Coordination of 3' mRNA Processing with Export to the Cytoplasm

9983378在复合(真核)细胞内生产蛋白质需要在细胞核中合成和处理mRNAs，然后将其运输到发生蛋白质合成的细胞质中。基因表达可以在大多数或所有这些步骤中进行调节。越来越多的证据表明，RNA合成(转录)和加工是耦合的。初步证据表明，核内信使核糖核酸的输出也与信使核糖核酸形成的其他步骤相关联。这项研究项目将确定这种耦合的机制。需要研究的模型是，一个或多个3‘加工因子在切割和多聚腺苷作用后仍与mRNA相关，然后介导mRNP与mRNA输出机制之间的重要相互作用。这些处理因子被提议与mRNP一起离开原子核，并在从mRNP中移除后穿梭回原子核。实验将使用萌芽酵母酿酒酵母进行。初步数据表明，当3‘处理的信号发生突变或产生突变的3’处理因子时，mRNAs在细胞核中积累。此外，一些遗传筛选已将3‘端加工因子与信使核糖核酸输出联系起来。当3‘端加工异常时，产生带有延长的3’端非翻译区和Poly(A)尾巴的mRNAs。至少有一个3‘端加工因子穿梭于细胞核和细胞质之间。这些数据与信使核糖核酸输出需要通过正常机制形成正确的3‘末端的观点一致。为了测试这种可能性，我们将研究通过顺式作用的锤头状核酶形成mRNA3‘端的效果。其他研究将确定哪些3‘处理因子穿梭，以及它们是否与mRNA一起离开细胞核。将分析延伸的mRNAs上的聚(A)尾巴，以寻找能够介导RNA核保持的序列。3‘端加工因子与REV(或MS2外壳蛋白)的RNA结合域的融合将被用来测试与mRNA捆绑的加工因子参与mRNA输出的能力。还将准备和鉴定更多的突变等位基因，以尝试分离特定因子的输出和3‘处理功能。其他初步研究表明，出口因素可能参与了3‘加工。在体外提取液中，当GST-Xpo1p加入到提取液中时，可以看到2-3倍的切割和多聚腺苷酸化的刺激。这将处理效率从25%-30%提高到~75%。将与克莱尔·摩尔博士合作进行几项体外实验，以确定观察到的Xpo1p刺激加工的基础，并确定是否也参与了Ran/Gsp1p。其他运输因素刺激3‘加工的能力将被确定。

Charles ColeMCB 9983378 The production of proteins within complex (eukaryotic) cells requires the synthesis and processing of mRNAs in the cell nucleus followed by their transport to the cytoplasm where protein synthesis takes place. Gene expression can be regulated at most or all of these steps. Evidence is accumulating that RNA synthesis (transcription) and processing are coupled. Preliminary evidence suggests that export of the mRNA from the nucleus is also coupled to other steps in mRNA formation. This research project will determine the mechanism of this coupling. The model to be examined is that one or more 3' processing factors remain associated with the mRNA following cleavage and polyadenylation, and then mediate important interactions between the mRNP and the mRNA export machinery. These processing factors are proposed to exit the nucleus with the mRNP and to shuttle back to the nucleus following their removal from the mRNP. Experiments will be performed using budding yeast, Saccharomyces cerevisiae. Preliminary data indicates that mRNAs accumulate in the nucleus when the signals for 3' processing are mutated or when mutant 3' processing factors are produced. In addition, several genetic screens have linked 3' processing factors to mRNA export. When 3' processing is aberrant, mRNAs are produced with extended 3' untranslated regions and a poly(A) tail. At least one 3' processing factor shuttles between nucleus and cytoplasm. These data are consistent with the idea that mRNA exportability requires formation of correct 3' ends through the normal mechanism. To test this possibility, the effect of forming an mRNA 3' end via a cis-acting, hammerhead ribozyme will be examined. Other studies will determine which 3' processing factors shuttle and whether they exit the nucleus with mRNA. The poly(A) tails on the extended mRNAs will be analyzed for sequences able to mediate nuclear retention of the RNA. Fusions of 3' processing factors to the RNA binding domain of Rev (or MS2 coat protein) will be used to test the ability of processing factors tethered to the mRNA to participate in mRNA export. Additional mutant alleles will be prepared and characterized in an attempt to separate the export and 3' processing functions of specific factors. Other preliminary studies suggest that export factors may participate in 3' processing. Using in vitro extracts, a consistent 2-3 fold stimulation of cleavage and polyadenylation was seen when GST-Xpo1p was added to the extract. This increased the processing efficiency from 25-30% to ~75%. Several in vitro experiments, in collaboration with Dr. Claire Moore, will be performed to determine the basis for the observed stimulation of processing by Xpo1p and to determine whether Ran/Gsp1p is also involved. The ability of other transport factors to stimulate 3' processing will be determined.