Title: Mechanisms coupling steps in gene expression
Description: Eukaryotic gene expression is a multi-step process beginning with transcription of pre-mRNA in the nucleus. The pre-mRNA undergoes several processing steps, including 5' capping, splicing, and 3' end processing. Finally, spliced mRNA is exported to the cytoplasm for protein synthesis. Although each of these steps requires distinct machineries, they are physically and functionally coupled to one another. This dissertation focuses on understanding the coupling among steps in gene expression from transcription to translation. In Chapter 2, I describe the development of a mini-nuclear extract method combined with RNA interference to determine the functions of specific proteins in the coupled RNAP II transcription/splicing reaction. The feasibility of this method was demonstrated by knocking down two model proteins, the conserved splicing factors U1C and Slu7. My data indicate that the knockdown mini-nuclear extract is a rapid and general in vitro strategy for determining the functions of specific proteins in gene expression, as well as in other cellular processes. In Chapter 3, I investigate the function of eIF4AIII, a translation initiation-like factor present in the nucleus. My work showed that eIF4AIII is recruited to spliced mRNPs and is a component of the exon junction complex, which is a protein complex recruited upstream of exon junctions during splicing. In addition, my work indicated that exon junction complexes are recruited to every exon junction present in the mRNA. Finally, eIF4AIII, as well as a translation factor DDX3, co-localizes with splicing factors in nuclear speckle domains. Thus, eIF4AIII and DDX3 may be recruited to mRNA during splicing in the nucleus, and then function in translation-related processes in the cytoplasm.