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      A Quantitative, High-Throughput Reverse Genetic Screen Reveals Novel Connections between Pre–mRNA Splicing and 5′ and 3′ End Transcript Determinants

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          Abstract

          Here we present the development and implementation of a genome-wide reverse genetic screen in the budding yeast, Saccharomyces cerevisiae, that couples high-throughput strain growth, robotic RNA isolation and cDNA synthesis, and quantitative PCR to allow for a robust determination of the level of nearly any cellular RNA in the background of 5,500 different mutants. As an initial test of this approach, we sought to identify the full complement of factors that impact pre–mRNA splicing. Increasing lines of evidence suggest a relationship between pre–mRNA splicing and other cellular pathways including chromatin remodeling, transcription, and 3′ end processing, yet in many cases the specific proteins responsible for functionally connecting these pathways remain unclear. Moreover, it is unclear whether all pathways that are coupled to splicing have been identified. As expected, our approach sensitively detects pre–mRNA accumulation in the vast majority of strains containing mutations in known splicing factors. Remarkably, however, several additional candidates were found to cause increases in pre–mRNA levels similar to that seen for canonical splicing mutants, none of which had previously been implicated in the splicing pathway. Instead, several of these factors have been previously implicated to play roles in chromatin remodeling, 3′ end processing, and other novel categories. Further analysis of these factors using splicing-sensitive microarrays confirms that deletion of Bdf1, a factor that links transcription initiation and chromatin remodeling, leads to a global splicing defect, providing evidence for a novel connection between pre–mRNA splicing and this component of the SWR1 complex. By contrast, mutations in 3′ end processing factors such as Cft2 and Yth1 also result in pre–mRNA splicing defects, although only for a subset of transcripts, suggesting that spliceosome assembly in S. cerevisiae may more closely resemble mammalian models of exon-definition. More broadly, our work demonstrates the capacity of this approach to identify novel regulators of various cellular RNAs.

          Author Summary

          The coding portions of most eukaryotic genes are interrupted by non-coding regions termed introns that must be excised prior to their translation. The excision of introns from precursor messenger RNA (pre–mRNA), is catalyzed by the spliceosome, a large macromolecule composed of both RNA and protein components. Several studies have uncovered connections between pre–mRNA splicing and other RNA processing pathways such as the remodeling of chromatin structure, transcription, and processing events that take place at the 3′ end of the transcript. To date, however, the full complement of factors that function to couple splicing to other processes in the cell remains unknown. Here, we have developed a novel screening methodology in the budding yeast, Saccharomyces cerevisiae, that allowed us to individually examine nearly all of the ∼6,000 genes to determine which factors functionally impact splicing. We identified mutations in components that function at either the 5′ or 3′ end of a gene. Most of these components have previously established roles in other aspects of gene expression, including chromatin remodeling and cleavage and polyadenylation processes, and their identification here provides the first evidence for their roles in coupling these pathways.

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          Gene Ontology: tool for the unification of biology

          Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.
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            Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

            A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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              The genetic landscape of a cell.

              A genome-scale genetic interaction map was constructed by examining 5.4 million gene-gene pairs for synthetic genetic interactions, generating quantitative genetic interaction profiles for approximately 75% of all genes in the budding yeast, Saccharomyces cerevisiae. A network based on genetic interaction profiles reveals a functional map of the cell in which genes of similar biological processes cluster together in coherent subsets, and highly correlated profiles delineate specific pathways to define gene function. The global network identifies functional cross-connections between all bioprocesses, mapping a cellular wiring diagram of pleiotropy. Genetic interaction degree correlated with a number of different gene attributes, which may be informative about genetic network hubs in other organisms. We also demonstrate that extensive and unbiased mapping of the genetic landscape provides a key for interpretation of chemical-genetic interactions and drug target identification.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                March 2012
                March 2012
                29 March 2012
                : 8
                : 3
                : e1002530
                Affiliations
                [1]Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America
                Yale University School of Medicine, United States of America
                Author notes

                Conceived and designed the experiments: L-O Albulescu, JA Pleiss. Performed the experiments: L-O Albulescu, N Sabet, M Gudipati, N Stepankiw, ZJ Bergman, JA Pleiss. Analyzed the data: L-O Albulescu, M Gudipati, N Stepankiw. Contributed reagents/materials/analysis tools: TC Huffaker. Wrote the paper: L-O Albulescu, JA Pleiss.

                Article
                PGENETICS-D-11-01856
                10.1371/journal.pgen.1002530
                3315463
                22479188
                fbafce82-45a7-4a49-a87c-39f09bb60018
                Albulescu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 1 September 2011
                : 19 December 2011
                Page count
                Pages: 16
                Categories
                Research Article
                Biology
                Genetics
                Gene Expression
                Genomics
                Chromosome Biology
                Genome Analysis Tools

                Genetics
                Genetics

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