Williams, Kathryn
(2021)
Tying the CNOT: CNOT1 regulates poly(A) tail length at the end and the beginning.
PhD thesis, University of Nottingham.
Abstract
Poly(A) tails affect multiple aspects of gene regulation: they help identify mRNAs for nuclear export, enhance translation efficiency, and are essential to regulating mRNA degradation rate. A poly(A) tail of 200-250 residues is thought to be uniformly added to newly synthesised mRNA and later gradually removed in the cytoplasm, allowing degradation of the mRNA itself.
Previous work in the lab showed that poly(A) tail addition is not uniform; soon after serum induction, transiently expressed mRNAs are exported with long poly(A) tails, but towards the end of the transcription pulse the tails of new transcripts are much shorter. In contrast, housekeeping mRNAs consistently receive only 30-70 adenosines both before and throughout the serum response and do not appear to be gradually deadenylated.
Given these controversial findings, the work presented here began by assessing the suitability of the PCR-based PAT assay for detecting differences in poly(A) tail length. This was achieved by comparing poly(A) length measurements obtained using the PAT assay with those using RNase H northern blots, which detected RNA directly. PAT assays of chromatin-associated, nucleoplasmic and cytoplasmic fractions then revealed that in NIH 3T3 cells, the poly(A) tail lengths of most mRNAs tested were determined before release from the chromatin. For the remainder of mRNAs, poly(A) tail lengths were determined in the nucleoplasm. Genome-wide analysis of poly(A) tails using adapted RNA-Seq (PQ-Seq) showed that in NIH 3T3 cells, nuclear regulation of polyadenylation was widespread and was not limited to the mRNAs previously selected for PAT. Short poly(A) tails were associated with reduced stability of transiently expressed transcripts, and it logically follows that nascent poly(A) regulation resulting in production of short-tailed transcripts at the end of the response may enhance the precision with which gene expression is controlled. Specifically, production of unstable transcripts at the end of the serum response would sharpen the peak in mature mRNA levels, limiting the time during which translation can occur.
Knockdown of the mRNA encoding the CCR4-NOT deadenylase subunit, CNOT1, increased chromatin and/or nucleoplasmic poly(A) tail size for all mRNAs tested, indicating it was involved in nuclear poly(A) tail regulation. Furthermore, the magnitudes of these changes were gene-specific. Preliminary data suggested that CCR4-NOT-dependent initial poly(A) regulation may also occur in human cells. As well as increasing initial poly(A) length (and therefore presumably enhancing transcript stability), Cnot1 knockdown caused decreases in pre-mRNA levels of all mRNAs tested. Together, these data suggested that the CCR4-NOT complex may mediate mRNA homeostasis in mammalian cells.
Several lines of enquiry were followed to explore the mechanism through which CNOT1 both limited initial poly(A) length and seemingly promoted mRNA production. Although the exact mechanism remained elusive, differential effects of RNAi-mediated depletion versus pharmacological inhibition of the complex’s CAF1 subunit suggested that the documented involvement of CCR4-NOT in both deadenylation and transcription elongation may have been significant.
The above findings complement work from other groups showing changes to CCR4-NOT subunit levels in different physiological conditions (e.g. nutrient deprivation, B cell activation). Specifically, levels of CCR4-NOT may be adjusted to simultaneously affect both mRNA production (through promoting transcription elongation) and nuclear determination of cytoplasmic mRNA stability (through promoting nuclear deadenylation). In this way, the mammalian CCR4-NOT complex may mediate high and low mRNA turnover states according to the state of the cell.
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