Caenorhabditis elegans embarks on a quiescent dauer state upon exposure to unfavourable conditions and can sustain for a very long period without food, but it returns to continuous life cycle upon arrival of suitable conditions. Thus, dauer state plays a critical role in its adaptive fitness and survival. ATP-independent endoribonuclease activity of XRN-2 has been implicated in dauer microRNA metabolism, perturbation of which causes their collapse within a very short span of time. Here, we present a detailed comparative analyses of dauer transcriptomes from a conditional mutant strain for the endoribonuclease activity of XRN-2, maintained under control and experimental conditions. We observed that even a limited disruption of microRNA homeostasis in experimental dauers results in deregulation of a large number of mRNA targets. Our bioinformatic analyses, supported by morphological, physiological, and behavioral evidence further demonstrate critical changes in metabolism leading to a state unsupportive of dauer maintenance, alongside potential defects in multiple neuronal activities, which might have caused an overall disruption of dauer plasticity. We explore a possible role of this endoribonuclease activity towards the maintenance of chromatin architecture and transposon expression that in turn might affect the transcriptional program critically required for the maintenance of non-aging, long-lived dauers. Finally, we also demonstrate that perturbation of the endoribonuclease activity during the dauer state exerts drastic adverse effects on the continuity of life cycle after dauer-exit. They not only fail to recapitulate the wild type events of germline development and embryogenesis, but also present traits of very old worms and formation of ‘tumor-like’ structures in the proximal gonad.

MicroRNAs are critical regulators of diverse developmental and physiological processes, and they themselves get regulated both at the level of biogenesis and turnover. We demonstrate that the ribonuclease XRN2 can degrade the mature forms of certain let-7 family members in multiple human cancer cell lines, without affecting their precursors. 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 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. We demonstrate that the miRNA is released by an as-yet unidentified proteinaceous ‘miRNA release factor’ from the grasp of Argonaute before its degradation. Our analyses of the patient-derived transcriptomics data also show that XRN2, via its regulation of let-7, affects multiple pathways in a consistent manner across epithelial and glial cell lineages, and thus, is of critical pathophysiological significance.

Piwi-interacting RNAs (piRNAs) are an animal-specific class of germline-enriched small non-coding RNAs that shape transcriptome, as well as ensure genomic integrity and fertility by regulating transposons and other selfish genetic elements. In Caenorhabditis elegans mature piRNAs are 21-nucleotides long, begin with a monophosphorylated uridine, and they associate with PRG-1 to form piRISCs that scan the transcriptome for ‘non-self’ sequences. However, these piRNAs are born as longer 5’-capped transcripts, where PARN-1, a 3’-5’ exoribonuclease, contributes to the formation of the mature 3’-end. But, till date, the 5’-processing events remain elusive. We demonstrate that the recently identified endoribonuclease activity of XRN-2 is involved in the processing of the 5’-end of precursor piRNAs in worms. Depletion of XRN-2 results in reduced mature piRNA levels, with concomitant increase in levels of the 5’-capped precursors. We also reveal that the piRNAs born as longer precursor molecules (≥60 nt), prior to 5’-end processing, undergo ENDU-1-mediated endoribonucleolytic processing of their 3’-ends. Our in vitro RNA-protein interaction studies unravel the mechanistic interactions between XRN-2 and PRG-1 towards the formation of mature 5’-ends of piRNAs. In vivo experiments employing prg-1 mutant worms indicate that XRN-2 has the potential to perform clearance of precursors that are not bound and protected by PRG-1. Finally, we also demonstrate that XRN-2 is not only important for the generation of mature piRNAs and piRNA-dependent endo-siRNAs, but through yet unknown pathways, it also affects piRNA-independent endo-siRNAs that shape transcriptome, as well as contribute to genomic integrity via regulation of transposable elements.

MicroRNAs (miRNAs) are critical regulators of diverse developmental and physiological processes, and they themselves get regulated both at the level of biogenesis and turnover. We demonstrate that the ribonuclease XRN2 can degrade the mature forms of certain let-7 family members in multiple human cancer cell lines, without affecting their precursors. XRN2 also affects the accumulation of several other tumor suppressor miRNAs known to play important roles in cancer metabolism. XRN2 depletion results in a reduction in the expression of many oncogenes and diminishes the proliferative and metastatic potential of cancer cells in vitro . These experimental cancer cells also show reduced capacity to form tumors in mice and regress over time. The clinical relevance of these observations is further verified in tumour 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. We demonstrate that the miRNA is released by an as-yet unidentified proteinaceous ‘miRNA release factor’ from the grasp of Argonaute before its degradation, which is more abundant in the nuclear fraction. Our analyses of the patient-derived transcriptomics data also show that XRN2, via its regulation of let-7, affects multiple pathways in a consistent manner across epithelial and glial cell lineages, and thus, is of critical pathophysiological significance.

microRNAs (miRNAs) are known to regulate a vast majority of the eukaryotic genes by post-transcriptional means, and multiple nucleases play critical roles in the biogenesis and turnover of these regulators. A number of studies have indicated that turnover is important for determining the abundance of miRNAs, and thus, in turn govern their functionality. Recent research in Caenorhabditis elegans has revealed an ATP-independent endoribonuclease activity of the ‘miRNase’-XRN-2. Here, we report the characterization of this new enzymatic activity of the fundamentally important XRN-2, and show that it is critical for miRNA turnover and survival of quiescent dauer worms. The dual enzymatic activity of XRN-2 capacitates the mechanism of miRNA turnover to be dynamic, which might confer adaptive advantage to the organism. In continuously growing worms, this new enzymatic activity does not act on miRNAs, but it is important for the generation of mature ribosomal RNAs, which in turn is critical for translation, and thus indispensable for the survival of worms.

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.

microRNAs are known to regulate expression of more than two third of all the eukaryotic genes by post-transcriptional means, and regulation of these tiny regulators play an important role in determining their activities. Here, we report a macromolecular microRNA turnover complex, whose components are crucial to microRNA homeostasis and development in Caenorhabditis elegans . Biochemical investigations with the purified complex in an isolated system not only unfolded the roles of the individual subunits critical for the functionality of the complex, but also unraveled the different modes of operations and regulatability of this biological machine. Our results reveal that this complex is highly receptive and capable of switching between an ATP-dependent and ATP-independent mode of operation depending on the availability of ATP in its environment, which might allow the complex to function dynamically during different physiological conditions.

MicroRNAs (miRNAs) are tightly regulated through transcriptional and posttranscriptional mechanisms, including degradation by nucleases. Here, we report that in C. elegans, target mRNAs can protect their cognate miRNAs from degradation in vivo. We show that the let-7(n2853) mutation destabilizes the mature let-7 miRNA by impairing this protection. Moreover, presence of a cognate target or depletion of the xrn-1 (XRN1) or xrn-2 (XRN2/Rat1p) exoribonucleases enforces accumulation of certain miRNA passenger (miR∗) strands. Thus, following biased miRNA strand loading into Argonaute, elimination of nonfunctional RNAs can further refine miRNA strand selection. Conversely, by aligning the levels of miRNAs with those of their targets, the opposing activities of mature miRNA degradation and target-mediated miRNA protection (TMMP) may enable dynamic expression of either mature strand of a pre-miRNA, and evolution of miRNAs. Thus, it seems that mRNAs are more than inert targets and function with miRNAs in a network of mutual regulation.

microRNAs (miRNAs) are small noncoding RNAs that regulate numerous target mRNAs through an antisense mechanism. Initially thought to be very stable with half-lives on the order of days, mature miRNAs have recently been shown to be subject to degradation by 'microRNases' (miRNases) in plants (the small RNA degrading nucleases, SDN) and animals (exoribonuclease 2/XRN-2/XRN2). Interference with these miRNA turnover pathways causes excess miRNA activity, consistent with an important contribution to miRNA homeostasis. Moreover, it is now emerging that long half-lives are not an invariant feature of miRNAs but that marked differences exist in the stabilities of individual miRNAs and that cellular states can further determine miRNA turnover rates. Although the means of regulation are still largely unclear, biochemical data suggest that target mRNA-binding can stabilize miRNAs within their Argonaute (AGO) effector complexes, providing one possible mechanism that may control miRNA half-lives. We will summarize here what is known about miRNA turnover in animals and how recent discoveries have established a new dynamic of miRNA-mediated gene regulation. We will highlight some of the open questions in this emerging area of research.

Recycling silencing complexes The class of small RNAs known as microRNAs play important roles in shaping gene expression profiles during development, by binding to and inhibiting, or silencing, the translation of certain messenger RNAs. It is thought that miRNAs were one factor responsible for the evolution of unicellular organisms into multicellular organisms. Saibal Chatterjee and Helge Groβhans report that after miRNAs have acted on a target mRNA and are released from the silencing complex, the ribonuclease XRN-2 promotes their degradation. In this way, XRN-2 acts as a homeostatic regulator of miRNA levels, which may be important in responding to new developmental cues.