Characterization of XRN2-mediated microRNA turnover mechanism and its pathophysiological significance in eukaryotes

Abstract

MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that are extensively involved in the posttranscriptional regulation of gene expression in eukaryotes. Comprehensive studies on miRNA expression and function have demonstrated a consistent link between the dysregulation of miRNA expression and various diseases, highlighting the importance of robust regulation of miRNA activity. The process of miRNA biogenesis and various regulatory mechanisms governing miRNA biogenesis are well-understood. However, molecular pathways regulating the miRNA functionality through the turnover of these small RNAs remain to be explored. Several attempts have been initiated to understand the potential of miRNA turnover pathways in maintaining miRNA homeostasis, leading to the discovery of several miRNA-degrading ribonucleases. The first report on the active turnover of miRNAs in animals demonstrated that ribonuclease XRN-2, which is known to perform vital roles in multiple RNA transaction pathways, is also involved in the degradation of several mature miRNAs, including let-7 miRNA in Caenorhabditis elegans (C. elegans). Interestingly, XRN2 and let-7 miRNAs are highly conserved throughout eukaryotic organisms. Reasonably, it raised a question of whether the miRNA degradation activity of C. elegans XRN-2 is conserved in higher animals like human cells. Objective 1: Elucidating the role of XRN2 in the regulation of the microRNAs in human cell lines and its pathophysiological significance. Here, we demonstrate that human XRN2 directly participates in executing the turnover of mature miRNAs in multiple human cancer cell lines and is thus referred to as ‘miRNase’. Further exploration of the capability of XRN2 as a ‘miRNase’ revealed that most of the miRNAs targeted by XRN2 belong to the tumor suppressor family of miRNAs. Substantial increase in the levels of tumor suppressor miRNAs upon XRN2 knockdown in different cancer cell lines, with a concomitant effect on the levels of their crucial target-oncogenes, is endorsed by a major impact on the tumorigenicity of the cancer cells. In vivo experiments in athymic mouse models illustrate a drastic reduction in tumor growth upon XRN2 depletion, which was more prominent in the case of glioblastoma. The clinical relevance of these observations is also verified in tumor transcriptomics data from public RNA-sequencing datasets, where XRN2 mRNA expression is inversely correlated with the levels of a large number of miRNAs, including let-7 members, and high XRN2 mRNA levels are associated with poor survival in hepatocellular carcinoma, lung adenocarcinoma, and glioblastoma. Together, our results elucidate the molecular mechanism underlying the oncogenic potential of XRN2 in relation to its ‘miRNase’ activity and thus explain the multifarious roles of XRN2 in cancer. Objective 2: Deciphering the molecular events that facilitate the XRN2-mediated turnover of AGO-bound miRNAs in human cells. Mature miRNAs are well protected from any enzymatic action due to their firm interactions and defined placement inside the AGO protein. Depletion of XRN2 leads to the accumulation of AGO-bound miRNAs that, in turn, downregulate their cognate targets, indicating that XRN2-mediated turnover of miRNAs occurs downstream of the process of disruption of the AGO2-miRNA complex. Ex vivo biochemical assays indicate that XRN2-mediated degradation of mature let-7 miRNAs happens upon the ‘release’ of miRNAs from AGO by proteinaceous factors without affecting AGO integrity, and these two steps are kinetically linked. Our results suggest that both the release of miRNAs from the grasp of AGO and its subsequent degradation by XRN2 occurs majorly in the nuclear compartment of the cell. Inside the nucleus, XRN2 associates with a nucleolar protein, NOP58, which functions as an RNA binding factor, thereby ensuring XRN2’s nuclease activity to act specifically on mature miRNAs. Collectively, these results reveal that human let-7 miRNAs are regulated by a two-step turnover pathway, wherein XRN2 plays the role of a ‘miRNase’ in various tissues. We further report that the NOP58 protein is also involved in facilitating the generation of mature let-7 miRNAs by directly interacting with the pre-let-7 transcript in the nucleus. Objective 3: Exploration of the function of miRNasome-1 complex during the larval development in Caenorhabditis elegans. Previous studies in our lab have revealed that in C. elegans, an XRN-2 containing multiprotein complex, which is referred to as miRNasome-1, is involved in the active turnover of several mature miRNAs, including the developmentally regulated miRNA let-7. Here we report that the miRNasome-1 mediated degradation, along with the LIN-28-mediated regulation, acts simultaneously but in an independent manner to ensure the complete clearance of mature let-7 miRNAs during the early-L3 stage of C. elegans development. In summary, we propose a model for the active turnover of mature miRNAs, which is a layered process involving multiple steps in human cancer cells. We furnish the role of XRN2, and its associatory proteins, as a critical regulator of miRNA stability with a prominent impact on the physiology of cancer cells, especially their tumorigenicity. Our findings suggest that the putative ‘miRNA release factor’ and XRN2-mediated miRNA turnover constitutes an essential pathway to regulate the stability and function of let-7 members. We also present an additional layer of regulation of functional mature let-7 during the initial development in C. elegans through the involvement of the XRN-2-mediated turnover mechanism.