UCLA

Production of RNAs, their processing, and the regulation thereof encompasses numerous biological pathways that work both on their own and in concert with one another to ensure proper gene expression and cellular function. This thesis will present four works, each exploring various RNA processing pathways, how they function, and how they may interconnect with one another in the production or processing of their given substrate RNAs.The first work presented examines the relationship between splicing and 3′ end processing in S. cerevisiae, processes that have been shown to be coupled in mammalian cells. To further explore this relationship, we set out to study the production of RNA in conditions where the cleavage and polyadenylation (CPA) or splicing pathways were inactivated through nuclear depletion of related proteins. We find that unlike in mammalian cells, the processes of splicing and 3′ end processing are not directly coupled in S. cerevisiae. However, we demonstrate that in the event of transcriptional read-through, many yeast mRNAs present extended 5′ ends due to termination failure of an upstream gene. These 5′ extensions are correlated with decreased splicing efficiency in comparison to unextended mRNAs in a length dependent manner. Furthermore, we find that in the event of transcriptional read-through due to CPA inactivation, novel intergenic and intragenic splicing events may occur utilizing splice sites found within the extended RNA products. Continuing this work exploring the effects of CPA inactivation on RNA production, we determined the existence of a population of cleavage and polyadenylation independent mRNAs, which are stably produced in the absence CPA. We provide evidence that this population of mRNAs may utilize novel mechanisms for 3′ end formation without the primary CPA components. First, we demonstrate that in the absence of CPA, transcriptional roadblocks are capable of producing stable mRNA 3′ ends. We also provide evidence for a novel mechanism of 3′ end formation which relies on genetically encoded poly(A) tracts. These poly(A) tracts are actively transcribed and correspond to the exact 3′ end of their given mRNAs, suggesting they may act as pseudo poly(A) tails. In the third work, we follow up on previous data from our lab, which established the use of RNase-III mediated decay (RMD) as a pathway utilized for the regulation of bromo-domain factor 2 (BDF2) mRNAs. The exact mechanism through which this RMD regulation occurred, however was not yet understood. We show that this RMD dependent degradation of BDF2 mRNAs is hyperactivated in conditions which are known to induce increased nuclear retention of global mRNAs, such as salt stress or nuclear depletion of 3′ end processing factors. Strikingly, this RMD dependent processing of BDF2 mRNAs can be prevented in these same conditions through the nuclear depletion of yeast RNase-III, Rnt1p. Together, these data suggest that the regulation of BDF2 by RMD is result of increased proximity with Rnt1p in the nucleus as a result of increased nuclear retention in stress conditions. Finally, we explore the relationships between proper splicing and the production of intron-encoded snoRNAs. We present evidence that when splicing is inactivated, a novel hybrid mRNA-snoRNA (hmsnoRNA) species is generated. Here, we explore the mechanisms which regulate the production and decay of these hmsnoRNAs, shedding light on the importance of proper splicing in the production of intron-encoded snoRNAs. Altogether, the research presented within this thesis furthers the understanding of the relationships between various RNA processing pathways and how these pathways may or may not come together for their proper function in the production of RNAs in the cell. Additionally, we elucidate the existence of potential novel RNA processing pathways in S. cerevisiae.