| dc.description.abstract | Eukaryotic gene expression begins with transcription of DNA into RNA by RNA polymerases and for protein coding genes is followed by translation into protein in the cytoplasm. Production of functioning mature RNA relies on proper processing events including 5’ capping, splicing and 3’ end processing. Endonucleases that cleave RNA are vital for these processing events and are involved in degradation pathways that may also be relevant for the turnover of aberrant transcripts. Studies investigating transcription, processing events and degradation pathways of RNA have generally focused on RNA polymerase II transcripts, which includes protein-coding genes. Many of these pathways were elucidated by studies in yeast due to the high conservation of the transcription process between yeast and metazoans. The discovery and development of CRISPR/Cas9 mediated genome editing techniques have led to a more complete and direct approach to study specific protein functions, within human cells, than previous methods such as RNAi. In this study, a combination of CRISPR/Cas9 with protein tags including the auxin inducible degron and small molecule assisted shut-off, allowed rapid and conditional protein depletion in human cell lines for three endonucleases, DIS3, INTS11 and CPSF73. These endonucleases cooperate with accessory proteins and actively transcribing polymerase complexes to target a broad range of RNA transcripts, to ensure proper RNA processing and integrity of the transcriptome. Generation of these cell lines, coupled with high-throughput RNA sequencing analysis of nuclear transcriptomes, helped to elucidate specific substrates for each endonuclease. The following work shows the effects of aberrant processing in a variety of transcripts, their subsequent potential degradation and what happens when a major degradation pathway is disrupted. A major finding in this study was disruption of 3’ end processing in protein coding mRNA resulted in extensive readthrough and termination defects, whereas 3’ end misprocessing in smaller RNA species including snRNAs and replication dependent histones results in a much smaller extension and termination that occurs relatively close to the gene transcription end site. Additionally, this works shows the importance of the exosome subunit, DIS3, in maintaining appropriate gene expression and RNA environment, whilst suggesting aberrant RNA processing may commonly occur in human cells. | en_GB |
