|dc.description.abstract||Ribonucleic acid (RNA) molecules play a central role in every pivotal process in the cell, and ribonucleases (RNases) are critical for their biogenesis, processing, and degradation. Therefore, RNases are indispensable for cellular RNA homeostasis.
MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that extensively regulate gene expression in eukaryotes. Any alteration in their expression profiles, as well as their steady-state levels, may lead to several pathological conditions, notably neurological disorders and cancer. Therefore, regulation of the levels of these regulators is of utmost importance. While the events leading to the biogenesis of a mature, functional miRNA are well lineated, little is known about the turnover pathways responsible for the maintenance of the functional levels of these RNAs.
Recent research in Caenorhabditis elegans (C. elegans), identified and characterized a multiprotein miRNA turnover complex, miRNasome-1. This biological machine is composed of four subunits (XRN-2, PAXT-1, NOL-58, B0024.11/ miRNasome-1.4), and it displays a dual mode of action on the substrate miRNAs in in vitro assays. The researchers also reported a previously unknown endoribonuclease activity of the fundamentally important enzyme, XRN-2, and surprisingly, this activity was found to be much more efficacious on the miRNAs than the previously known exoribonuclease activity. It was shown that miRNasome-1 residing XRN-2’s activity and specificity is governed by two of the newly identified members. The RNA-binding receptor component of the complex, NOL-58, was not only found to be crucial for worm development but also conferred in vivo substrate specificity to the complex, which corroborated with that of the complex’s activity, in vitro. The researchers demonstrated that miRNasome-1 residing XRN-2 cleaves the substrate through an endoribonucleolytic mode at low substrate concentrations, in the absence of ATP. In the presence of ATP, miRNasome-1.4 binds ATP and exerts an inhibitory effect on this endoribonuclease activity. Whereas, at optimal miRNA concentration, NOL-58 binds miRNA and stimulates ATP hydrolysis by miRNasome-1.4 through conformational changes, and the energy is utilized towards the transfer of the miRNA to the ‘miRNase-XRN-2’ for its exoribonucleolysis. Thus, miRNasome-1.4, allowed the complex to switch between two alternative mechanisms of turnover (energy-independent endoribonucleolysis vs energy-dependent exoribonucleolysis). This study clearly demonstrated that the ‘miRNase’ XRN-2, the core protein of miRNasome-1, which was previously known only as an exoribonuclease, harbours a previously unknown, energy-independent endoribonuclease activity.
XRN-2 is a nuclear-localized ribonuclease that is indispensable in all organisms as its deficiency leads to severe developmental defects. It is known to play imperative roles in both maturation and turnover of a cohort of significant RNAs, such as ribosomal RNA, snoRNA, tRNA, etc. It is also known to function in specialized processes integral to RNA metabolism, like transcription termination and gene silencing. Since, all these deductions were through genetic screens and most notably, did not involve a null mutant for the exoribonuclease activity, it is ambiguous whether these roles can be exclusively assigned to the already characterized exoribonuclease activity.
In this study, I report the functional characterization of this novel endoribonuclease activity of XRN-2 in C. elegans. I show that the endoribonuclease active site is formed by five negatively charged amino acid residues, in comparison to the two invariant aspartates of the exoribonuclease active site. Further, it was found that XRN-2 undergoes a significant conformational change upon substrate binding to assemble the active site. I could demonstrate the in vivo importance of this highly efficacious activity in the energy deficient, alternate life stage of the worms called dauer stage. Perturbation of the endoribonuclease activity of XRN-2 in the dauers leads to severe alterations in the steady-state levels of miRNAs, which ultimately leads to the fall of these otherwise sturdy organisms. However, it does not affect the miRNA homeostasis in the continuous life cycle of the worms, where the exoribonuclease activity, in all likelihood, performs the task of miRNA turnover. Moreover, this study also reveals the role of this novel activity in the biogenesis and maturation of the fundamentally important ribosomal RNAs, thus, signifying that further study of this endoribonuclease activity can bring about a major change in the holistic view of RNA metabolism.||en_US