Coupling Transcription, Splicing and mRNA Export

描述（由申请人提供）：蛋白质编码基因的表达是一个多步骤过程，从细胞核中RNA聚合酶II的转录开始。在转录过程中，新生的前mRNA经历几个加工步骤，包括加帽、剪接和聚腺苷酸化。然后将成熟的mRNA输出到细胞质进行翻译。虽然基因表达的每一步都是由不同的细胞机器完成的，但越来越多的证据表明，每一个机器之间都有一个广泛的耦合相互作用网络。然而，目前人们对这种耦合所涉及的机制知之甚少。本提案的目标是确定耦合转录，剪接和mRNA输出的机制。在特异性目的1中，将使用功能测定来确定RNA聚合酶II的转录是否促进mRNA输出。此外，最近确定的TREX复合物，这可能是耦合所有三个过程的功能，将进行详细研究。将克隆、表达所有全长人TREX组分并生成抗体。这些试剂将用于测试TREX复合物的功能，并确定该复合物如何与活性基因及其转录物结合。在特定目的2中，将使用RNA聚合酶II的转录与剪接偶联的体外系统研究转录与剪接偶联的机制。还将进行研究以确定剪接因子，包括U1和U2 snRNP，是否被招募到活跃转录的基因中。最后，在特定目标3中，将通过分离和确定促进输出的剪接mRNP的组成来研究剪接与mRNA输出的偶联。此外，保守的DEAD盒解旋酶蛋白UAP 56的作用，其在输出中起作用并且也存在于TREX复合物和剪接体两者中，将使用该蛋白中的特定突变结合用于偶联的功能测定来确定。

DESCRIPTION (provided by applicant): Expression of protein-coding genes is a multi-step process beginning with transcription by RNA polymerase II in the nucleus. During transcription, the nascent pre-mRNA undergoes several processing steps including capping, splicing, and polyadenylation. The mature mRNA is then exported to the cytoplasm for translation. Although each of the steps in gene expression is carried out by a distinct cellular machine, growing evidence indicates that there is an extensive network of coupled interactions between each machine. At present, however, little is understood about the mechanisms involved in the coupling. The goal of this proposal is to determine the mechanisms for coupling transcription, splicing, and mRNA export. In Specific Aim 1, a functional assay will be used to determine whether transcription by RNA polymerase II promotes mRNA export. In addition, the recently identified TREX complex, which may function in coupling all three processes, will be investigated in detail. All of the full-length human TREX components will be cloned, expressed, and antibodies generated. These reagents will be used to test the function of the TREX complex and to determine how this complex associates with active genes and their transcripts. In Specific Aim 2, the mechanism for coupling transcription to splicing will be investigated using an in vitro system for coupling transcription by RNA polymerase II to splicing. Studies will also be carried out to determine whether splicing factors, including U1 and U2 snRNPs, are recruited to actively transcribed genes. Finally, in Specific Aim 3, coupling of splicing to mRNA export will be investigated by isolating and determining the composition of the spliced mRNP that promotes export. In addition, the role of the conserved DEAD box helicase protein UAP56, which functions in export and is also present in both the TREX complex and spliceosome, will be determined using specific mutations in this protein combined with functional assays for coupling.