As nascent pre-mRNAs emerge from elongating RNA polymerase II (pol II), their post-synthetic fate is determined by a large number of RNA-binding proteins. Primary among these proteins are the heterogeneous nuclear ribonucleoproteins (hnRNPs) which have been shown to play multiple roles in mRNA processing and transport. The broad objective of this project is to understand how a subset of yeast hnRNPs interact with the transcription machinery to regulate specific gene expression. The central hypothesis of this project is that a subset of hnRNPs and processing factors form a complex that binds specific cis-acting sites in some nascent pol II transcripts. The formation of such hnRNP-RNA complexes is proposed to lead to transcription pausing, arrest, or termination through interaction with the pol II C-terminal domain (CTD). CTD kinase I (CTDK-I) may regulate this process by phosphorylating the CTD and changing the nature of the interaction with the hnRNP complex. This hypothesis will be tested in the yeast Saccharomyces cerevisiae. The first objective is to determine whether interaction between the Nrd1p/Nab3p hnRNP complex and the CTD is regulated by phosphorylation and, if so, whether CTDK-I carries out this phosphorylation. In vitro binding experiments will test the role of CTD phosphorylation in regulating this interaction. Genetic experiments will be used to test the role of CTDK-I. A second objective is to identify cis-acting RNA elements in genes that are regulated by Nrd1p and/or Nab3p. Both genetic and biochemical approaches will be used to characterize an autoregulatory element in the NRD1transcript. Similar elements will be sought in other genes that are regulated by NRD1 and NAB3. The final objective is to determine the stage in the transcription cycle regulated by Nrd1p and Nab3p. The autoregulation of NRD1 expression will be studied in vivo and in vitro to determine the mechanism of NRD1 function. Reaching these objectives will help to define a novel regulatory pathway in yeast. While RNA-binding proteins have been shown to regulate transcription in procaryotes and in HIV infection, no similar regulatory mechanism has been described in yeast. This project draws together the areas of hnRNP function and transcription regulation in an organism amenable to both genetic and
