Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado 80045, USA.
Genes Dev. 2010 Aug 1;24(15):1645-58. doi: 10.1101/gad.1940010. Epub 2010 Jul 12.
Spliced leader (SL) trans-splicing in Caenorhabditis elegans attaches a 22-nucleotide (nt) exon onto the 5' end of many mRNAs. A particular class of SL, SL2, splices mRNAs of downstream operon genes. Here we use an embryonic extract-based in vitro splicing system to show that SL2 specificity information is encoded within the polycistronic pre-mRNA, and that trans-splicing specificity is recapitulated in vitro. We define an RNA sequence required for SL2 trans-splicing, the U-rich (Ur) element, through mutational analysis and bioinformatics as a short stem-loop followed by a sequence motif, UAYYUU, located approximately 50 nt upstream of the trans-splice site. Furthermore, this element is predicted in intercistronic regions of numerous operons of C. elegans and other species that use SL2 trans-splicing. We propose that the UAYYUU motif hybridizes with the 5' splice site on the SL2 RNA to recruit the SL to the pre-mRNA. In this way, the UAYYUU motif in the pre-mRNA would serve an analogous function to the similar sequence in the U1 snRNA, which binds to the 5' splice site of introns, effectively reversing the roles of snRNP and pre-mRNA in trans-splicing.
秀丽隐杆线虫中的拼接 leader (SL) 反式拼接将 22 个核苷酸 (nt) 的外显子附加到许多 mRNA 的 5' 端。一类特殊的 SL，SL2，拼接下游操纵子基因的 mRNA。在这里，我们使用基于胚胎提取物的体外 splicing 系统表明，SL2 特异性信息编码在多顺反子前 mRNA 中，并且体外反式拼接特异性被再现。我们通过突变分析和生物信息学定义了一个 RNA 序列，该序列对于 SL2 反式拼接是必需的，即富含 U 的 (Ur) 元件，该序列是一个短茎环结构，随后是一个序列基序 UAYYUU，位于反式拼接位点上游约 50nt。此外，该元件在 C. elegans 和其他使用 SL2 反式拼接的物种的众多操纵子的顺式间区中被预测。我们提出 UAYYUU 基序与 SL2 RNA 上的 5' 剪接位点杂交，从而将 SL 招募到 pre-mRNA。通过这种方式，pre-mRNA 中的 UAYYUU 基序将发挥类似于 U1 snRNA 中相似序列的功能，后者结合到内含子的 5' 剪接位点，有效地在反式拼接中反转 snRNP 和 pre-mRNA 的作用。
