Regulation of let-7 microRNAs by a two-step turnover pathway is of critical pathophysiological significance in humans

MicroRNAs (miRNAs) are critical regulators of diverse developmental and physiological processes in animals, and their dysregulation has been linked to various disorders and diseases. Not only multiple regulatory mechanisms acting at different levels of human miRNA biogenesis determine miRNA abundance and function, but a few recently identified factors by facilitating miRNA turnover also make critical contributions. We demonstrate that the ribonuclease XRN2, whose worm ortholog had previously been shown to actively degrade let-7 family of miRNAs, can degrade the mature forms of certain let-7 family members in multiple human cancer cell lines, without affecting their precursors. The XRN2mediated turnover of let-7 has patho-physiological significance as XRN2 depletion results in a reduction in the expression of a number of oncogenes, and diminishes the proliferative and metastatic potential of cancer cells. The clinical relevance of these observations is also verified in tumour transcriptomics data from public RNA-sequencing datasets, where we observe that higher XRN2 mRNA expression is inversely correlated with the levels of mature let-7 miRNAs and associated with poor survival in hepatocellular carcinoma, lung adenocarcinoma, and glioblastoma. We also demonstrate that miRNA turnover is a step-wise process, where a miRNA is released from the grasp of Argonaute before its degradation. This yet unidentified ‘miRNA releasing factor’ is proteinaceous in nature and its activity is kinetically linked with XRN2mediated turnover of miRNAs. Our analyses of the patient-derived transcriptomics data also show that XRN2, via its regulation of let-7, affects pathways related to cellular proliferation, development, and signalling in a consistent manner across epithelial and glial cell lineages. Collectively, our studies suggest an important role of XRN2 in regulating cancer physiology through degradation of the let-7 family of miRNAs. Introduction miRNAs function as critical regulators of diverse developmental and physiological processes in animals. Extensive studies on miRNA abundance and function have demonstrated a consistent link between dysregulation of miRNAs and various diseases, highlighting the importance of robust regulation of miRNA activity. Many miRNAs exhibit tissueand/or stage-specific expression patterns 3 and dramatic changes in the abundance of a number of miRNAs during different developmental stages of animals have also been reported. Global kinetic studies of miRNA metabolism have revealed that regardless of high biogenesis, several miRNAs exhibit reasonably low steady-state levels. It indicated that additional determinants, other than the factors of biosynthesis, play critical roles for the establishment of cellular miRNA homeostasis. A substantial amount of knowledge has been acquired on the different steps of miRNA biogenesis and their regulation, but much less is known about the miRNA turnover pathways and the constituent molecular machineries. Active turnover of miRNAs in animals was first reported in Caenorhabditis elegans (C. elegans), where 5’-3’ exoribonuclease XRN-2 was demonstrated to mediate the degradation of several miRNAs. Later, the paralogous protein, XRN-1, was also described as a 'miRNase’ in worms. In mammalian cells, a number of nucleases have been described to act on a given miRNA or a handful of miRNAs. XRN1 has been implicated in the turnover of miR-382 in HEK293 cells, and polynucleotide phosphorylase degrades miR-221, miR-222, miR-106b in human melanoma cells. Tudor-SN was demonstrated to target several functional mature miRNAs, including miR-31 and miR-29b, which control specific mRNAs encoding cell cycle regulatory proteins. Interestingly, Argonaute1 (Ago1)-bound miRNAs in Drosophila are known to be tailed by terminal nucleotidyl transferase and trimmed by the 3’ – 5’ exoribonuclease Nibbler upon binding an exogenously introduced target that harbours extensively complementary binding-sites for the given miRNA. Notably, very few unusual extensively complementary endogenous targets have also been reported that lead to the 3’-end trimming-mediated destabilization of the cognate miRNA upon their interaction. The terminal uridyl transferases TUT4 and TUT7 are known to participate in tailing of miRNAs across species from C. elegans to humans. A recent work using HEK293T cells has identified a machinery consisting of TUTs and DIS3L2, which could execute the decay of a subset of Argonaute (AGO)-bound mature miRNAs that have exposed 3′ ends. Very recently, two simultaneous reports have substantially accentuated our understanding on the mechanisms of target-directed miRNA degradation (TDMD), where it was described that pairing of RISC/AGO-loaded miRNA to certain unusual highly complementary targets triggers ZSWIM8 ubiquitin ligase-mediated degradation of AGO, rendering the miRNA susceptible to decay by unknown nuclease(s). Perturbation of ZSWIM8 led to the accumulation of several miRNAs in different mammalian cells and several other systems (worms and flies). Intriguingly, half-lives of AGO proteins are much longer than majority of the miRNAs, which suggested that ZSWIM8-mediated TDMD may not be the predominant miRNA turnover mechanism, rather, it is dedicated towards miRNAs that interact with unusual highly complementary targets. Thus, it is quite apparent that identification of yet unknown ‘miRNases’, followed by understanding of the pathways in which they are constituents, and their mechanistic details warrant further investigation. Notably, although, knowledge about miRNA metabolism emanated from the worm system have been found to be largely conserved, but the role of the human ortholog of the worm ‘miRNase’-XRN-2 remains unclear. Here, we explore the capacity of human XRN2 as a ‘miRNase’, but we decided to focus on the fundamentally important let-7 family of miRNAs that are known to regulate important target mRNAs in multiple tissues, whose perturbation leads to different disease states. We demonstrate that XRN2 specifically acts on the mature forms of most of the let-7 family members. Rapid and efficient depletion of XRN2 leads to the accumulation of AGO-bound let-7 members that in turn downregulate their cognate targets, including proto-oncogenes. Depletion of XRN2 in different cancer cells affect their cellular physiology by reversing critical cancer-parameters, including epithelial-to-mesenchymal transition. Our ex vivo biochemical assays indicate that XRN2 mediated degradation of mature let-7 miRNAs happens upon ‘release’ of miRNAs from AGO by a proteinaceous factor without affecting AGO integrity, and these two steps are kinetically linked. We also demonstrate that our cell line-based observations are of critical significance as they correlate with the clinical data of cancer patients from public transcriptomics datasets. Here, our analyses partially explain the multifarious roles of XRN2 in cancer through its likely participation in miRNA turnover, with the observation that XRN2 influences multiple pathways related to cellular proliferation, development, and signalling, especially related to extracellular matrix (ECM) development, and that these influences are mostly consistent across both epithelial and glial lineages. These analyses also partially explain previous observations of elevated XRN2 mRNA expression being associated with worse survival in lung cancer patients, and extend such observations into the more expansive Cancer Genome Atlas (TCGA) datasets for lung adenocarcinoma, hepatocellular carcinoma, and glioblastoma. Our analyses also suggest that for most of the let-7 family members, XRN2 demonstrates a stronger inverse relationship with them than some of the recently identified factors implicated in miRNA turnover, at an RNA level. Collectively, our study reveals that human let-7 miRNAs are regulated by a two-step turnover pathway, wherein, XRN2 plays the role of a ‘miRNase’ in various tissues.