title: Investigating the mechanisms of nuclear RNA quality control in Saccharomyces cerevisiae
Creators: name: Fulton, Stuart
Publisher: name: University of Liverpool Repository
Year: 2028

description: Quality control of mRNA biogenesis is crucial for eukaryotic gene expression and central to cellular homeostasis. Deregulated RNA quality control has been associated with numerous human diseases, including cancer. Evidence indicates that RNA quality control is closely linked to the transcriptional process. Cryptic and alternative initiation by RNA Polymerase II (RNAPII) leads to differential transcript isoform expression, while elongating RNAPII aborts and terminates transcription prematurely to prevent aberrant mRNA synthesis, with resulting transcripts rapidly degraded in the nucleus. However, the co-transcriptional mechanisms preventing expression of aberrant or non-functional transcripts remain poorly understood. 
The DNA Replication Checkpoint Kinase, Mec1, the yeast homolog of human Ataxia telangiectasia and Rad3-related kinase, ATR, is activated by DNA damage and replication stress. Mec1 is recruited to sites of DNA damage by Replication protein A (RPA) and classically functions in the stabilization of stalled replication forks which can form following collisions between the DNA replication, and transcriptional machinery. Recent publications involve Mec1 in RNAPII degradation. Mec1 has been shown to interact with the chromatin remodelling complex INO80, facilitating the eviction of RNAPII after replication stress. Recent work from our lab reveals a role for INO80C in non-coding transcription termination. This suggests that there may be an undiscovered role for Mec1 in transcription regulation and RNA quality control.
Here I pursue the hypothesis that in budding yeast, Mec1 may act to modulate transcription. The evidence I present suggests that Mec1 deletion causes sensitivity to transcriptional elongation stress, transcriptional read-through, inefficient non-coding Nrd1-Nab3-Sen1(NNS) dependent transcription termination and accumulation of terminated transcripts. Additionally, I report an unanticipated role for the ssDNA binding protein and replication checkpoint factor, RPA, in the inhibition of non-coding transcription termination. 
In this thesis, I report the development of 3'-5' RACE-Seq (Rapid Amplification of cDNA Ends), a novel genome-wide assay that can detect alternative initiation and termination events both quantitatively and qualitatively. Using 3’-5’ RACE-Seq, I analyse the transcriptome landscape of S.cerevisiae and map transcript isoforms at nucleotide resolution. I present analysis indicating a role for Mec1 in premature transcription termination and the suppression of cryptic transcription initiation within a proportion of genes. RACE-Seq analysis in the absence of Rrp6, a 3'-5' nuclear exosome subunit or the 5′-3′ cytoplasmic exonuclease Xrn1 reveals extensive premature termination across the yeast protein coding genome. My analysis demonstrates that while Xrn1 degrades transcripts which terminate across the gene body, transcripts degraded by the Rrp6 are terminated at specific genomic sites. I find that premature termination of mRNA genes is associated with binding motifs for specific RNA processing and DNA regulatory factors. Notably, this evidence suggests that both nuclear and cytoplasmic RNA degradation pathways preferentially target transcript isoforms initiated throughout the gene body, or at alternative promoter sites. I report that Nrd1, a component of NNS non-coding termination complex is responsible for sense promoter proximal termination, and antisense TES proximal termination. I also uncover a novel role for Nrd1 in suppression of cryptic transcription initiation. This work reveals the dynamic and complex nature of RNA quality control regulation.