A quantitative, high-throughput reverse genetic screen reveals novel connections between Pre-mRNA splicing and 5' and 3' end transcript determinants.
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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DOI：
10.1371/journal.pgen.1002530
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发表时间：
2012
期刊：
影响因子：
4.5
通讯作者：
Pleiss JA
中科院分区：
文献类型：
--
作者：
Albulescu LO;Sabet N;Gudipati M;Stepankiw N;Bergman ZJ;Huffaker TC;Pleiss JA
在这里，我们提出了一个全基因组反向遗传筛选的芽殖酵母，酿酒酵母，耦合高通量菌株生长，机器人RNA分离和cDNA合成，定量PCR的发展和实施，以允许一个强大的测定几乎任何细胞RNA的水平在5,500个不同的突变体的背景。作为这种方法的初步测试，我们试图确定影响前mRNA剪接的全部因素。越来越多的证据表明，前体mRNA剪接和其他细胞途径之间的关系，包括染色质重塑，转录和3 '端加工，但在许多情况下，负责功能连接这些途径的特定蛋白质仍然不清楚。此外，目前还不清楚是否所有的途径，耦合到剪接已被确定。正如预期的那样，我们的方法灵敏地检测了绝大多数含有已知剪接因子突变的菌株中的前mRNA积累。然而，值得注意的是，发现几个额外的候选物引起前mRNA水平的增加，类似于典型剪接突变体所观察到的，其中没有一个先前涉及剪接途径。相反，这些因子中的一些先前已经被牵连到染色质重塑，3′端加工和其他新类别中发挥作用。使用剪接敏感的微阵列进一步分析这些因素证实，Bdf 1，一个连接转录起始和染色质重塑的因子的缺失，导致全局剪接缺陷，为前体mRNA剪接和SWR 1复合物的该组分之间的新联系提供了证据。相比之下，3′端加工因子如Cft 2和Yth 1的突变也会导致前mRNA剪接缺陷，尽管只是针对一部分转录本，这表明S.酿酒酵母可能更接近于哺乳动物模型的外显子定义。更广泛地说，我们的工作证明了这种方法识别各种细胞RNA的新型调节因子的能力。大多数真核基因的编码部分被称为内含子的非编码区中断，内含子必须在翻译前切除。前体信使RNA（pre-mRNA）中内含子的切除由剪接体催化，剪接体是由RNA和蛋白质组分组成的大分子。一些研究已经揭示了前体mRNA剪接和其他RNA加工途径之间的联系，例如染色质结构的重塑，转录和发生在转录物3′端的加工事件。然而，迄今为止，在细胞中将剪接与其他过程偶联的完整因子仍是未知的。在这里，我们开发了一种新的筛选方法，在芽殖酵母，酿酒酵母，使我们能够单独检查几乎所有的106000个基因，以确定哪些因素在功能上影响剪接。我们鉴定了在基因的5′或3′末端起作用的组分中的突变。这些成分中的大多数先前已经在基因表达的其他方面确定了作用，包括染色质重塑和切割以及多聚腺苷酸化过程，并且它们的鉴定在这里为它们在耦合这些途径中的作用提供了第一个证据。
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. 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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