The Roles of Rnt1 and Putative Endoribonucleases in Eukaryotic mRNA Degradation

Lee-Ann Notice-Sarpaning (Advisor: Ambro van Hoof, PhD)

          Endoribonucleases initiate degradation by cleaving RNAs internally. Mutations in these enzymes have been shown to cause diseases such as cancer, developmental disorders, and neurodegenerative diseases. Yet, eukaryotic endoribonucleases have not been well studied, especially with regard to their role in nuclear mRNA degradation. Rnt1 is a Saccharomyces cerevisiae (budding yeast) nuclear endoribonuclease and homolog of human Drosha with well-characterized roles in the processing of ncRNAs. The enzyme recognizes and cleaves double-stranded RNA stems containing a terminal tetraloop with an AGNN consensus sequence. However, the scope and consequence of its function in mRNA degradation has heretofore been unclear. Previous studies have identified a few mRNAs cleaved by Rnt1 in vitro, but most of these cleavage sites have not been confirmed in vivo or by other studies. Those using genome-wide approaches have relied on changes in overall mRNA levels that include indirect effects. To identify precise Rnt1 cleavage sites throughout the yeast transcriptome in vivo, I compared RNT1 and rnt1Δ strains using an RNA sequencing approach called parallel analysis of RNA ends (PARE). This analysis revealed many previously undetected Rnt1 cleavage sites in protein-coding regions, as well as in other transcripts not previously described as Rnt1 substrates. I showed that identified mRNA targets meet the sequence and structural criteria for cleavage by Rnt1, and using a rnt1-D247R catalytic mutant, I demonstrated that the molecular features of Rnt1 cleavage are identical between mRNA and ncRNA substrates. Working with collaborators who performed Rnt1 in vitro cleavage reactions, I used PARE of in vitro-cleaved RNA to confirm direct cleavage of targets. However, since a substantially larger number of Rnt1 sites were detected in vitro, compared to in vivo, I hypothesized that Rnt1 nuclear localization may limit its access to mRNAs in vivo.

          By performing PARE using rnt1-ΔNLS and rnt1-K45I mutants that relocalized to the cytoplasm or nucleolus, respectively, I demonstrated that Rnt1 localization indeed functions as an additional layer of mRNA target selection control. By re-analyzing published data, I additionally showed that cleaved mRNAs are likely exported to the cytoplasm for further degradation by Xrn1 and that Rnt1 may play a role in regulating gene expression. In fact, several mRNAs rely heavily on Rnt1 for turnover. To further explore the biological relevance of Rnt1 mRNA cleavage, I performed mutational analysis and experimental evolution to show that Rnt1 cleavage of the YDR514C mRNA is essential for maintaining normal cell growth. While Rnt1 recognizes specific molecular signatures to cleave its mRNA targets, the putative cytoplasmic endonucleases Nmd4, Mkt1, Esl1, and Esl2 appear to cleave a wide range of mRNAs with no obvious sequence or structure conservation, possibly implicating these enzymes as broad-spectrum RNases. Overall, my work uncovers a wider target range for the yeast RNase III enzyme, exposing a vital role for the well-known endonuclease in regulating protein-coding genes, and defining a novel pathway of nuclear mRNA decay. Additionally, it investigates the possibility of four previously undescribed endonucleases in mRNA decay and highlights the possibility that other eukaryotic nucleases may participate in the degradation of non-classical targets.

Advisory Committee:

• Ambro van Hoof, PhD, Chair
• Swathi Arur, PhD
• Nayun Kim, PhD
• Anne-Marie Krachler, PhD
• Kevin Morano, PhD

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