McNally L M, McNally M T
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
J Virol. 1999 Mar;73(3):2385-93. doi: 10.1128/JVI.73.3.2385-2393.1999.
Retroviruses require both spliced and unspliced RNA for replication. Accumulation of unspliced Rous sarcoma virus RNA is facilitated in part by a negative cis element in the gag region, termed the negative regulator of splicing (NRS), which serves to repress splicing of viral RNA but can also block splicing of heterologous introns. The NRS binds components of the splicing machinery including SR proteins, U1 and U2, small nuclear ribonucleoproteins (snRNPs) of the major splicing pathway, and U11 snRNP of the minor pathway, yet splicing does not normally occur from the NRS. A mutation that abolishes U11 binding (RG11) also abrogates NRS splicing inhibition, indicating that U11 is functionally important for NRS activity and suggesting that the NRS is recognized as a minor-class 5' splice site (5' ss). We show here, using specific NRS mutations to disrupt U11 binding and coexpression of U11 snRNA genes harboring compensatory mutations, that the NRS U11 site is functional when paired with a minor-class 3' ss from the human P120 gene. Surprisingly, the expectation that the same NRS mutants would be defective for splicing inhibition proved false; splicing inhibition was as good as, if not better than, that for the wild-type NRS. Comparison of these new mutations with RG11 indicated that the latter may disrupt binding of a factor(s) other than U11. Our data suggest that this factor is U1 snRNP and that a U1 binding site that overlaps the U11 site is also disrupted by RG11. Analysis of mutations which selectively disrupted U1 or U11 binding indicated that splicing inhibition by the NRS correlates most strongly with U1 snRNP. Additionally, we show that U1 binding is facilitated by SR proteins that bind to the 5' half of the NRS, confirming an earlier proposal that this region is involved in recruiting snRNPs to the NRS. These data indicate a functional role for U1 in NRS-mediated splicing inhibition.
逆转录病毒复制需要剪接和未剪接的RNA。未剪接的劳氏肉瘤病毒RNA的积累部分得益于gag区域中的一个负性顺式元件，称为剪接负调控因子（NRS），它可抑制病毒RNA的剪接，但也能阻断异源内含子的剪接。NRS结合剪接机制的组分，包括SR蛋白、U1和U2（主要剪接途径的小核核糖核蛋白（snRNP））以及次要途径的U11 snRNP，但剪接通常不会从NRS处发生。一个消除U11结合的突变（RG11）也消除了NRS的剪接抑制作用，这表明U11对NRS活性具有重要功能，并且提示NRS被识别为一个次要类别的5'剪接位点（5' ss）。我们在此表明，使用特定的NRS突变来破坏U11结合，并共表达携带补偿性突变的U11 snRNA基因，当NRS的U11位点与来自人类P120基因的一个次要类别的3' ss配对时，该位点具有功能。令人惊讶的是，认为相同的NRS突变体在剪接抑制方面存在缺陷的预期被证明是错误的；剪接抑制效果与野生型NRS一样好，甚至更好。将这些新突变与RG11进行比较表明，后者可能破坏了除U11之外的一种或多种因子的结合。我们的数据表明，这种因子是U1 snRNP，并且与U11位点重叠的一个U1结合位点也被RG11破坏。对选择性破坏U1或U11结合的突变的分析表明，NRS的剪接抑制作用与U1 snRNP的相关性最强。此外，我们表明，与NRS 5' 半段结合的SR蛋白促进了U1的结合，证实了之前的一个提议，即该区域参与将snRNP招募到NRS。这些数据表明U1在NRS介导的剪接抑制中具有功能作用。
