Trans-splicing in C. elegans

线虫中的反式剪接

• 批准号: 7053326
• 负责人: Thomas Blumenthal
• 金额: $ 34.92万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7053326
• 关键词: Caenorhabditis elegans; RNA binding protein; RNA splicing; antibody; chemical binding; crosslink; functional /structural genomics; gel mobility shift assay; genetic regulation; genetic regulatory element; genetic screening; glycerol; green fluorescent proteins; helminth genetics; immunoprecipitation; laboratory rabbit; nucleic acid sequence; operon; posttranscriptional RNA processing; precursor mRNA; small nuclear ribonucleoproteins; transcription factor

DESCRIPTION (provided by applicant): C. elegans has polycistronic transcription units, similar to bacterial operons, that make polycistronic pre-mRNAs. These pre-mRNAs are processed by 3' end formation and trans-splicing, which occur in close proximity. What are the mechanims involved in production of mRNAs from polygenic precursors? Genes are separated by ~100 bp and the downstream genes are trans-spliced to SL2. We use a test operon to investigate the roles of sequences in the 3' end formation site and the intercistronic region in polycistronic pre-mRNA processing. We have firmly established a mechanistic and physical connection between the 3' end formation machinery and SL2 trans-splicing. We have discovered a U-rich sequence, Ur, near the 3' end of the upstream gene, that is required for SL2 utilization. We will determine whether CstF interacts with the Ur element to facilitate SL2 trans-splicing, or if not, what does. To determine what sequences on the SL2 snRNP are needed for trans-splicing, we have created a marked SL2 RNA gene which we have shown is correctly trans-spliced to downstream genes in operons. We have shown that C. elegans embryo extracts contain a complex containing the SL2 snRNP and CstF-64. Using a variety of mutant SL2 RNA genes, we have found that this complex is crucial for SL2 identity. Using glycerol gradients and co-immunoprecipitation experiments, we will determine additional protein components of this complex. We will also do genetic and RNAi screens for mutants in proteins involved in polycistronic pre-mRNA processing using an operon in which the downstream gene is fused to the gfp gene. We have recently performed a whole genome microarray analysis that has enabled us to identify ~90% of all C. elegans operons. Our preliminary results show that only genes encoding certain types of proteins are included in operons and that proteins that function in the same process are often co-expressed in the same operon. We will continue our bioinformatic analysis of the worm operons as a way of understanding the evolution and current function of this unusual mechanism of gene expression and regulation. We will also investigate whether SL2 trans-splicing is absolutely restricted to downstream operon trans-splice sites, and if so, how. If not, we will study the special class of non-operon trans-splice site that can be trans-spliced to SL2. Finally, we will determine whether the pre-mRNA cap attracts the SL1snRNP to non-operon trans-splice sites.

描述（由申请人提供）：C。线虫具有多顺反子转录单位，类似于细菌操纵子，其产生多顺反子前mRNA。这些前体mRNA通过3'端形成和反式剪接进行加工，这两种方式非常接近。从多基因前体产生mRNA的机制是什么？基因间隔约100 bp，下游基因反式剪接至SL 2。我们使用一个测试操纵子来研究多顺反子前体mRNA加工中3'端形成位点和顺反子间区域序列的作用。我们已经牢固地建立了3'端形成机制和SL 2反式剪接之间的机制和物理联系。我们已经发现了一个富含U的序列，Ur，靠近上游基因的3'端，这是SL 2利用所必需的。我们将确定CstF是否与Ur元件相互作用以促进SL 2反式剪接，或者如果不是，是什么。为了确定SL 2 snRNP上的哪些序列是反式剪接所需的，我们已经创建了标记的SL 2 RNA基因，我们已经证明该基因正确地反式剪接到操纵子中的下游基因。我们证明了C.线虫胚胎提取物含有含有SL 2 snRNP和CstF-64的复合物。使用各种突变的SL 2 RNA基因，我们已经发现，这种复合物是至关重要的SL 2身份。使用甘油梯度和免疫共沉淀实验，我们将确定该复合物的其他蛋白质组分。我们还将使用操纵子（其中下游基因与gfp基因融合）对参与多顺反子前mRNA加工的蛋白质中的突变体进行遗传和RNAi筛选。我们最近进行了一项全基因组微阵列分析，使我们能够识别约90%的C。线虫操纵子我们的初步结果表明，只有编码某些类型的蛋白质的基因被包括在操纵子中，并且在同一过程中起作用的蛋白质通常在同一操纵子中共表达。我们将继续对蠕虫操纵子进行生物信息学分析，以了解这种不寻常的基因表达和调控机制的进化和当前功能。我们还将调查是否SL 2的反式剪接是绝对限制下游操纵子反式剪接位点，如果是这样，如何。如果没有，我们将研究一类特殊的非操纵子反式剪接位点，可以反式剪接到SL 2。最后，我们将确定前mRNA帽是否吸引SL 1 snRNP非操纵子反式剪接位点。