice per experimental group All data are represented as mean ± S.E.M. Statistical significance was determined by logrank test in (B


Mice were superinfected as described in ( A ) ( C ) Pneumococcal cfu in BAL of mice Data information Dat are representative of at least two independent experiments with 3 - mice per experimental group data are represented as mean ± S . E . M Statistical significance wa determined b Mann - Whitney U in (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in (A) (C) Pneumococcal cfu in BAL of mice Data information Dat are representative of at least two independent experiments with 3- mice per experimental group All data are represented as mean ± S.E.M Statistical significance wa determined b Mann-Whitney U test in (C


Mice were superinfected as described in ( A ) ( D ) Absolute immune in lungs , assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3 - mice per experimental group . data are represented as mean ± S . E . M . Statistical significanc determined b unpaired student ' s T - in (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in (A) (D) Absolute immune cell counts in lungs, assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3- mice per experimental group. All data are represented as mean ± S.E.M. Statistical significanc determined b unpaired student's T-test in (D


Mice were superinfected as described in ( A ) ( E ) Pneumococcal cfu in BAL of mice 12 h after infection with 2x105 cfu of S . pneumoniae serotype 19F alone ( - Flu ) or following influenza infection ( + Flu ) Data information Dat are representative of at least two independent experiments wit 5 - mice per experimental group . data are represented as mean ± S . E . M . Statistical significance was determined b Mann - Whitney U tes n . s . = not

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in (A) (E) Pneumococcal cfu in BAL of mice 12 h after infection with 2x105 cfu of S. pneumoniae serotype 19F alone (-Flu) or following influenza virus infection (+Flu) Data information Dat are representative of at least two independent experiments wit 5- mice per experimental group. All data are represented as mean ± S.E.M. Statistical significance was determined b Mann-Whitney U tes n.s. = not significant


Mice were superinfected as described in ( A ) ( F ) Pneumococcal cfu in BAL 36 h after infection of naïve mice with 2x105 cfu of S . pneumoniae serotype 19F alone Data information Dat are representative of at least two independent experiments wit 5 - mice per experimental group . data are represented as mean ± S . E .

Mice were superinfected as described in (A) (F) Pneumococcal cfu in BAL 36 h after infection of naïve mice with 2x105 cfu of S. pneumoniae serotype 19F alone Data information Dat are representative of at least two independent experiments wit 5- mice per experimental group. All data are represented as mean ± S.E.M


Mice were superinfected as described in ( A ) ( G ) Absolute neutrophil eosinophil in lungs infected as described in ( E ) , assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3 - mice per experimental group data are represented as mean ± S . E . M . Statistical significance wa determined b ) , unpaired student ' s T - i ( G ( E ) . n . s . = not

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in (A) (G) Absolute neutrophil and eosinophil cell count in lungs infected as described in (E), assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3- mice per experimental group All data are represented as mean ± S.E.M. Statistical significance wa determined b ), unpaired student's T-test i (G (E). n.s. = not significant


( A ) Concentrations of cytokines or chemokines in BAL of days 5 9 post sub - lethal influenza infection as determined by bead array Data information : Data in ( A ) are obtained from one independent experiment with 4 mice per group , results of selected analytes IFNγ , CXCL10 , IL - 6 , IL - 13 IL - 17 are reconfirmed in a separate independent experiment by a customized bead array Significance i was determined by unpaired student ' s T - . n . s . = not

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Concentrations of cytokines or chemokines in the BAL of days 5 and 9 post sub-lethal influenza virus infection as determined by protein bead array Data information: Data in (A) are obtained from one independent experiment with 4 mice per group, and results of selected analytes IFNγ, CXCL10, IL-6, IL-13 and IL-17 are reconfirmed in a separate independent experiment by a customized protein bead array Significance i was determined by unpaired student's T-test. n.s. = not significant


Mice were superinfected as described in Fig 3A . ( B ) Concentrations of IL - 17 IL - 22 in BAL of mice , as determined by ELISA Data informatio Data in ( B are representative of two independent experiments with 3 - 4 mice per group . data are represented as mean ± S . E . M . Significance i was determined by unpaired student ' s T - . n . s . = not

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in Fig 3A. (B) Concentrations of IL-17 and IL-22 in BAL of mice, as determined by ELISA Data informatio Data in (B are representative of two independent experiments with 3-4 mice per group. All data are represented as mean ± S.E.M. Significance i was determined by unpaired student's T-test. n.s. = not significant


Mice were superinfected as described in Fig 3A ( C ) Concentrations of IL - 17 IL - 22 in BAL of mice 12 h after with 2x105 cfu of S . pneumoniae serotype 19F alone ( - Flu ) , or following superinfection ( + Flu ) , assessed by ELISA Data information Dat i ( C ) are representative of two independent experiments with 3 - 4 mice per group . data are represented as mean ± S . E . M . Significanc was determined by unpaired student ' s T - . n . s . = not

O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in Fig 3A (C) Concentrations of IL-17 and IL-22 in BAL of mice 12 h after with 2x105 cfu of S. pneumoniae serotype 19F alone (-Flu), or following superinfection (+Flu), assessed by ELISA Data information Dat i (C) are representative of two independent experiments with 3-4 mice per group. All data are represented as mean ± S.E.M. Significanc was determined by unpaired student's T-test. n.s. = not significant


Mice were superinfected as described in Fig 3A . ( A ) load as determined by enumeration of cfu in BAL following administration of αIL - 17 or αIL - 22 blocking antibody . Control wild - type T - bet - / - groups were administered with IgG isotype control antibody Data information Dat are combined from two independent experiments ( n = 8 - 12 mice per group ) data are represented as mean ± S . E . M . Statistical significance was determined with Kruskal - Wallis followed by Dunn " s multiple comparisons n . s . = not

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in Fig 3A. (A) Bacterial load as determined by enumeration of cfu in BAL following administration of αIL-17 or αIL-22 blocking antibody. Control wild-type and T-bet-/- groups were administered with IgG isotype control antibody Data information Dat are combined from two independent experiments (n = 8-12 mice per group) All data are represented as mean ± S.E.M. Statistical significance was determined with Kruskal-Wallis test followed by Dunn"s multiple comparisons test n.s. = not significant


Mice were superinfected as described in Fig 3A ( B ) T - bet - / - mice were administered αIL - 17 or IgG isotype control antibody every other day from day - 2 post - secondary infection monitored for survival Data information : Dat are combined from two independent experiments ( n = 8 - 12 mice per group ) data are represented as mean ± S . E . M . Statistical significance was determined wit logrank in (

O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in Fig 3A (B) T-bet-/- mice were administered αIL-17 or IgG isotype control antibody every other day from day -2 post-secondary bacterial infection and monitored for survival Data information: Dat are combined from two independent experiments (n = 8-12 mice per group) All data are represented as mean ± S.E.M. Statistical significance was determined wit logrank test in (B


Mice were superinfected as described in Fig 3A ( C ) Absolute neutrophil in lungs following administration of αIL - 17 or IgG isotype control antibody , as determined by flow cytometry Data information Dat are representative of two independent experiments with 3 - 4 in ( C mice per group data are represented as mean ± S . E . M . Statistical significance was determined wit unpaired - students t - in (

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice were superinfected as described in Fig 3A (C) Absolute neutrophil counts in the lungs following administration of αIL-17 or IgG isotype control antibody, as determined by flow cytometry Data information Dat are representative of two independent experiments with 3-4 in (C mice per group All data are represented as mean ± S.E.M. Statistical significance was determined wit unpaired-students t-test in (C


Mice were superinfected as described in Fig 3A ( D ) Pneumococcal cfu in BAL following αLy6G antibody mediated neutrophil depletion . Control T - bet - / - mice were administered with IgG isotype control antibody Data information : Dat are combined from two independent experiments ( n = 8 - 12 mice per group ) data are represented as mean ± S . E . M . Statistical significance was determined wit Mann - Whitney U in (

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Mice were superinfected as described in Fig 3A (D) Pneumococcal cfu in BAL following αLy6G antibody mediated neutrophil depletion. Control T-bet-/- mice were administered with IgG isotype control antibody Data information: Dat are combined from two independent experiments (n = 8-12 mice per group) All data are represented as mean ± S.E.M. Statistical significance was determined wit Mann-Whitney U test in (D


Mice were superinfected as described in Fig 3A ( E ) mRNA expression of antimicrobial peptides in lungs following administration of αIL - 17 , IL - 22 or IgG control antibody , as determined by qPCR Data information Dat are representative of two independent experiments wit 4 - 6 in ( E ) mice per group . data are represented as mean ± S . E . M . Statistical significance was determined wit one - way ANOVA followed by Tukey " s multiple comparisons in ( E ) . n . s . = not

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Mice were superinfected as described in Fig 3A (E) mRNA expression of antimicrobial peptides in lungs following administration of αIL-17, IL-22 or IgG control antibody, as determined by qPCR Data information Dat are representative of two independent experiments wit 4-6 in (E) mice per group. All data are represented as mean ± S.E.M. Statistical significance was determined wit one-way ANOVA followed by Tukey"s multiple comparisons test in (E). n.s. = not signif


Mice were superinfected as described in Fig 3A ( A ) Frequency of IFNγ + IL - 17 + immune following intracellular cytokine flow cytometry Data information : Data are representative of at least two independent experiments with 3 mice per group . data are represented as mean ± S . E . M . Statistical significanc was determined by unpaired student ' s T -

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Mice were superinfected as described in Fig 3A (A) Frequency of IFNγ+ and IL-17+ immune cells following intracellular cytokine staining and flow cytometry Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M. Statistical significanc was determined by unpaired student's T-test


Mice were superinfected as described in Fig 3A ( B ) Representative flow cytometry plots of total IL - 17 + lymphocyte subsets on day 3 post - secondary infected lungs following intracellular cytokine . Numbers in plots indicate frequency of each IL - 17 + subset over total numbers of respective - types Data information : Data are representative of at least two independent experiments with 3 mice per group . data are represented as mean ± S . E . M

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Mice were superinfected as described in Fig 3A (B) Representative flow cytometry plots of total and IL-17+ lymphocyte subsets on day 3 post-secondary bacterial infected lungs following intracellular cytokine staining. Numbers in plots indicate the frequency of each IL-17+ cell subset over total numbers of respective cell-types Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M


Mice were superinfected as described in Fig 3A ( C ) Frequency over total IL - 17 + absolute numbers of each IL - 17 producing lymphocyte subset on days 0 , 1 3 post - secondary superinfected lungs as assessed by flow cytometry of intracellular cytokine stained Data information : Data are representative of at least two independent experiments with 3 mice per group . data are represented as mean ± S . E . M . Statistical significanc i ( C ) was determined by unpaired student ' s T -

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Mice were superinfected as described in Fig 3A (C) Frequency over total IL-17+ cells and absolute numbers of each IL-17 producing lymphocyte subset on days 0, 1 and 3 post-secondary bacterial superinfected lungs as assessed by flow cytometry of intracellular cytokine stained cells Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M. Statistical significanc i (C) was determined by unpaired student's T-test


Clustered of whole chemical genomics screen ; each subsection belongs to an ' s KO library . Grey indicate missing values .

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Clustered heatmap of the whole chemical genomics screen; each subsection belongs to an individual strain's KO library. Grey cells indicate missing values.


S - score scatterplots for each pairwise comparison , highlighting conserved ( black points ) , significant changes ( red points ) gene - condition relationships for which no call can reliably be made ( grey points ) . " r " , pearson ' s r value .

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S-score scatterplots for each pairwise strain comparison, highlighting conserved phenotypes (black points), significant changes (red points) and gene-condition relationships for which no call can reliably be made (grey points). "r", pearson's r value.


Fraction of deletion in each conserved with other stains in pairwise , three - way four - way comparisons . Error bars represent standard deviation for pairwise three - way comparisons . Only a four - way comparison is possible for each so no error bars are represented in these cases .

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Fraction of deletion phenotypes in each strain conserved with other stains in pairwise, three-way and four-way comparisons. Error bars represent standard deviation for all pairwise and three-way comparisons. Only a four-way comparison is possible for each strain so no error bars are represented in these cases.


Gene exclusiveness : a measure of each gene ' s propensity to change its chemical genomics profile across strains . top 10 genes ' names are reported .

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Gene exclusiveness: a measure of each gene's propensity to change its chemical genomics profile across strains. The top 10 genes' names are reported.


Barplots reporting average number of gain sick that are specific to each across pairwise comparisons .

Barplots reporting the average number of gain and sick phenotypes that are specific to each strain across all pairwise comparisons.


Wild type fitness of each relative to S288C across same conditions as in panel A ; each dot represents a specific replicate where colony sizes were measured .

Wild type fitness of each strain relative to S288C across the same conditions as in panel A; each dot represents a specific replicate where colony sizes were measured.


Relationship between wild type fitness relative to S288C number of conditionally essential genes relative to S288C ; each dot represents a - condition replicate as in panel B . conditions maltose , glycerol , NaCl are highlighted .

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Relationship between the wild type fitness relative to S288C and the number of conditionally essential genes relative to S288C; each dot represents a strain-condition replicate as in panel B. The conditions maltose, glycerol, and NaCl are highlighted.


Changes in gene deletion for growth on osmotic stress conditions . top contains genes belonging to HOG pathway , while bottom one those genes whose growth varies most between Y55 YPS . Significant growth are marked with " - " .

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Changes in gene deletion phenotypes for growth on osmotic stress conditions. The top heatmap contains genes belonging to the HOG pathway, while the bottom one those genes whose growth phenotypes varies the most between Y55 and YPS. Significant growth phenotypes are marked with "-".


Changes in deletion for growth on glycerol maltose . top contains MAL genes , while bottom one those genes whose growth vary most between S288C other three strains . Significant growth are marked with " - " .

Changes in deletion phenotypes for growth on glycerol and maltose. The top heatmap contains the MAL genes, while the bottom one those genes whose growth phenotypes vary the most between S288C and the other three strains. Significant growth phenotypes are marked with "-".


S - score of yeast natural isolates across 34 conditions that were also used in KO screening . B ) Number of variants significantly associated ( p - value < 1E - 6 ) with phenotypic variation in each growth condition . C ) Number of associated variants that overlap ( i . e . are in a 3kbp window ) with a conditionally essential gene in same condition , in any of four KO libraries .

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S-score heatmap of the yeast natural isolates across 34 conditions that were also used in the KO screening. B) Number of variants significantly associated (p-value < 1E-6) with phenotypic variation in each growth condition. C) Number of associated variants that overlap (i.e. are in a 3kbp window) with a conditionally essential gene in the same condition, in any of the four KO libraries.


Manhattan plots showing examples of overlaps between associated variants KO screening . top plot shows associations between variants growth in natural isolates as a function of - log10 of association p - value , while four bottom plots show strength of KO across four yeast strains , as a function of - log10 of corrected S - score p - value . Sections shaded in grey indicate overlap between associations KO data . Position in yeast chromosome is reported in kilobase units .

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Manhattan plots showing examples of overlaps between associated variants and the KO screening. The top plot shows the associations between variants and growth in the natural isolates as a function of the -log10 of the association p-value, while the four bottom plots show the strength of the KO phenotypes across the four yeast strains, as a function of the -log10 of the corrected S-score p-value. Sections shaded in grey indicate the overlap between associations and KO data. Position in the yeast chromosome is reported in kilobase units.


C . RADX interacts with ssDNA . Extracts of U2OS stably expressing GFP - RADX WT or mutants were incubated with biotin - coupled ssDNA oligo immobilized on Streptavidin beads , washed extensively immunoblotted with GFP RPA1 antibodies .

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C. RADX interacts with ssDNA. Extracts of U2OS cells stably expressing GFP-RADX WT or mutants were incubated with biotin-coupled ssDNA oligo immobilized on Streptavidin beads, washed extensively and immunoblotted with GFP and RPA1 antibodies.


D . ssDNA - bound Streptavidin beads incubated with extracts as in ( C ) were washed with buffer containing indicated salt concentrations prior to immunoblotting .

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D. ssDNA-bound Streptavidin beads incubated with cell extracts as in (C) were washed with buffer containing indicated salt concentrations prior to immunoblotting.


E . Lysates of untransfected HCT116 were incubated with immobilized ssDNA or dsDNA probes as in ( C ) immunoblotted with RADX RPA1 antibodies .

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E. Lysates of untransfected HCT116 cells were incubated with immobilized ssDNA or dsDNA probes as in (C) and immunoblotted with RADX and RPA1 antibodies.


F . Immunoblot of RADX expression in human lines . See also Fig EV1B .

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F. Immunoblot analysis of RADX expression in human cell lines. See also Fig EV1B.


G . Representative from in situ proximity ligation assays ( PLAs ) in BrdU - labeled U2OS GFP / GFP - RADX lines ( Fig EV1E , F ) using GFP BrdU antibodies under native conditions . Scale bar , 10 μm .

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G. Representative images from in situ proximity ligation assays (PLAs) in BrdU-labeled U2OS and GFP/GFP-RADX cell lines (Fig EV1E,F) using GFP and BrdU antibodies under native conditions. Scale bar, 10 μm.


I . Soluble - enriched fractions of U2OS lines stably expressing GFP - RADX alleles were immunoblotted with indicated antibodies .

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I. Soluble and chromatin-enriched fractions of U2OS cell lines stably expressing GFP-RADX alleles were immunoblotted with indicated antibodies.


A . Immunoblot of fractions of U2OS transfected with indicated siRNAs treated or not with HU .

A. Immunoblot analysis of chromatin fractions of U2OS cells transfected with indicated siRNAs and treated or not with HU.


B . U2OS treated with HU in presence or absence of ATR inhibitor ( ATRi ) were fractionated immunoblotted with indicated antibodies . Relative RADX on were quantified normalized to histone H3 . See also Fig EV2A .

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B. U2OS cells treated with HU in the presence or absence of ATR inhibitor (ATRi) were fractionated and immunoblotted with indicated antibodies. Relative RADX levels on chromatin were quantified and normalized to histone H3. See also Fig EV2A.


C . Representative of U2OS stably expressing indicated GFP - RADX alleles that were subjected to microirradiation , fixed with methanol / acetone immediately afterwards immunostained with γH2AX antibody .

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C. Representative images of U2OS cells stably expressing indicated GFP-RADX alleles that were subjected to laser microirradiation, fixed with methanol/acetone immediately afterwards and immunostained with γH2AX antibody.


D . As in ( C ) , except were fixed with paraformaldehyde 20 min after damage infliction immunostained with PCNA antibody .

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D. As in (C), except cells were fixed with paraformaldehyde 20 min after DNA damage infliction and immunostained with PCNA antibody.


E . Representative from PLAs with GFP RPA2 antibodies in U2OS U2OS / GFP - RADX lines labeled with EdU for 30 min then fixed or exposed to HU or CPT for 4 h before fixation .

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E. Representative images from PLAs with GFP and RPA2 antibodies in U2OS and U2OS/GFP-RADX cell lines labeled with EdU for 30 min and then fixed or exposed to HU or CPT for 4 h before fixation.


A . Immunoblot of HCT116 transfected with indicated RADX siRNAs RADX knockout lines ( RADXΔ ) .

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A. Immunoblot analysis of HCT116 cells transfected with indicated RADX siRNAs and RADX knockout cell lines (RADXΔ).


C . in ( A ) were labeled with consecutive pulses of CldU IdU as shown in ( B ) . Replication fork speeds were calculated as of labeled track divided by time ( bars , mean ; n = 400 , pooled from two independent experiments , per condition ) .

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C. Cells in (A) were labeled with consecutive pulses of CldU and IdU as shown in (B). Replication fork speeds were calculated as length of labeled track divided by pulse time (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


D . Bidirectional replication fork symmetry was calculated as of shorter divided by longer tracks from ( C ) . Concordance is 100 % , representing equal rates of bidirectional elongation for both daughter forks ( bars , mean ; n = 50 bidirectional forks , pooled from two independent experiments , per condition ) .

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D. Bidirectional replication fork symmetry was calculated as percentage of shorter divided by longer tracks from (C). Concordance is 100%, representing equal rates of bidirectional elongation for both daughter forks (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition).


E . Proportion of stalled forks ( CldU - only tracks ) among from ( C ) ( bars , mean ; 200 analyzed per condition ; n = 2 independent experiments ) .

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E. Proportion of stalled forks (CldU-only tracks) among DNA fibers from (C) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


F . Proportion of new origins ( IdU - only tracks ) among in ( C ) ( bars , mean ; 20 fields of view quantified per condition ; n = 2 independent experiments ) .

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F. Proportion of new origins (IdU-only tracks) among DNA fibers in (C) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).


H . Proportion of stalled forks ( CldU - only tracks ) among from labeled as in ( G ) ( bars , mean ; 200 analyzed per condition ; n = 2 independent experiments ) .

H. Proportion of stalled forks (CldU-only tracks) among DNA fibers from cells labeled as in (G) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


I . Proportion of restarted forks ( CldU - IdU - positive tracks ) among in ( G ) ( bars , mean ; 200 analyzed per condition ; n = 2 independent experiments ) .

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I. Proportion of restarted forks (CldU- and IdU-positive tracks) among DNA fibers in (G) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


A . Replication fork speeds in U2OS or U2OS / GFP - RADX transfected with control ( − ) or RADX ( # 6 ) siRNA targeting 3 ' UTR labeled with CldU IdU as in Fig 3B ( bars , mean ; n = 400 , pooled from two independent experiments , per condition ) .

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A. Replication fork speeds in U2OS or U2OS/GFP-RADX cells transfected with control (−) or RADX(#6) siRNA targeting the 3'UTR and labeled with CldU and IdU as in Fig 3B (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


B . Fork symmetry in treated as in ( A ) ( bars , mean ; n = 50 bidirectional forks , pooled from two independent experiments , per condition ) .

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B. Fork symmetry in cells treated as in (A) (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition).


C . Proportion of stalled forks ( CldU - only tracks ) among in ( A ) ( bars , mean ; 200 analyzed per condition ; n = 2 independent experiments ) .

C. Proportion of stalled forks (CldU-only tracks) among DNA fibers in (A) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


D . Proportion of new origins ( IdU - only tracks ) among in ( A ) ( bars , mean ; 20 fields of view quantified per condition ; n = 2 independent experiments ) .

D. Proportion of new origins (IdU-only tracks) among DNA fibers in (A) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).


E . Parental U2OS derivative lines stably expressing GFP - RADX alleles or three RPA isoforms at near - endogenous ( U2OS / Super - RPA ) ( Fig EV4A ) were transfected with control ( − ) or RADX siRNA . Replication fork speeds were determined as in ( A ) ( bars , mean ; n = 400 , pooled from two independent experiments , per condition ) .

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E. Parental U2OS and derivative lines stably expressing GFP-RADX alleles or all three RPA isoforms at near-endogenous levels (U2OS/Super-RPA) (Fig EV4A) were transfected with control (−) or RADX siRNA. Replication fork speeds were determined as in (A) (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


F . Replication fork speeds in HCT116 with indicated genotypes transfected with control ( − ) , RPA1 ( 0 . 2 nM [ 17 ] ) or RAD51 siRNAs ( Fig EV4B ) labeled with CldU IdU as in Fig 3B ( bars , mean ; n = 400 , pooled from two independent experiments , per condition ) .

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F. Replication fork speeds in HCT116 cells with indicated genotypes transfected with control (−), RPA1 (0.2 nM concentration [17]) or RAD51 siRNAs (Fig EV4B) and labeled with CldU and IdU as in Fig 3B (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


G . Fork symmetry among in ( F ) ( bars , mean ; n = 50 bidirectional forks , pooled from two independent experiments , per condition ) .

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G. Fork symmetry among DNA fibers in (F) (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition).


H . New origin firing among in ( F ) ( bars , mean ; 20 fields of view quantified per condition ; n = 2 independent experiments ) .

H. New origin firing among DNA fibers in (F) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).


B . Replication fork degradation ( IdU / CldU ) in U2OS transfected with indicated siRNAs processed as in ( A ) ( bars , mean ; n = 200 , pooled from two independent experiments , per condition ) .

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B. Replication fork degradation (IdU/CldU ratio) in U2OS cells transfected with indicated siRNAs and processed as in (A) (bars, mean; n=200 fibers, pooled from two independent experiments, per condition).


C . As in ( B ) , except fork degradation was analyzed in U2OS / GFP - RADX lines .

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C. As in (B), except fork degradation was analyzed in U2OS/GFP-RADX cell lines.


D . U2OS or U2OS / GFP - RADX transfected with indicated siRNAs exposed to HU for 4 h were co - immunostained with RPA1 γ - H2AX antibodies analyzed by QIBC . Proportion of displaying maximal RPA loading accompanied by H2AX hyperphosphorylation ( Fig EV5A ) , reflecting replication fork catastrophe [ 17 ] , is indicated ( bars , mean ; n = 5 independent experiments ( ≥ 3000 analyzed per condition ) ; * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , unpaired t - ) .

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D. U2OS or U2OS/GFP-RADX cells transfected with indicated siRNAs and exposed to HU for 4 h were co-immunostained with RPA1 and γ-H2AX antibodies and analyzed by QIBC. Proportion of cells displaying maximal RPA chromatin loading accompanied by H2AX hyperphosphorylation (Fig EV5A), reflecting replication fork catastrophe [17], is indicated (bars, mean; n=5 independent experiments (≥3000 cells analyzed per condition); *P≤0.05,**P≤0.01,***P≤0.001,****P≤0.0001, unpaired t-test).


E . As in ( D ) , but using U2OS or U2OS / Super - RPA exposed or not to HU ( n = 6 independent experiments ) . See also Fig EV5B .

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E. As in (D), but using U2OS or U2OS/Super-RPA cells exposed or not to HU (n=6 independent experiments). See also Fig EV5B.