Characterization of Rous sarcoma virus polyadenylation site use in vitro.
Characterization of Rous sarcoma virus polyadenylation site use in vitro.
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DOI：
10.1016/j.virol.2008.01.012
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发表时间：
2008-05
期刊：
影响因子：
3.7
通讯作者：
Nicole L Maciolek;M. McNally
中科院分区：
文献类型：
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作者：
Nicole L Maciolek;M. McNally
Rous肉瘤病毒（RSV） RNA的多聚腺苷化是低效的，因为大约15%的RSV RNA代表使用下游细胞多聚腺苷化位点（poly(a)位点）的可读转录本。通读转录对病毒和宿主都有影响，因为它与致癌基因捕获和肿瘤诱导有关。为了探索RSV RNA 3 '端低效形成的基础，我们利用HeLa细胞核提取物和HEK293全细胞提取物在体外对RSV多聚腺苷化进行了表征。由病毒RNA的天然3′端和不同长度的上游序列组成的RSV多聚腺苷化底物很少或没有多聚腺苷化，表明RSV聚(A)位点是次优的。有效使用的聚(A)位点通常在靠近保守的AAUAAA信号的位置具有可识别的上游和下游元件（USEs和DSEs）。RSV聚(A)位点的上游和下游序列与有效利用的聚(A)位点的序列不同，这可能解释了RSV聚腺苷酸化效率低的原因。为了评估RSV的use和dse的质量，我们将特征良好的SV40晚期use和/或dse替换为RSV元素，反之亦然，这表明RSV的use和dse是次优的，但功能良好。CstF与RSV聚腺苷化底物的相互作用很差，而RSV聚(A)位点的失活至少部分是由于CstF与RSV底物的结合较差，因为将CstF系在RSV底物上激活了聚腺苷化。我们的数据与USE和DSE中较差的聚腺苷化因子结合位点一致，这是RSV聚(A)位点低效使用的基础，并指出RSV RNA中其他元件在促进3 '端形成方面的重要性。
Polyadenylation of Rous sarcoma virus (RSV) RNA is inefficient, as approximately 15% of RSV RNAs represent read-through transcripts that use a downstream cellular polyadenylation site (poly(A) site). Read-through transcription has implications for the virus and the host since it is associated with oncogene capture and tumor induction. To explore the basis of inefficient RSV RNA 3′-end formation, we characterized RSV polyadenylation in vitro using HeLa cell nuclear extracts and HEK293 whole cell extracts. RSV polyadenylation substrates composed of the natural 3′ end of viral RNA and various lengths of upstream sequence showed little or no polyadenylation, indicating that the RSV poly(A) site is suboptimal. Efficiently used poly(A) sites often have identifiable upstream and downstream elements (USEs and DSEs) in close proximity to the conserved AAUAAA signal. The sequences upstream and downstream of the RSV poly(A) site deviate from those found in efficiently used poly(A) sites, which may explain inefficient RSV polyadenylation. To assess the quality of the RSV USEs and DSEs, the well-characterized SV40 late USEs and/or DSEs were substituted for the RSV elements and vice versa, which showed that the USEs and DSEs from RSV are suboptimal but functional. CstF interacted poorly with the RSV polyadenylation substrate, and the inactivity of the RSV poly(A) site was at least in part due to poor CstF binding since tethering CstF to the RSV substrate activated polyadenylation. Our data are consistent with poor polyadenylation factor binding sites in both the USE and DSE as the basis for inefficient use of the RSV poly(A) site and point to the importance of additional elements within RSV RNA in promoting 3′ end formation.