Rapid identification of mRNA processing defects with a novel single-cell yeast reporter

Sorenson, Matthew; Stevens, Scott W.

doi:10.1261/rna.042663.113

Cited by 12 publications

(17 citation statements)

References 75 publications

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“…Our clustering analysis pipeline vectorizes the data to compare the shape of the 2-dimensional reporter data in a non-directed manner. 44 As with the deletion collection, the majority of histone H3 and H4 mutants did not differ significantly from wild-type (Fig. S1).…”

Section: Resultsmentioning

confidence: 82%

“…44 Briefly, our reporter results in expression of the fluorescent proteins mCherry and GFP, serving as a proxy for in vivo levels of reporter pre-mRNA and spliced mRNA, respectively (Fig. 1E).…”

Section: Resultsmentioning

confidence: 99%

“…Given the aforementioned findings, as well as the growing amount of work describing links between histone modifications and pre-mRNA splicing, we analyzed a collection of 486 histone H3 and H4 substitution and deletion mutants with our reporter via highthroughput flow cytometry. 45 The resulting histone mutant data were incorporated into the deletion collection dataset consisting of nearly 5000 unique deletion mutants 44 and re-clustered (raw data and processed data provided as Supporting Data sets S1 and S2-S4, respectively). Our clustering analysis pipeline vectorizes the data to compare the shape of the 2-dimensional reporter data in a non-directed manner.…”

Section: Resultsmentioning

confidence: 99%

“…For instance we observe a redshift in mutants of nonsense mediated decay and green-shifts in mutants of mRNA export and specific mRNA decay/pathways. 44 Therefore, orthogonal assays are necessary to confirm the specific gene expression pathway(s) affected.…”

Section: Resultsmentioning

confidence: 99%

“…These studies suggested that deletion of several transcription factors and histone modifiers may cause a pre-mRNA splicing defect. 44 Here, we sought to further characterize the role of histone modification in RNA splicing. Utilizing the reporter to probe for splicing defects in a library consisting of hundreds of synthetic histone point mutants, 45 we identified several histone point mutations displaying splicinglike defects.…”

Section: Introductionmentioning

confidence: 99%

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Histone H3K36 methylation regulates pre-mRNA splicing inSaccharomyces cerevisiae

et al. 2016

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Co-transcriptional splicing takes place in the context of a highly dynamic chromatin architecture, yet the role of chromatin restructuring in coordinating transcription with RNA splicing has not been fully resolved. To further define the contribution of histone modifications to pre-mRNA splicing in Saccharomyces cerevisiae, we probed a library of histone point mutants using a reporter to monitor pre-mRNA splicing. We found that mutation of H3 lysine 36 (H3K36) -a residue methylated by Set2 during transcription elongation -exhibited phenotypes similar to those of pre-mRNA splicing mutants. We identified genetic interactions between genes encoding RNA splicing factors and genes encoding the H3K36 methyltransferase Set2 and the demethylase Jhd1 as well as point mutations of H3K36 that block methylation. Consistent with the genetic interactions, deletion of SET2, mutations modifying the catalytic activity of Set2 or H3K36 point mutations significantly altered expression of our reporter and reduced splicing of endogenous introns. These effects were dependent on the association of Set2 with RNA polymerase II and H3K36 dimethylation. Additionally, we found that deletion of SET2 reduces the association of the U2 and U5 snRNPs with chromatin. Thus, our study provides the first evidence that H3K36 methylation plays a role in co-transcriptional RNA splicing in yeast.Abbreviations: (H3K36), histone H3 lysine 36; (ChIP), chromatin immunoprecipitations; (RT-qPCR), reverse transcription PCR; (snRNP), small nuclear ribonucleprotein; (RNA Pol II), RNA polymerase II

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Section: Resultsmentioning

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Histone H3K36 methylation regulates pre-mRNA splicing inSaccharomyces cerevisiae

et al. 2016

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H3K36 Methylation and the Chromodomain Protein Eaf3 Are Required for Proper Cotranscriptional Spliceosome Assembly

et al. 2019

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SUMMARY In the eukaryotic cell, spliceosomes assemble onto pre-mRNA cotranscriptionally. Spliceosome assembly takes place in the context of the chromatin environment, suggesting that the state of the chromatin may affect splicing. The molecular details and mechanisms through which chromatin affects splicing, however, are still unclear. Here, we show a role for the histone methyltransferase Set2 and its histone modification, H3K36 methylation, in pre-mRNA splicing through high-throughput sequencing. Moreover, the effect of H3K36 methylation on pre-mRNA splicing is mediated through the chromodomain protein Eaf3. We find that Eaf3 is recruited to intron-containing genes and that Eaf3 interacts with the splicing factor Prp45. Eaf3 acts with Prp45 and Prp19 after formation of the precatalytic B complex around the time of splicing activation, thus revealing the step in splicing that is regulated by H3K36 methylation. These studies support a model whereby H3K36 facilitates recruitment of an “adapter protein” to support efficient, constitutive splicing.

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High-throughput single-molecule screen for small-molecule perturbation of splicing and transcription kinetics

Day

Chen

Coulon

et al. 2016

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In eukaryotes, mRNA synthesis is catalyzed by RNA polymerase II and involves several distinct steps, including transcript initiation, elongation, cleavage, and transcript release. Splicing of RNA can occur during (co-transcriptional) or after (post-transcriptional) RNA synthesis. Thus, RNA synthesis and processing occurs through the concerted activity of dozens of enzymes, each of which is potentially susceptible to perturbation by small molecules. However, there are few, if any, high-throughput screening strategies for identifying drugs which perturb a specific step in RNA synthesis and processing. Here we have developed a high-throughput fluorescence microscopy approach in single cells to screen for inhibitors of specific enzymatic steps in RNA synthesis and processing. By utilizing the high affinity interaction between bacteriophage capsid proteins (MS2, PP7) and RNA stem loops, we are able to fluorescently label the intron and exon of a β-globin reporter gene in human cells. This approach allows one to measure the kinetics of transcription, splicing and release in both fixed and living cells using a tractable, genetically encoded assay in a stable cell line. We tested this reagent in a targeted screen of molecules that target chromatin readers and writers and identified three compounds that slow transcription elongation without changing transcription initiation.

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Rapid identification of mRNA processing defects with a novel single-cell yeast reporter

Cited by 12 publications

References 75 publications

Histone H3K36 methylation regulates pre-mRNA splicing inSaccharomyces cerevisiae

Histone H3K36 methylation regulates pre-mRNA splicing inSaccharomyces cerevisiae

H3K36 Methylation and the Chromodomain Protein Eaf3 Are Required for Proper Cotranscriptional Spliceosome Assembly

High-throughput single-molecule screen for small-molecule perturbation of splicing and transcription kinetics

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