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

锥虫体内所有的蛋白质编码基因都以多顺反电子初级转录本的形式表达，这些转录本必须经过偶联的反式剪接和聚腺苷酸化才能产生成熟的mrna。此外，已知只有很少的基因经历内部内含子的顺式剪接。反式剪接特别需要Spliced Leader (SL) RNA，它将其5 '端39个核苷酸的小外显子添加到每个蛋白质编码外显子上。另一方面，U1小核核糖核蛋白（snRNP）被认为是在极少数内部内含子的顺式剪接过程中特异性作用的成分。我们最近对来自布鲁氏锥虫的U1 snRNP进行了表征，这是一种罕见的RNP，具有最小的U1 snRNA， U1- 70k， U1C和其他系统中未发现的蛋白质成分（U1- 24k）。包括我们自己在内的几项研究积累的证据表明，U1 snRNA存在几种不同的RNP复合物，它们的蛋白质成分具有与U1无关的作用，连接顺式和反式剪接以及聚腺苷化。我们建议使用系统表位标记、RNAi敲低、全基因组rna结合（iCLIP）和深度测序方法，详细研究单个U1 snRNP蛋白对锥虫中这些主要mRNA加工反应的贡献及其之间的潜在联系。

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.