Position-dependent inhibition of the cleavage step of pre-mRNA 3'-end processing by U1 snRNP.
Position-dependent inhibition of the cleavage step of pre-mRNA 3'-end processing by U1 snRNP.
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DOI：
10.1017/s1355838200991854
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发表时间：
2000-02
期刊：
影响因子：
4.5
通讯作者：
Stéphan Vagner;Ursula Rüegsegger;Samuel I. Gunderson;Walter Keller;I. Mattaj
中科院分区：
文献类型：
--
作者：
Stéphan Vagner;Ursula Rüegsegger;Samuel I. Gunderson;Walter Keller;I. Mattaj
大多数真核生物前mrna的3′端是通过3′核内裂解和随后的聚腺苷化产生的。3′端形成受到多种因素的积极或消极影响。特别是，当U1 snRNP结合到牛乳头瘤病毒（BPV）晚期转录本的3‘端形成信号上游或HIV-1前病毒5’ LTR的3‘端加工信号下游的5’剪接位点时，它可以作为抑制剂。先前的研究表明，在BPV中，受U1 snRNP影响的不是第一步，即3'切割，而是第二步，即聚腺苷酸化。由于HIV-1的生物学要求是产生穿过5' LTR切割位点的转录本，而不是在那里被切割，因此这种机制似乎不太可能适用。这两个例子的明显区别在于3'端形成信号和U1 snrnp结合位点的相对取向。因此，我们使用体外实验来评估U1 snRNP在相对于切割/聚腺苷化位点的不同位置结合对3'切割反应的影响。在HIV-1 LTR中，当U1 snRNP结合到裂解/聚腺苷化位点下游的一个5'剪接位点时，发现它可以抑制裂解。U1 snRNP结合在这个位置，不影响多个裂解/聚腺苷化因子向裂解底物的募集，表明抑制不太可能是由于位阻。U1A、U1 70K和聚(A)聚合酶之间的相互作用介导了U1 snRNP对其他前mrna聚腺苷化的影响，而这并不是切割抑制所必需的。因此，结合在5‘剪接位点上的U1 snRNP，根据5’剪接位点位于剪接位点的上游还是下游，能够以两种机制不同的方式抑制裂解和聚腺苷酸化。
The 3' ends of most eukaryotic pre-mRNAs are generated by 3' endonucleolytic cleavage and subsequent polyadenylation. 3'-end formation can be influenced positively or negatively by various factors. In particular, U1 snRNP acts as an inhibitor when bound to a 5' splice site located either upstream of the 3'-end formation signals of bovine papilloma virus (BPV) late transcripts or downstream of the 3'-end processing signals in the 5' LTR of the HIV-1 provirus. Previous work showed that in BPV it is not the first step, 3' cleavage, that is affected by U1 snRNP, but rather the second step, polyadenylation, that is inhibited. Since in HIV-1 the biological requirement is to produce transcripts that read through the 5' LTR cleavage site rather than being cleaved there, this mechanism seemed unlikely to apply. The obvious difference between the two examples was the relative orientation of the 3'-end formation signals and the U1 snRNP-binding site. In vitro assays were therefore used to assess the effect of U1 snRNP bound at various locations relative to a cleavage/polyadenylation site on the 3' cleavage reaction. U1 snRNP was found to inhibit cleavage when bound to a 5' splice site downstream of the cleavage/polyadenylation site, as in the HIV-1 LTR. U1 snRNP binding at this location was shown not to affect the recruitment of multiple cleavage/polyadenylation factors to the cleavage substrate, indicating that inhibition is unlikely to be due to steric hindrance. Interactions between U1A, U1 70K, and poly(A) polymerase, which mediate the effect of U1 snRNP on polyadenylation of other pre-mRNAs, were shown not to be required for cleavage inhibition. Therefore, U1 snRNP bound to a 5' splice site can inhibit cleavage and polyadenylation in two mechanistically different ways depending on whether the 5' splice site is located upstream or downstream of the cleavage site.