Trans-splicing in C. elegans

描述（由申请人提供）：秀丽隐杆线虫具有多顺反子转录单位，类似于细菌的操纵子，可产生多顺反子前mrna。这些前mrna通过3'端形成和反式剪接进行加工，这两个过程发生在很近的距离。多基因前体产生mrna的机制是什么？基因间隔约100 bp，下游基因反接到SL2上。我们使用一个测试操纵子来研究3'端形成位点和顺反子间区域序列在多顺反子pre-mRNA加工中的作用。我们已经牢固地建立了3'端形成机制和SL2反式剪接之间的机械和物理联系。我们在上游基因的3'端附近发现了一个富含u的序列Ur，这是SL2利用所必需的。我们将确定CstF是否与Ur元素相互作用以促进SL2反式剪接，如果不是，则是什么作用。为了确定SL2 snRNP上的哪些序列需要进行反式剪接，我们创建了一个标记的SL2 RNA基因，我们已经证明它正确地反式剪接到下游的操纵子基因上。我们已经证明秀丽隐杆线虫胚胎提取物含有一个包含SL2 snRNP和CstF-64的复合物。利用多种突变SL2 RNA基因，我们发现该复合体对SL2身份至关重要。使用甘油梯度和共免疫沉淀实验，我们将确定该复合物的其他蛋白质成分。我们还将使用下游基因与gfp基因融合的操纵子，对参与多顺反子前mrna加工的蛋白质突变体进行遗传和RNAi筛选。我们最近进行了全基因组微阵列分析，使我们能够识别约90%的秀丽隐杆线虫操纵子。我们的初步结果表明，只有编码特定类型蛋白质的基因被包含在操纵子中，并且在相同过程中起作用的蛋白质通常在相同的操纵子中共表达。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。我们还将研究SL2反式剪接是否绝对局限于下游的操纵子反式剪接位点，如果是，如何限制。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SL2。最后，我们将确定前mrna帽是否将SL1snRNP吸引到非操纵子反式剪接位点。

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.