Volume 167, Issue 5, Pages e15 (November 2016)

Slides:



Advertisements
Similar presentations
Figure 1. Cdc48 is cotranscriptionally recruited on active genes
Advertisements

Cotranscriptional Recruitment of the mRNA Export Factor Yra1 by Direct Interaction with the 3′ End Processing Factor Pcf11  Sara Ann Johnson, Gabrielle.
Volume 55, Issue 1, Pages (July 2014)
Volume 28, Issue 4, Pages (November 2007)
DELLAs Modulate Jasmonate Signaling via Competitive Binding to JAZs
Telomerase RNA TLC1 Shuttling to the Cytoplasm Requires mRNA Export Factors and Is Important for Telomere Maintenance  Haijia Wu, Daniel Becker, Heike.
Volume 26, Issue 1, Pages (April 2007)
Volume 18, Issue 6, Pages (June 2005)
Volume 37, Issue 1, Pages (January 2010)
Volume 68, Issue 1, Pages e5 (October 2017)
Human Senataxin Resolves RNA/DNA Hybrids Formed at Transcriptional Pause Sites to Promote Xrn2-Dependent Termination  Konstantina Skourti-Stathaki, Nicholas J.
John T. Arigo, Kristina L. Carroll, Jessica M. Ames, Jeffry L. Corden 
Volume 30, Issue 3, Pages (May 2008)
Volume 58, Issue 6, Pages (June 2015)
Volume 135, Issue 2, Pages (October 2008)
Volume 23, Issue 2, Pages (July 2006)
The Intracellular Domain of the Frazzled/DCC Receptor Is a Transcription Factor Required for Commissural Axon Guidance  Alexandra Neuhaus-Follini, Greg J.
Volume 27, Issue 2, Pages (July 2007)
Volume 27, Issue 21, Pages e5 (November 2017)
John T. Arigo, Daniel E. Eyler, Kristina L. Carroll, Jeffry L. Corden 
Glucose-Induced β-Catenin Acetylation Enhances Wnt Signaling in Cancer
Phosphorylation of Serine 2 within the RNA Polymerase II C-Terminal Domain Couples Transcription and 3′ End Processing  Seong Hoon Ahn, Minkyu Kim, Stephen.
Ashton Breitkreutz, Lorrie Boucher, Mike Tyers  Current Biology 
Jungmook Lyu, Vicky Yamamoto, Wange Lu  Developmental Cell 
Volume 60, Issue 3, Pages (November 2015)
Antonin Morillon, Nickoletta Karabetsou, Anitha Nair, Jane Mellor 
Young-Hee Cho, Sang-Dong Yoo, Jen Sheen  Cell 
Yutian Peng, Lois S. Weisman  Developmental Cell 
Andrew W Snowden, Philip D Gregory, Casey C Case, Carl O Pabo 
Volume 28, Issue 3, Pages e3 (February 2018)
Targeted Proteomic Study of the Cyclin-Cdk Module
Hyunsuk Suh, Dane Z. Hazelbaker, Luis M. Soares, Stephen Buratowski 
A Single Templating RNA in Yeast Telomerase
Volume 48, Issue 4, Pages (November 2012)
Nuclear Retention Prevents Premature Cytoplasmic Appearance of mRNA
Volume 26, Issue 4, Pages (May 2007)
Arabidopsis MSBP1 Is Activated by HY5 and HYH and Is Involved in Photomorphogenesis and Brassinosteroid Sensitivity Regulation  Shi Qiu-Ming , Yang Xi.
Regulation of Ribonucleotide Reductase in Response to Iron Deficiency
Xudong Wu, Jens Vilstrup Johansen, Kristian Helin  Molecular Cell 
Dimethylation of H3K4 by Set1 Recruits the Set3 Histone Deacetylase Complex to 5′ Transcribed Regions  TaeSoo Kim, Stephen Buratowski  Cell  Volume 137,
Cotranscriptional Recruitment of the mRNA Export Factor Yra1 by Direct Interaction with the 3′ End Processing Factor Pcf11  Sara Ann Johnson, Gabrielle.
Volume 26, Issue 1, Pages (April 2007)
Distinct Pathways for snoRNA and mRNA Termination
Volume 68, Issue 5, Pages e3 (December 2017)
Volume 20, Issue 5, Pages (November 2016)
Volume 12, Issue 5, Pages (November 2003)
Arabidopsis WRKY45 Interacts with the DELLA Protein RGL1 to Positively Regulate Age-Triggered Leaf Senescence  Ligang Chen, Shengyuan Xiang, Yanli Chen,
Two Functional Modes of a Nuclear Receptor-Recruited Arginine Methyltransferase in Transcriptional Activation  María J. Barrero, Sohail Malik  Molecular.
Arabidopsis NF-YCs Mediate the Light-Controlled Hypocotyl Elongation via Modulating Histone Acetylation  Yang Tang, Xuncheng Liu, Xu Liu, Yuge Li, Keqiang.
Mst1 Is an Interacting Protein that Mediates PHLPPs' Induced Apoptosis
Xiang Han, Hao Yu, Rongrong Yuan, Yan Yang, Fengying An, Genji Qin
Volume 30, Issue 4, Pages (May 2008)
Volume 116, Issue 1, Pages (January 2004)
Volume 60, Issue 5, Pages (December 2015)
Short Telomeres in ESCs Lead to Unstable Differentiation
Volume 45, Issue 3, Pages (February 2012)
Volume 61, Issue 2, Pages (January 2016)
CNOT3-Dependent mRNA Deadenylation Safeguards the Pluripotent State
H2B Ubiquitylation Controls the Formation of Export-Competent mRNP
Growth Factor-Dependent Trafficking of Cerebellar NMDA Receptors via Protein Kinase B/Akt Phosphorylation of NR2C  Bo-Shiun Chen, Katherine W. Roche 
Adelina A. Davies, Andrea Neiss, Helle D. Ulrich  Cell 
Volume 25, Issue 11, Pages e5 (December 2018)
Mahesh Ramamoorthy, Susan Smith  Cancer Cell 
Volume 26, Issue 12, Pages e4 (March 2019)
Volume 55, Issue 1, Pages (July 2014)
Volume 15, Issue 16, Pages (August 2005)
Condensin and Hmo1 Mediate a Starvation-Induced Transcriptional Position Effect within the Ribosomal DNA Array  Danni Wang, Andres Mansisidor, Gayathri.
Volume 41, Issue 4, Pages (February 2011)
Volume 62, Issue 6, Pages (June 2016)
Volume 9, Issue 5, Pages (May 2002)
Presentation transcript:

Volume 167, Issue 5, Pages 1201-1214.e15 (November 2016) The Chromatin Remodeler ISW1 Is a Quality Control Factor that Surveys Nuclear mRNP Biogenesis  Anna Babour, Qingtang Shen, Julien Dos-Santos, Struan Murray, Alexandre Gay, Drice Challal, Milo Fasken, Benoît Palancade, Anita Corbett, Domenico Libri, Jane Mellor, Catherine Dargemont  Cell  Volume 167, Issue 5, Pages 1201-1214.e15 (November 2016) DOI: 10.1016/j.cell.2016.10.048 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cell 2016 167, 1201-1214.e15DOI: (10.1016/j.cell.2016.10.048) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 The Chromatin-Associated ISW1 Complex Controls Nuclear Accumulation of Poly(A) RNA (A) Co-immunoprecipitation of Isw1-13Myc and Mex67. Mock, pre-immune serum. (B) Inactivation of the ISW1 complex does not affect poly (A) RNA localization as observed by oligo dT FISH analyses (n = 3, mean ± SD). (C) Deletion of ISW1 rescues the growth of the mex67ΔUBA mutant. The indicated strains containing pRS316-MEX67 were grown at 30°C on 5-FOA plates to counter select pRS316-MEX67. (D) Inactivation of the ISW1 complex rescues the poly(A) RNA nuclear accumulation defect of the mex67ΔUBA mutant. FISH analysis and quantification was performed as in (B) in the indicated strains grown for 2 hr at 30°C. (E) Deletion of each subunit of the ISW1 complex rescues the growth of the npl3-1 mutant at 30°C on YPD. (F) Inactivation of the ISW1 complex reduces the poly(A) nuclear accumulation defect of the npl3-1 mutant. Subcellular localization of poly(A) RNA was analyzed as in (B) in the different strains grown overnight at 25°C in YPD and shifted for 3 hr at 30°C prior to fixation. Scale bar, 5 μm. See also Figure S1. (G) Overexpression of Isw1 inhibits the growth of npl3-1 cells. Left: serial dilutions of strains grown on selective media. Right: total protein extracts from WT ISW1-3FL or npl3-1 ISW1-3FL cells transformed with pRS415GPD or pRS415GPD3FL-ISW1 were analyzed by western blot with anti-FLAG and anti-Mex67 (loading control) antibodies. See also Figure S1. Cell 2016 167, 1201-1214.e15DOI: (10.1016/j.cell.2016.10.048) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 The Function of ISW1 in Nuclear mRNP Biogenesis Depends on Its Recruitment onto Chromatin, but Not on Its Nucleosome Sliding Activity (A) Deletion of ISW1 but not of other chromatin remodeling complexes rescues the growth of the npl3-1 mutant at 30°C. Growth of single mutants is shown on Figure S2A. (B) Schematic representation of the domains organization of Isw1. The SANT and SLIDE domains of Isw1 facilitate its interaction with chromatin via Set1-dependent H3K4 methylation whereas Ioc4 is recruited by the interaction of its PWWP domain with Set2-dependent H3K36 methylation. (C–F) Preventing ISW1 complex association to chromatin (D–F) but not inactivating its catalytic activity (C) recapitulates the effect of its inactivation on the growth of the npl3-1 mutant. ISW1 chromatin association was prevented by deleting Isw1 SANT and SLIDE domains (D), inhibiting H3K4me3 (E) or H3K36me3 (F). Expression of Isw1 mutant proteins is shown on Figure S2B. See also Figure S2. Cell 2016 167, 1201-1214.e15DOI: (10.1016/j.cell.2016.10.048) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Inactivation of ISW1 Rescues Nuclear Export of Improper mRNPs and Resulting Genetic Instability (A and B) Deletion of ISW1 does not affect transcription elongation. Serial dilutions of the indicated strains were grown with or without MPA. (C) LYS2 transcription shut off experimental setting: npl3-1 and npl3-1 isw1Δ cells were shifted from 25°C to 30°C for 1 hr and transcription was blocked by addition of phenanthroline (t = 0). Samples were collected for analysis at t = 0, 30′,and 60′. (D) Similar CTD recruitment to LYS2 in npl3-1 and npl3-1 isw1Δ cells analyzed by chromatin immunoprecipitation (ChIP) and normalized to the value at t = 0 (n = 3, mean ± SD). See Figure S3C for non-normalized values. (E) npl3-1 and npl3-1 isw1Δ show similar LYS2 mRNA levels as analyzed by qRT-PCR and normalized to ACT1 mRNA expression (n = 5, mean ± SD). (F) Deletion of ISW1 releases the LYS2 transcripts accumulated in a nuclear dot of npl3-1 cells. The subcellular localization of the LYS2 transcript after blocking transcription with phenanthroline in npl3-1 and npl3-1 isw1Δ cells was analyzed by FISH using Quasar570 -LYS2 probes. For each time point, the percentage of cells showing a nuclear dot was scored (n = 3, mean ± SD). White arrows point to nuclear localized LYS2 transcripts. Scale bar, 5 μm. (G) ISW1 inactivation reduces the number of spontaneous Rad52 foci in npl3-1 cells grown for 3 hr at 30°C. Fluorescence microscopic examination of the indicated cells transformed with a pRS415-Rad52-YFP plasmid. White arrows highlight Rad52 foci in npl3-1 unbudded cells. For each cell type, an average of 300 budded and unbudded cells were examined (n = 3, mean ± SD). (H) ISW1 inactivation partially rescues the hyperrecombination phenotype of the npl3-1 mutant. Recombination was analyzed in the indicated strains carrying pL or pLYΔN plasmids, grown for 3 hr at 30°C and plated at 25°C. Average and standard deviation of three fluctuation tests consisting of the median value of 12 independent colonies for each condition are shown. See also Figure S3. Cell 2016 167, 1201-1214.e15DOI: (10.1016/j.cell.2016.10.048) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Isw1 Participates in mRNP Surveillance (A) ISW1 deletion displays synthetic negative genetic interaction with XRN1 and MLP1 MLP2 but is neutral toward RRP6. (B) ISW1 and RRP6 deletions rescue the growth of the npl3-1 mutant and show additive effects. (C) A catalytically inactive version of Rrp6 rescues the growth of npl3-1 cells at 30°C. For (C), serial dilutions of the npl3-1, npl3-1 rrp6Δ, and npl3-1 rrp6Δ isw1Δ strains transformed with an empty pRS415 (−), with pRS415-RRP6 or pRS415-rrp6D238A. (D) Deletions of ISW1 and RRP6 synergistically restore the cytoplasmic localization of the LYS2 transcript in npl3-1 cells grown overnight at 30°C as analyzed by FISH using Quasar570 -LYS2 probes. Noteworthy, while npl3-1 cells are misshapen, inactivation of ISW1 or RRP6 partially restores their shape and npl3-1 isw1Δ rrp6Δ cells are indistinguishable from WT. Scale bar, 5 μm. For each strain, the number of cells containing only one nuclear dot (black box), no signal (white), less (gray), or more (hatched) than four cytoplasmic dots was quantified on at least 300 cells (n = 2, mean ± SD). (E) Isw1 interacts with the Rrp6 and Rrp4 subunits of the nuclear exosome in WT cells and this interaction is fostered in npl3-1 cells shifted for 3 hr at 30°C. Cell lysates (Input) and immunoprecipitates (IP) were analyzed by immunoblotting with anti-tags or anti-Mex67 antibodies. (−), HA, untagged strain. The ratio of co-immunoprecipitated Isw1-13myc relative to immunoprecipitated Rrp4-3HA (n = 3, mean ± SD). See also Figure S4. Cell 2016 167, 1201-1214.e15DOI: (10.1016/j.cell.2016.10.048) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Inactivation of ISW1 Rescues the Nuclear Export of the lys2-370 Transcript (A) ISW1 and RRP6 deletions rescue the growth of the lys2-370 mutant on DO-LYS and have additive effects. (B) Effect of ISW1 and RRP6 deletions on the total level of the LYS2/lys2-370 transcript, analyzed by qRT-PCR and normalized by the expression of ACT1 transcript (n = 3, mean ± SD). (C) FISH analysis of the subcellular localization of the LYS2 transcript, using Quasar570 -LYS2 probes in strains grown overnight at 30°C in YPD. Scale bar, 5 μm. For each strain, the number of cells containing only one nuclear dot (black box), less (white), or more (hatched) than four cytoplasmic dots was quantified on at least 300 cells (n = 2, mean ± SD). (D) The lys2-370 transcript interacts with Isw1. RNA immunoprecipitation experiments were performed with PrA-tagged Isw1 in LYS2 and lys2-370 strains. The ratio of co-immunoprecipitated ACT1 or LYS2 RNA relative to the total RNA present in each strain quantified by qRT-PCR is represented. (n = 3, mean ± SD). Untagged WT cells were used as negative (−) control. Every IP is significant (p < 0.01) compared to the untagged strain. See also Figure S5. Cell 2016 167, 1201-1214.e15DOI: (10.1016/j.cell.2016.10.048) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Isw1 Interacts with Nuclear mRNPs (A) Venn diagram for transcripts statistically enriched in WT, npl3-1, and no tag strains. (B) The interaction between Isw1 and its mRNA targets is increased in the npl3-1 mutant compared to WT. RNA immunoprecipitation experiments were performed with PrA-tagged Isw1 in WT and npl3-1 cells grown for 3 hr at 30°C. The ratio of five co-immunoprecipitated mRNA targets identified in the genome-wide analysis (IOC2, IOC3, INO80, MDN1, HAP1) relative to the total RNA present in each strain was quantified by qRT-PCR (n = 3, mean ± SD). One representative and not normalized experiment is shown in Figure S6C. (C, D, and F) Average levels of sense transcript (C), nucleosome occupancy (D), and Isw1 (F) at those genes whose transcripts are statistically enriched for, in an Isw1-PrA immunoprecipitate. Shown are the classes of genes enriched in WT (blue) and in npl3-1 (red) strains, compared to all protein-coding, non-dubious genes in the yeast genome (green). (E) Histograms showing length distributions of transcripts enriched in WT (blue) and in npl3-1 (red) strains, compared to all protein-coding transcripts in the yeast genome (green). See also Figure S6 and Table S1. (G) Isw1 UV cross-links to RNA in vivo. HTP tagged Isw1 was cross-linked (+) or not (−) and purified from cell extracts. A total of 2.5% of the nickel eluate was resolved by SDS-PAGE after (lanes 5–6) or not (lanes 3–4) RNase treatment and detected by autoradiography (upper panel) or anti-HIS western blot (lower panel). An untagged strain was used as a control (lanes 1–2). The red asterisk indicates a contaminant band. See also Figure S6. Cell 2016 167, 1201-1214.e15DOI: (10.1016/j.cell.2016.10.048) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure S1 Related to Figure 1 (A) Left: The interaction between Isw1 and Mex67 is partially sensitive to RNase. Co-immunoprecipitation of Isw1-13Myc and Mex67 were assayed from lysates treated or not with 200 μg/mL RNaseA. ∗ indicates the position of IgG heavy chain. Right: Isw2-HA does not co-precipitate with Mex67. Mock = pre-immune serum. (B) Deletion of each subunit of the ISW1 complex does not affect cell growth at 30°C. Fivefold serial dilutions of strains grown for 2 days on YPD. (C) Inactivation of ISW1 complex restores the growth of some mRNA export mutants. Fivefold serial dilutions of strains grown at the indicated temperatures on YPD (mft1Δ, thp1Δ, rna15-58, pap1-1, fip1-206, sen 1-1, ref2Δ and nup159-1) or DO-LEU (GFP-yra1-8, ΔN-nab2). (D) ISW1 deletion does not affect bulk poly(A) tail length. Bulk poly(A) tail length was analyzed in indicated strains grown 3 hr at 30°C. (E) Upper panel: Fivefold serial dilutions of strains grown for 4 days on selective media at 25°C. Lower panel: ChIP analysis of Isw1-3FL recruitment to PMA1 in the indicated strains. Cell 2016 167, 1201-1214.e15DOI: (10.1016/j.cell.2016.10.048) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure S2 Related to Figure 2 (A) Effect of the inactivation of one subunit of various chromatin remodelers on cell growth. Fivefold serial dilutions of strains grown for 2 days on YPD. (B) The WT and mutant forms of Isw1 have comparable expression levels. Total protein extracts from npl3-1 isw1Δ cells transformed with pRS416, pRS416-ISW1-2FL, pRS416-isw1K227R-2FL, pRS416-isw1ΔSANT-2FL or pRS416-isw1ΔSLIDE-2FL were analyzed by western blot with anti-FLAG and anti-Mex67 (loading control) antibodies. Cell 2016 167, 1201-1214.e15DOI: (10.1016/j.cell.2016.10.048) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure S3 Related to Figure 3 (A) Deletion of ISW1 or DST1 does not affect cell growth at 30°C. (B) Mycophenolic acid (MPA) does not affect the growth of isw1Δ cells. Fivefold serial dilutions of the indicated strains grown on YPD plates (A) or on MPA containing YPD plates (B). (C) Similar CTD recruitment to LYS2 in npl3-1 and npl3-1 isw1Δ cells analyzed by ChIP (n = 3, mean ± sd). (D) IMD2 induction/chase experimental setting: the IMD2 gene was induced in npl3-1 and npl3-1 isw1Δ cells grown for 1h at 30°C in SC-URA by addition of 75 μg/mL 6-Azauracil (+6AU) during 1h (Induction). At t = 0, cells were washed in SC media free of 6AU and samples were collected for analysis (Repression). (E) Similar CTD recruitment to IMD2 in npl3-1 and npl3-1 isw1Δ cells. Error bars, sd of n = 5 independent biological repeats. (F) Similar IMD2 mRNA level in npl3-1 and npl3-1 isw1Δ cells as analyzed by RT-qPCR and normalized to ACT1 mRNA expression (n = 3, mean ± sd). (G) The IMD2 transcript is nuclear retained in npl3-1 cells and exported upon ISW1 deletion. The fate of the IMD2 transcript was compared between npl3-1 and npl3-1 isw1Δ cells by analyzing the subcellular localization of the IMD2 transcripts by FISH with an IMD2-Quasar 570 probe. Scale bar 5 μm. For each time point, the percentage of cells showing a nuclear dot was scored. At least 200 Hoechst-stained cells per condition were examined in 3 independent experiments. Error bars, sd. Cell 2016 167, 1201-1214.e15DOI: (10.1016/j.cell.2016.10.048) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure S4 Related to Figure 4 (A) ISW1 deletion is neutral toward RRP6 deletion at all tested temperatures. (B) RRP6 inactivation impairs the growth of npl3-1 cells at 25°C but rescues their growth at 30°C. (C) The WT and mutant forms of Rrp6 have comparable expression levels. Total protein extracts from npl3-1, npl3-1 rrp6Δ cells transformed with pRS415, pRS415-RRP6, pRS415-rrp6D238A, were analyzed by western blot with anti-Rrp6 and anti-Mex67 (loading control) antibodies. (D) Effect of TRF4 and ISW1 inactivation on the growth of WT cells. (E) ISW1 and TRF4 deletions rescue the growth at 30°C of the npl3-1 mutant and show additive effects. Opposite effects are observed at 25°C. (A, B, D, E) Cell growth of indicated strains was analyzed at indicated temperatures using fivefold serial dilutions. (F) The Isw1 Rrp4 interaction is insensitive to RNase. The interaction between Rrp4-HA and Isw1-13Myc or Mex67 was analyzed by co-immunoprecipitation as in Figure 4E with or without treatment with 200 μg/mL RNase A. (G) Isw2-HA does not co-immunoprecipitate with Rrp6. Mock = unrelated Ab. (H) Increased co-localization of Isw1 and Rrp6 in npl3-1 cells. Localization of genomically tagged Isw1-GFP and Rrp6-mRFP was analyzed in WT and npl3-1 cells shifted for 3 hr at 30°C. Bar, 5 μm. For quantification of the Isw1-GFP/Rrp6-mRFP co-localization, the overlapping area between the Isw1-GFP and Rrp6-mRFP signals was determined using the “image calculator” function of the ImageJ software on binary images and measured with the “Analyze particles” function. 16 classes of size of overlapping areas were arbitrary defined. The frequency of cells per class was calculated for 5 independent experiments in each of which 75 to 150 cells were analyzed. Error bars, sem. (I) Rrp6 delocalizes from the nucleolus in npl3-1 cells. Localization of genomically tagged Isw1-mRFP and Nop1-GFP (left panel) or Rrp6-mRFP and Nop1-GFP was analyzed in WT and npl3-1 cells shifted to 30°C for 3 hr. Cell 2016 167, 1201-1214.e15DOI: (10.1016/j.cell.2016.10.048) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure S5 Related to Figure 5 (A) ISW1 deletion restores the growth of the lys2-370 mutant on DO-LYS. (B) Effect of ISW1 and RRP6 deletions on the lys2-370 transcripts distribution as shown in Figure 5C. Quantification of the number of LYS2 transcripts for one representative experiment: for each cell type the percentage of cells containing only a nuclear dot (N), 1, to more than 10 cytoplasmic transcripts was scored. (C) RIP assay allows detecting the interaction of Cbp20 with ACT1 and LYS2 transcripts. RNA immunoprecipitation experiments were performed with CBP20-TAP or untagged strains. The ratio of co-immunoprecipitated ACT1 or LYS2 RNA relative to the total RNA present in each strain quantified by RT-qPCR is represented as a mean of 3 replicates (mean ± sd). Significance of the observed differences was evaluated using Student’s t test (∗P 0.01–0.05; ∗∗P 0.001–0.01; ∗∗∗p < 0.001). Cell 2016 167, 1201-1214.e15DOI: (10.1016/j.cell.2016.10.048) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure S6 Related to Figure 6 (A) Isw1 specifically interacts with its identified targets. RNA immunoprecipitation experiments were performed with PrA-tagged Isw1 and TAP-tagged Cbp20 cells. The ratio of co-immunoprecipitated PMA1 (non Isw1 target) or IOC3 (Isw1 target) RNA relative to the total RNA present in each strain quantified by RT-qPCR is represented as a mean of 3 biological replicates (mean ± sd). Untagged WT cells (No Tag) were used as negative control. Significance of the observed differences was evaluated using Student’s t test (∗P 0.01–0.05; ∗∗P 0.001–0.01; ∗∗∗p < 0.001). (B) The interaction between Isw1 and its mRNA targets is increased in the npl3-1 mutant compared to WT. RNA immunoprecipitation experiments were performed with PrA-tagged Isw1 in WT and npl3-1 cells grown for 3 hr at 30°C. The ratio of 5 co-immunoprecipitated mRNA targets identified in the genome-wide analysis (IOC2, IOC3, INO80, MDN1, HAP1) relative to the total RNA present in each strain was quantified by RT-qPCR. Here is shown a representative experiment. C-F Average levels of X at those genes whose transcripts are statistically enriched for, in an Isw1-PrA immunoprecipitate. Shown are the classes of genes enriched in WT (blue) and in npl3-1 (red) strains, compared to all protein-coding, non-dubious genes in the yeast genome (green). (G) Ioc2 but not Rpb3 UV cross-links to RNA in vivo. Left: HTP tagged Ioc2 was cross-linked (+) or not (-) and purified from cell extracts. 2.5% of the nickel eluates were resolved by SDS-PAGE after (lanes 3-4) or not (lanes 1-2) RNase treatment and detected by autoradiography (upper panel) or anti-HIS western blot (lower panel). The red asterisk indicates a contaminant band. Right: HTP tagged Rpb3 (1) was cross-linked and purified from cell extracts. 2.5% of the nickel eluates were resolved by SDS-PAGE and detected by autoradiography (upper panel) or anti-Rpb3 western blot (lower panel). An untagged WT strain served as a negative control (2). Cell 2016 167, 1201-1214.e15DOI: (10.1016/j.cell.2016.10.048) Copyright © 2016 Elsevier Inc. Terms and Conditions