Wilusz Jeremy E, Beemon Karen L
Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
J Virol. 2006 Oct;80(19):9634-40. doi: 10.1128/JVI.00845-06.
The Rous sarcoma virus gag gene contains a cis-acting negative regulator of splicing (NRS) element that is implicated in viral polyadenylation regulation. To study the mechanism of polyadenylation promotion at the viral poly(A) site located over 8 kb downstream, we performed in vitro polyadenylation analysis. RNA containing only the poly(A) site and flanking sequences in the 3' long terminal repeat (LTR) was not polyadenylated detectably in vitro; however, if the transcript contained the NRS upstream of the LTR, polyadenylation was observed. Insertion of the viral env 3' splice site sequence between the NRS and the LTR did not alter the level of polyadenylation appreciably. We conclude that the NRS promotes polyadenylation in vitro and can do so without formation of a splicing complex with a 3' splice site. We then explored the roles of several cellular factors in NRS-mediated polyadenylation. Mutation of the binding sites of U1 and U11 snRNPs to the NRS did not affect polyadenylation, whereas hnRNP H strongly inhibited polyadenylation. We propose a model in which hnRNP H and SR proteins compete for binding to the NRS. Bound SR proteins may bridge between the NRS and the 3' LTR and aid in the recruitment of the 3'-end processing machinery.
劳氏肉瘤病毒gag基因包含一个顺式作用的剪接负调控元件（NRS），该元件与病毒的聚腺苷酸化调控有关。为了研究位于下游超过8 kb处的病毒聚腺苷酸化位点上聚腺苷酸化促进的机制，我们进行了体外聚腺苷酸化分析。仅含有聚腺苷酸化位点以及3'长末端重复序列（LTR）侧翼序列的RNA在体外无法检测到聚腺苷酸化；然而，如果转录本在LTR上游包含NRS，则可观察到聚腺苷酸化。在NRS和LTR之间插入病毒env 3'剪接位点序列不会明显改变聚腺苷酸化水平。我们得出结论，NRS在体外促进聚腺苷酸化，并且无需与3'剪接位点形成剪接复合体即可实现。然后，我们探究了几种细胞因子在NRS介导的聚腺苷酸化中的作用。U1和U11 snRNP与NRS的结合位点发生突变并不影响聚腺苷酸化，而hnRNP H强烈抑制聚腺苷酸化。我们提出了一个模型，其中hnRNP H和SR蛋白竞争与NRS的结合。结合的SR蛋白可能在NRS和3' LTR之间形成桥梁，并有助于募集3'末端加工机制。
