Investigation into the subcellular localisation and function of miR-122

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

Eukaryotic translation initiation factor 4All contributes to microRNA-122 regulation of hepatitis C virus replication

Hepatitis C virus (HCV) is a positive sense RNA virus that persistently infects human liver, leading to cirrhosis and hepatocellular carcinoma. HCV replication requires the liver-specific microRNA-122 (miR-122). In contrast to canonical miRNA-mediated repression via 3’UTR sites, miR-122 positively regulates HCV replication by a direct interaction with the 5’ untranslated region (UTR) of the viral RNA. The protein factor requirements for this unusual miRNA regulation remain poorly understood. Here, we identify eIF4AII, previously implicated in miRNA-mediated repression via 3’UTR sites, as a host factor that is important for HCV replication. We demonstrate that eIF4AII interacts with HCV RNA and that this interaction is miR-122-dependent. We show that effective miR-122 binding to, and regulation of, HCV RNA are reduced following eIF4AII depletion. We find that the previously identified HCV co-factor CNOT1, which has also been implicated in miRNA-mediated repression via 3’UTR sites, contributes to regulation of HCV by eIF4AII. Finally, we show that eIF4AI knockdown alleviates the inhibition of HCV replication mediated by depletion of either eIF4AII or CNOT1. Our results suggest a competition effect between the eIF4A proteins to influence HCV replication by modulation of miR-122 function

Investigation into the subcellular localisation and function of miR-122

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

Eukaryotic translation initiation factor 4All contributes to microRNA-122 regulation of hepatitis C virus replication

Hepatitis C virus (HCV) is a positive sense RNA virus that persistently infects human liver, leading to cirrhosis and hepatocellular carcinoma. HCV replication requires the liver-specific microRNA-122 (miR-122). In contrast to canonical miRNA-mediated repression via 3’UTR sites, miR-122 positively regulates HCV replication by a direct interaction with the 5’ untranslated region (UTR) of the viral RNA. The protein factor requirements for this unusual miRNA regulation remain poorly understood. Here, we identify eIF4AII, previously implicated in miRNA-mediated repression via 3’UTR sites, as a host factor that is important for HCV replication. We demonstrate that eIF4AII interacts with HCV RNA and that this interaction is miR-122-dependent. We show that effective miR-122 binding to, and regulation of, HCV RNA are reduced following eIF4AII depletion. We find that the previously identified HCV co-factor CNOT1, which has also been implicated in miRNA-mediated repression via 3’UTR sites, contributes to regulation of HCV by eIF4AII. Finally, we show that eIF4AI knockdown alleviates the inhibition of HCV replication mediated by depletion of either eIF4AII or CNOT1. Our results suggest a competition effect between the eIF4A proteins to influence HCV replication by modulation of miR-122 function

Thymosin beta 4 prevents systemic lipopolysaccharide-induced plaque load in middle-age APP/PS1 mice

Lipopolysaccharide (LPS) produced by the gut during systemic infections and inflammation is thought to contribute to Alzheimer's disease (AD) progression. Since thymosin beta 4 (Tβ4) effectively reduces LPS-induced inflammation in sepsis, we tested its potential to alleviate the impact of LPS in the brain of the APPswePS1dE9 mouse model of AD (APP/PS1) and wildtype (WT) mice. 12.5-month-old male APP/PS1 mice (n = 30) and their WT littermates (n = 29) were tested for baseline food burrowing performance, spatial working memory and exploratory drive in the spontaneous alternation and open-field tests, prior to being challenged with LPS (100ug/kg, i.v.) or its vehicle phosphate buffered saline (PBS). Tβ4 (5 mg/kg, i.v.) or PBS, was administered immediately following and at 2 and 4 h after the PBS or LPS challenge, and then once daily for 6 days (n = 7-8). LPS-induced sickness was assessed though monitoring of changes in body weight and behaviour over a 7-day period. Brains were collected for the determination of amyloid plaque load and reactive gliosis in the hippocampus and cortex. Treatment with Tβ4 alleviated sickness symptoms to a greater extent in APP/PS1 than in WT mice by limiting LPS-induced weight loss and inhibition of food burrowing behaviour. It prevented LPS-induced amyloid burden in APP/PS1 mice but increased astrocytic and microglial proliferation in the hippocampus of LPS-treated WT mice. These data show that Tβ4 can alleviate the adverse effects of systemic LPS in the brain by preventing exacerbation of amyloid deposition in AD mice and by inducing reactive microgliosis in aging WT mice

Pramipexole induced place preference after L-dopa therapy and nigral dopaminergic loss: linking behavior to transcriptional modifications

International audienc

Specifically neuropathic Gaucher's mutations accelerate cognitive decline in Parkinson's

International audienc