Investigation into the subcellular localisation and function of miR-122

Winlow, Poppy (2021) Investigation into the subcellular localisation and function of miR-122. PhD thesis, University of Nottingham.

PDF (Thesis - as examined) - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader
Download (7MB) | Preview


MicroRNAs are key post-transcriptional regulators of gene expression and function either by translational repression or degradation of target mRNAs. To do so, microRNAs must form an RNA-induced silencing complex (RISC) and direct its binding to the 3’ UTR of their target mRNAs. Whilst the understanding of microRNA function has been extensively investigated, there has been little research on whether this differs in subcellular locations.

Although the majority of mature microRNA and their targets are concentrated in the cytoplasm, there is growing evidence for microRNA localisation and function in different subcellular compartments. As there has been no direct comparison of microRNA function in different subcellular sites in human cells, this project aims to address this question by applying subcellular fractionation methods and by generating luciferase reporters to compare regulation between subcellular compartments. This project specifically aimed to investigate microRNA regulation at the Endoplasmic Reticulum (ER), as they may have a direct role in silencing transcripts encoding secreted or membrane-localised proteins, and in the nucleus where they may have a range of functions including the regulation of microRNA biogenesis and regulation of nascent transcripts at the chromatin.

The overall aim was to investigate the subcellular localisation and function of miR-122. It is liver-specific and one of the most highly expressed microRNAs, accounting for roughly 70% of the total microRNA pool in Huh7 hepatocellular carcinoma cells, making it an ideal target for study. Furthermore, the role of miR-122 in the positive regulation of Hepatitis C Virus (HCV) RNA in liver cells provides the opportunity to examine if there are any differences in regulation between the ER and cytoplasm in the context of up-regulation of translation by miR-122.

To establish how microRNA regulation occurs at the ER versus cytoplasm, a series of ER-translated luciferase reporters for miR-122 regulation at the 3’ UTR were successfully generated and their regulation by miR-122 was compared with that of equivalent reporters translated in the cytoplasm. This approach also enabled the comparison of miR-122 repression of the 3’ UTR to miR-122 activation of translation via 5’ UTR sites from HCV RNA. In summary, there was evidence for differential regulation via 3’ UTR sites at the ER and cytoplasm in some, but not all, tested reporters.

Next, regulation of endogenous miR-122 targets that are known to associate to different subcellular sites were investigated using a membrane fractionation method to isolate ER and cytoplasm-localised mRNAs. The effects of miR-122 inhibition and overexpression on known miR-122 mRNA targets was compared in these fractions and some differences in regulation of these endogenous targets was observed between the ER and cytoplasm.

Finally, to examine the localisation of miR-122 within the nucleus a fractionation method was used to isolate chromatin and nucleoplasmic fractions from the cytoplasm which demonstrated the presence of miR122 specifically in the chromatin fractions. To investigate the role of miR122 in the chromatin, CRISPR/Cas9n genome modification was designed to disrupt a potential miR-122 seed match downstream of the pre-miR-122 encoding gene with the aim of investigating whether miR122 autoregulates in a similar fashion to let-7 in C.elegans.

Ultimately these investigations provide new understanding of the subcellular localisation of miR-122 in Huh7 cells, demonstrating differences in miR-122 regulation at the ER and cytoplasm, and generated tools for the further analysis of miR-122 activity at different subcellular sites.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Catherine, Jopling
Hilary, Collins
Keywords: MicroRNAs, subcellular compartments, Endoplasmic Reticulum, cytoplasm
Subjects: Q Science > QH Natural history. Biology > QH426 Genetics
Q Science > QH Natural history. Biology > QH573 Cytology
Q Science > QP Physiology > QP501 Animal biochemistry
Faculties/Schools: UK Campuses > Faculty of Science > School of Pharmacy
Item ID: 66832
Depositing User: Winlow, Poppy
Date Deposited: 08 Dec 2021 04:40
Last Modified: 08 Dec 2021 04:40

Actions (Archive Staff Only)

Edit View Edit View