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Unravelling the pathways and functions of the RNA exosome

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandlingForskning

  • Evdoxia Karadoulama
Cellular RNA homeostasis and gene expression regulation, which are vital to cell fitness, are tightly controlled by transcription, co- and post-transcriptional processes. Mammalian genomes are pervasively transcribed, producing a plethora of coding and noncoding RNA molecules which require extensive processing and intense surveillance to ensure proper cellular function. RNA decay is indispensable in ensuring regular cellular function by sorting out and regulating the vast transcriptome. The RNA exosome complex is an essential 3’-5’, highly conserved degradation machinery, functioning in both nuclear and cytoplasmic compartments in various configurations. Its cytoplasmic activity, which is mainly the quality control of protein coding mRNAs, has long been in the research spotlight, leaving the nuclear RNA exosome less explored.

This thesis has aimed to enrich our understanding of the nuclear RNA exosome, its functions and associated adaptors. To reach these aims, computational analysis and modelling of the nuclear activities of the RNA exosome and its adaptors was performed utilizing various types of high-throughput sequencing data. More precisely, total and nascent RNA sequencing data was used to estimate gene degradation levels, along with 5’ end transcriptomic data and data mapping active transcription sites, to assess the effect of the exosome on the detected transcription start sites and gene transcription levels, respectively. The estimation of degradation levels was done in exosome and accessory protein depleted conditions.

As part of investigating nuclear RNA exosome decay, we explored the network of the RNA exosome and their substrates. We experimentally described a novel nuclear exosome decay pathway, the PAXT connection, consisting of ZFC3H1 and PABPN1, which was further supported by the high-throughput sequencing data. Genome investigation of PAXT’s substrates revealed the pathway’s specificity for longer and predominantly polyadenylated transcripts in relation to the previously characterized NEXT complex, which mainly targets unprocessed transcripts with short half-lives. Both PAXT and NEXT utilized hMTR4 to connect to the RNA exosome complex in a mutually exclusive fashion. This suggested that the nuclear RNA exosome acquires its targets specificity by swapping its associated partners.

We further explored the polyadenylated substrates of the nuclear RNA exosome by disrupting the exosome’s nuclear function. We observed that nuclear polyadenylated transcripts accumulated in distinct nuclear foci in the RNA exosome’s absence. Additional experiments identified the PAXT component ZFC3H1 as a retention factor of nuclear polyadenylated RNA exosome targets. We discovered that a portion of the nuclear retained RNA was spliced, full length mRNA.

To explore possible regulatory functions of the nuclear RNA exosome, we investigated its contribution during spontaneous stem cell differentiation. More specifically, we evaluated the contribution of transcription versus nuclear RNA exosome decay, in the transcriptomic expression changes observed, between embryonic stem cells (ESCs) and embryoid bodies. We observed an anticorrelation between RNA stabilization and RNA abundance in exosome depleted conditions, which also applied to RNAs differentially expressed between the two cell states. We demonstrated that the nuclear RNA exosome plays a regulatory role in accomplishing the immense and rapid changes in gene expression required during the differentiation process, functioning both independently and in coupling with transcriptional changes.
OriginalsprogEngelsk
ForlagAarhus Universitet
Antal sider140
StatusUdgivet - 2020

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