Gotowa bibliografia na temat „Endoribonuclease activity”

Las1 is a recently discovered endoribonuclease that collaborates with Grc3–Rat1–Rai1 to process precursor ribosomal RNA (rRNA), yet its mechanism of action remains unknown. Disruption of the mammalian Las1 gene has been linked to congenital lethal motor neuron disease and X-linked intellectual disability disorders, thus highlighting the necessity to understand Las1 regulation and function. Here, we report that the essential Las1 endoribonuclease requires its binding partner, the polynucleotide kinase Grc3, for specific C2 cleavage. Our results establish that Grc3 drives Las1 endoribonuclease cleavage to its targeted C2 site both in vitro and in Saccharomyces cerevisiae. Moreover, we observed Las1-dependent activation of the Grc3 kinase activity exclusively toward single-stranded RNA. Together, Las1 and Grc3 assemble into a tetrameric complex that is required for competent rRNA processing. The tetrameric Grc3/Las1 cross talk draws unexpected parallels to endoribonucleases RNaseL and Ire1, and establishes Grc3/Las1 as a unique member of the RNaseL/Ire1 RNA splicing family. Together, our work provides mechanistic insight for the regulation of the Las1 endoribonuclease and identifies the tetrameric Grc3/Las1 complex as a unique example of a protein-guided programmable endoribonuclease.

ABSTRACTWe previously established that cells infected with herpes simplex virus 2 (HSV-2) are disrupted in their ability to form stress granules (SGs) in response to oxidative stress and that this disruption is mediated by virion host shutoff protein (vhs), a virion-associated endoribonuclease. Here, we test the requirement for vhs endoribonuclease activity in disruption of SG formation. We analyzed the ability of HSV-2 vhs carrying the point mutation D215N, which ablates its endoribonuclease activity, to disrupt SG formation in both transfected and infected cells. We present evidence that ablation of vhs endoribonuclease activity results in defects in vhs-mediated disruption of SG formation. Furthermore, we demonstrate that preformed SGs can be disassembled by HSV-2 infection in a manner that requires vhs endoribonuclease activity and that, befitting this ability to promote SG disassembly, vhs is able to localize to SGs. Together these data indicate that endoribonuclease activity must be maintained in order for vhs to disrupt SG formation. We propose a model whereby vhs-mediated destruction of SG mRNA promotes SG disassembly and may also prevent SG assembly.IMPORTANCEStress granules (SGs) are transient cytoplasmic structures that form when a cell is exposed to stress. SGs are emerging as potential barriers to viral infection, necessitating a more thorough understanding of their basic biology. We identified virion host shutoff protein (vhs) as a herpes simplex virus 2 (HSV-2) protein capable of disrupting SG formation. As mRNA is a central component of SGs and the best-characterized activity of vhs is as an endoribonuclease specific for mRNAin vivo, we investigated the requirement for vhs endoribonuclease activity in disruption of SG formation. Our studies demonstrate that endoribonuclease activity is required for vhs to disrupt SG formation and, more specifically, that SG disassembly can be driven by vhs endoribonuclease activity. Notably, during the course of these studies we discovered that there is an ordered departure of SG components during their disassembly and, furthermore, that vhs itself has the capacity to localize to SGs.

ABSTRACT Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in Escherichia coli has endoribonuclease activity that preferentially cleaved 5′ of uridylates of RNAs. Blocking either the 5′ or 3′ terminus did not affect cleavage. Double- and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn2+ at 2 to 10 mM was needed for optimal endoribonuclease activity, but Mg2+ and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn2+ needed for endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus endoribonuclease.

Conditions were established for the assay of three nucleolytic enzymes: a Mg2+-independent endoribonuclease, a Mg2+-dependent endonuclease, and a Mg2+-dependent 5′-exonuclease in Saccharomyces cerevisiae cell extracts. The changes in the activities of these enzymes were determined throughout the life cycle of the organism. As the cells progressed from the exponential to the stationary growth phase, the specific activities of the Mg2+-independent endoribonuclease and of the Mg2+-dependent 5′-exonuclease increased, whereas the Mg2+-dependent endonuclease decreased. During sporulation the Mg2+-independent endoribonuclease and the Mg2+-dependent 5′-exonuclease increased several-fold over the first 10 h, but, since a similar increase was seen in nonsporulating control cells, the increases did not appear to be related to sporulation. However, the specific activity of the Mg2+-dependent endonuclease showed a sporulation-related increase during the first 3 h of sporulation, with a subsequent decline to very low levels. The specific activity of this enzyme increased again during germination to the levels seen in exponential phase cells. The Mg2+-independent endoribonuclease and the Mg2+-dependent 5′-exonuclease showed little change during germination of the ascospores. The high specific activity of the Mg2+-independent endoribonuclease during periods of nutrient deprivation is in agreement with the proposed role for this enzyme in the degradation of rRNA under these conditions.

NIPP1 (351 residues) is a major regulatory and RNA-anchoring subunit of protein phosphatase 1 in the nucleus. Using recombinant and synthetic fragments of NIPP1, the RNA-binding domain was mapped to the C-terminal residues 330-351. A synthetic peptide encompassing this sequence equalled intact NIPP1 in RNA-binding affinity and could be used to dissociate NIPP1 from the nuclear particulate fraction. An NIPP1 fragment consisting of residues 225-351 (Ard1/NIPP1γ), that may be encoded by an alternatively spliced transcript in transformed B-lymphocytes, displayed a single-strand Mg2+-dependent endoribonuclease activity. However, full-length NIPP1 and NIPP1143-351 were not able to cleave RNA, indicating that the endoribonuclease activity of NIPP1 is restrained by its central domain. The endoribonuclease activity was also recovered in the RNA-binding domain, NIPP1330-351, but with a 30-fold lower specific activity. Thus, the endoribonuclease catalytic site and the RNA-binding site both reside in the C-terminal 22 residues of NIPP1. The latter domain does not conform to any known nucleic-acid binding motif.

45 S RNP (ribonucleoprotein) particles from calf thymus or L5178y mouse lymphoma cells contain the poly(A)-modulated and oligo(U)-binding endoribonuclease VII [Bachmann, Zahn & Müller (1983) J. Biol. Chem. 258, 7033-7040]. From these particles a 4.5 S RNA was isolated that possesses an oligo(U) sequence. By using monospecific and non-cross-reacting antibodies directed against the La or Ro antigen, both proteins were identified in the endoribonuclease VII-RNP complex after phosphorylation in vitro. In a second approach, endoribonuclease VII activity was identified in immunoaffinity-purified Ro RNPs after preparative isoelectric focusing. Therefore we conclude that the 4.5 S RNA belongs to the Ro RNAs. The results indicate a possible function of endoribonuclease VII in activating stored mRNAs.

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.