Heidelberg University Centre for Molecular Biology (ZMBH), Heidelberg, Germany.
Department of Infectious Diseases/Virology, Section Viral Vector Technologies, Medical Faculty, University of Heidelberg, BioQuant, Heidelberg, Germany.
PLoS Negl Trop Dis. 2022 Oct 26;16(10):e0010876. doi: 10.1371/journal.pntd.0010876. eCollection 2022 Oct.
Spliced leader trans splicing is the addition of a short, capped sequence to the 5' end of mRNAs. It is widespread in eukaryotic evolution, but factors that influence trans splicing acceptor site choice have been little investigated. In Kinetoplastids, all protein-coding mRNAs are 5' trans spliced. A polypyrimidine tract is usually found upstream of the AG splice acceptor, but there is no branch point consensus; moreover, splicing dictates polyadenylation of the preceding mRNA, which is a validated drug target.
We here describe a trans splicing reporter system that can be used for studies and screens concerning the roles of sequences and proteins in processing site choice and efficiency. Splicing was poor with poly(U) tracts less than 9 nt long, and was influenced by an intergenic region secondary structure. A screen for signals resulted in selection of sequences that were on average 45% U and 35% C. Tethering of either the splicing factor SF1, or the cleavage and polyadenylation factor CPSF3 within the intron stimulated processing in the correct positions, while tethering of two possible homologues of Opisthokont PTB inhibited processing. In contrast, tethering of SR-domain proteins RBSR1, RBSR2, or TSR1 or its interaction partner TSR1IP, promoted use of alternative signals upstream of the tethering sites. RBSR1 interacts predominantly with proteins implicated in splicing, whereas the interactome of RBSR2 is more diverse.
Our selectable constructs are suitable for screens of both sequences, and proteins that affect mRNA processing in T. brucei. Our results suggest that the functions of PTB and SR-domain proteins in splice site definition may already have been present in the last eukaryotic common ancestor.
拼接 leader 转位拼接是在 mRNA 的 5' 端添加一个短的、加帽序列。它在真核生物进化中广泛存在，但影响转位拼接接受位点选择的因素尚未得到充分研究。在动基体目生物中，所有的蛋白质编码 mRNA 都在 5' 端进行转位拼接。在 AG 剪接受体位点的上游通常会发现一个多嘧啶序列，但没有分支点的共识；此外，拼接决定了前一个 mRNA 的多聚腺苷酸化，这是一个经过验证的药物靶点。
我们在这里描述了一个转位拼接报告系统，可用于研究和筛选序列和蛋白质在加工位点选择和效率中的作用。聚 U 序列小于 9 个核苷酸时，拼接效果较差，并且受到基因间区二级结构的影响。信号筛选导致选择的序列平均含有 45%的 U 和 35%的 C。在内含子中 tethering 拼接因子 SF1 或剪接和多聚腺苷酸化因子 CPSF3 可刺激正确位置的加工，而 tethering 两个可能的后生动物 PTB 同源物则抑制加工。相比之下， tethering SR 结构域蛋白 RBSR1、RBSR2 或 TSR1 或其相互作用伙伴 TSR1IP 可促进在 tethering 位点上游使用替代信号。RBSR1 主要与参与拼接的蛋白质相互作用，而 RBSR2 的相互作用组则更加多样化。
我们的可选择构建体适合于在 T. brucei 中筛选影响 mRNA 加工的序列和蛋白质。我们的结果表明，PTB 和 SR 结构域蛋白在剪接位点定义中的功能可能已经存在于最后一个真核生物共同祖先中。
