Spliceosomal small nuclear ribonucleoproteins in trypansomes: Genomwide analysis of role of U1 snRPN proteins in mRNA processing

锥虫中的所有蛋白质编码基因表达为多顺反子初级转录物，其必须通过偶联的反式剪接和聚腺苷酸化加工以产生成熟的mRNA。此外，已知只有极少数基因经历内部内含子的顺式剪接。反式剪接特别需要剪接前导（SL）RNA，其将其5 '末端39个核苷酸的小外显子添加到每个蛋白质编码外显子。另一方面，U1小核核糖核蛋白（snRNP）被认为是一个组成部分，特别是在顺式剪接的非常少的内部内含子。我们最近的特点是U1 snRNP从布氏锥虫，一个不寻常的RNP与最小的U1 snRNA，U1- 70 K，U1 C，和蛋白质组分（U1- 24 K）没有发现在良好的表征U1 snRNP从其他系统。包括我们自己的几项研究的证据已经积累，认为存在几种不同的RNP复合物的U1 snRNA和U1独立的作用，其蛋白质组分，连接顺式和反式剪接以及聚腺苷酸化。我们建议详细研究单个U1 snRNP蛋白的贡献和这些主要的mRNA加工反应之间的潜在联系，在锥虫，使用系统表位标记，RNAi敲除，以及全基因组RNA结合（iCLIP）和深度测序方法。

All protein-coding genes in trypanosomes are expressed as polycistronic primary transcripts that have to be processed by coupled trans splicing and polyadenylation to yield mature mRNAs. In addition, only very few genes are known to undergo cis splicing of an internal intron. Trans splicing requires specifically the Spliced Leader (SL) RNA, which adds its 5’-terminal 39-nucleotide miniexon to each of the protein-coding exons. On the other hand, the U1 small nuclear ribonucleoprotein (snRNP) is considered a component acting specifically during cis splicing of the very few internal introns. We have recently characterized the U1 snRNP from Trypanosoma brucei, an unusual RNP with a minimal U1 snRNA, U1-70K, U1C, and a protein component (U1-24K) not found in well-characterized U1 snRNPs from other systems. Evidence from several studies including our own has accumulated that argues for the existence of several different RNP complexes of the U1 snRNA and for U1-independent roles of their protein components, linking cis and trans splicing as well as polyadenylation. We propose to study in detail the contributions of individual U1 snRNP proteins to and the potential links between these principal mRNA processing reactions in trypanosomes, using systematic epitope tagging, RNAi knockdown, as well as genomewide RNA-binding (iCLIP) and deep-sequencing approaches.