Статті в журналах: "Endoribonuclease activity"

1

Pillon, Monica C., Mack Sobhany, Mario J. Borgnia, Jason G. Williams, and Robin E. Stanley. "Grc3 programs the essential endoribonuclease Las1 for specific RNA cleavage." Proceedings of the National Academy of Sciences 114, no. 28 (June 26, 2017): E5530—E5538. http://dx.doi.org/10.1073/pnas.1703133114.

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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.

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2

Finnen, Renée L., Mingzhao Zhu, Jing Li, Daniel Romo, and Bruce W. Banfield. "Herpes Simplex Virus 2 Virion Host Shutoff Endoribonuclease Activity Is Required To Disrupt Stress Granule Formation." Journal of Virology 90, no. 17 (June 22, 2016): 7943–55. http://dx.doi.org/10.1128/jvi.00947-16.

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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.

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3

Bhardwaj, Kanchan, Linda Guarino, and C. Cheng Kao. "The Severe Acute Respiratory Syndrome Coronavirus Nsp15 Protein Is an Endoribonuclease That Prefers Manganese as a Cofactor." Journal of Virology 78, no. 22 (November 15, 2004): 12218–24. http://dx.doi.org/10.1128/jvi.78.22.12218-12224.2004.

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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.

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4

Van Ryk, Donald I., and Richard G. von Tigerstrom. "Activity changes of three nucleolytic enzymes during the life cycle of Saccharomyces cerevisiae." Canadian Journal of Microbiology 31, no. 12 (December 1, 1985): 1095–102. http://dx.doi.org/10.1139/m85-207.

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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.

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5

JIN, Qiming, Monique BEULLENS, Izabela JAGIELLO, Aleyde VAN EYNDE, Veerle VULSTEKE, Willy STALMANS, and Mathieu BOLLEN. "Mapping of the RNA-binding and endoribonuclease domains of NIPP1, a nuclear targeting subunit of protein phosphatase 1." Biochemical Journal 342, no. 1 (August 10, 1999): 13–19. http://dx.doi.org/10.1042/bj3420013.

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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.

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6

Sorokin, Alexey V., and Lev P. Ovchinnikov. "Novel findings on endoribonuclease activity of proteasomes." Cell Cycle 9, no. 6 (March 15, 2010): 1025–30. http://dx.doi.org/10.4161/cc.9.6.11241.

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7

Bachmann, M., W. J. Mayet, H. C. Schröder, K. Pfeifer, K. H. Meyer zum Büschenfelde, and W. E. G. Müller. "Identification of the Ro and La antigens in the endoribonuclease VII–ribonucleoprotein complex." Biochemical Journal 243, no. 1 (April 1, 1987): 189–94. http://dx.doi.org/10.1042/bj2430189.

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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.

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8

Kuznetsova, A. A., A. A. Gavrilova, D. S. Novopashina, O. S. Fedorova, and N. A. Kuznetsov. "Mutational and Kinetic Analysis of APE1 Endoribonuclease Activity." Molecular Biology 55, no. 2 (March 2021): 211–24. http://dx.doi.org/10.1134/s0026893321020102.

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9

Fedorova, O. A., T. N. Moiseeva, A. G. Mittenberg, and N. A. Barlev. "Recombinant proteasome alpha-type subunits exhibit endoribonuclease activity." Cell and Tissue Biology 5, no. 2 (April 2011): 123–26. http://dx.doi.org/10.1134/s1990519x11020064.

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10

Larson, Amy S., and Paul J. Hergenrother. "Light Activation ofStaphylococcus aureusToxin YoeBSa1Reveals Guanosine-Specific Endoribonuclease Activity." Biochemistry 53, no. 1 (December 23, 2013): 188–201. http://dx.doi.org/10.1021/bi4008098.

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11

Mittenberg, A. G., T. N. Moiseeva, V. O. Kuzyk, E. P. Podolskaya, I. N. Evteeva, and N. A. Barlev. "Mass-spectrometric analysis of proteasome subunits exhibiting endoribonuclease activity." Cell and Tissue Biology 8, no. 5 (September 2014): 423–40. http://dx.doi.org/10.1134/s1990519x14050058.

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12

Jabouille, Arnaud, Maylis Delugin, Raphaël Pineau, Alexandre Dubrac, Fabienne Soulet, Stéphanie Lhomond, Nestor Pallares-Lupon та ін. "Glioblastoma invasion and cooption depend on IRE1α endoribonuclease activity". Oncotarget 6, № 28 (23 липня 2015): 24922–34. http://dx.doi.org/10.18632/oncotarget.4679.

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13

Nedialkova, Danny D., Rachel Ulferts, Erwin van den Born, Chris Lauber, Alexander E. Gorbalenya, John Ziebuhr, and Eric J. Snijder. "Biochemical Characterization of Arterivirus Nonstructural Protein 11 Reveals the Nidovirus-Wide Conservation of a Replicative Endoribonuclease." Journal of Virology 83, no. 11 (March 18, 2009): 5671–82. http://dx.doi.org/10.1128/jvi.00261-09.

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ABSTRACT Nidoviruses (arteriviruses, coronaviruses, and roniviruses) are a phylogenetically compact but diverse group of positive-strand RNA viruses that includes important human and animal pathogens. Nidovirus RNA synthesis is mediated by a cytoplasmic membrane-associated replication/transcription complex that includes up to 16 viral nonstructural proteins (nsps), which carry common enzymatic activities, like the viral RNA polymerase, but also unusual and poorly understood RNA-processing functions. Of these, a conserved endoribonuclease (NendoU) is a major genetic marker that is unique to nidoviruses. NendoU activity was previously verified in vitro for the coronavirus nsp15, but not for any of its distantly related orthologs from other nidovirus lineages, like the arterivirus nsp11. Here, we show that the bacterially expressed nsp11 proteins of two arteriviruses, equine arteritis virus and porcine respiratory and reproductive syndrome virus, possess pyrimidine-specific endoribonuclease activity. RNA cleavage was independent of divalent cations in vitro and was greatly reduced by replacement of residues previously implicated in catalysis. Comparative characterization of the NendoU activity in arteriviruses and severe acute respiratory syndrome coronavirus revealed common and distinct features of their substrate requirements and reaction mechanism. Our data provide the first biochemical evidence of endoribonuclease activity associated with arterivirus nsp11 and support the conclusion that this remarkable RNA-processing enzyme, whose substrate in the infected cell remains to be identified, distinguishes nidoviruses from all other RNA viruses.

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14

Canal, Berta, Ryo Fujisawa, Allison W. McClure, Tom D. Deegan, Mary Wu, Rachel Ulferts, Florian Weissmann, et al. "Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp15 endoribonuclease." Biochemical Journal 478, no. 13 (July 2, 2021): 2465–79. http://dx.doi.org/10.1042/bcj20210199.

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SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed. SARS-CoV-2 encodes 16 non-structural proteins (nsp) that possess different enzymatic activities with important roles in viral genome replication, transcription and host immune evasion. One key aspect of host immune evasion is performed by the uridine-directed endoribonuclease activity of nsp15. Here we describe the expression and purification of nsp15 recombinant protein. We have developed biochemical assays to follow its activity, and we have found evidence for allosteric behaviour. We screened a custom chemical library of over 5000 compounds to identify nsp15 endoribonuclease inhibitors, and we identified and validated NSC95397 as an inhibitor of nsp15 endoribonuclease in vitro. Although NSC95397 did not inhibit SARS-CoV-2 growth in VERO E6 cells, further studies will be required to determine the effect of nsp15 inhibition on host immune evasion.

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15

Babu, Vignesh M. P., Siva Sankari, James A. Budnick, Clayton C. Caswell, and Graham C. Walker. "Sinorhizobium meliloti YbeY is a zinc-dependent single-strand specific endoribonuclease that plays an important role in 16S ribosomal RNA processing." Nucleic Acids Research 48, no. 1 (November 28, 2019): 332–48. http://dx.doi.org/10.1093/nar/gkz1095.

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Abstract Single-strand specific endoribonuclease YbeY has been shown to play an important role in the processing of the 3′ end of the 16S rRNA in Escherichia coli. Lack of YbeY results in the accumulation of the 17S rRNA precursor. In contrast to a previous report, we show that Sinorhizobium meliloti YbeY exhibits endoribonuclease activity on single-stranded RNA substrate but not on the double-stranded substrate. This study also identifies the previously unknown metal ion involved in YbeY function to be Zn2+ and shows that the activity of YbeY is enhanced when the occupancy of zinc is increased. We have identified a pre-16S rRNA precursor that accumulates in the S. meliloti ΔybeY strain. We also show that ΔybeY mutant of Brucella abortus, a mammalian pathogen, also accumulates a similar pre-16S rRNA. The pre-16S species is longer in alpha-proteobacteria than in gamma-proteobacteria. We demonstrate that the YbeY from E. coli and S. meliloti can reciprocally complement the rRNA processing defect in a ΔybeY mutant of the other organism. These results establish YbeY as a zinc-dependent single-strand specific endoribonuclease that functions in 16S rRNA processing in both alpha- and gamma-proteobacteria.

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16

Dutta, Tanmay, Arun Malhotra, and Murray P. Deutscher. "Exoribonuclease and Endoribonuclease Activities of RNase BN/RNase Z both Function in Vivo." Journal of Biological Chemistry 287, no. 42 (August 14, 2012): 35747–55. http://dx.doi.org/10.1074/jbc.m112.407403.

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Escherichia coli RNase BN, a member of the RNase Z family of endoribonucleases, differs from other family members in that it also can act as an exoribonuclease in vitro. Here, we examine whether this activity of RNase BN also functions in vivo. Comparison of the x-ray structure of RNase BN with that of Bacillus subtilis RNase Z, which lacks exoribonuclease activity, revealed that RNase BN has a narrower and more rigid channel downstream of the catalytic site. We hypothesized that this difference in the putative RNA exit channel might be responsible for the acquisition of exoribonuclease activity by RNase BN. Accordingly, we generated several mutant RNase BN proteins in which residues within a loop in this channel were converted to the corresponding residues present in B. subtilis RNase Z, thus widening the channel and increasing its flexibility. The resulting mutant RNase BN proteins had reduced or were essentially devoid of exoribonuclease activity in vitro. Substitution of one mutant rbn gene (P142G) for wild type rbn in the E. coli chromosome revealed that the exoribonuclease activity of RNase BN is not required for maturation of phage T4 tRNA precursors, a known specific function of this RNase. On the other hand, removal of the exoribonuclease activity of RNase BN in a cell lacking other processing RNases leads to slower growth and affects maturation of multiple tRNA precursors. These findings help explain how RNase BN can act as both an exo- and an endoribonuclease and also demonstrate that its exoribonuclease activity is capable of functioning in vivo, thus widening the potential role of this enzyme in E. coli.

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17

Lu, Patricia, Holly A. Saffran, and James R. Smiley. "The vhs1 Mutant Form of Herpes Simplex Virus Virion Host Shutoff Protein Retains Significant Internal Ribosome Entry Site-Directed RNA Cleavage Activity." Journal of Virology 75, no. 2 (January 15, 2001): 1072–76. http://dx.doi.org/10.1128/jvi.75.2.1072-1076.2001.

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ABSTRACT The virion host shutoff (vhs) protein of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated turnover of host and viral mRNAs during HSV infection. As well, it induces endoribonucleolytic cleavage of RNA substrates when produced in a rabbit reticulocyte lysate (RRL) in vitro translation system. The vhs1 point mutation (Thr 214→Ile) eliminates vhs function during virus infection and in transiently transfected mammalian cells and was therefore previously considered to abolish vhs activity. Here we demonstrate that the vhs1 mutant protein induces readily detectable endoribonuclease activity on RNA substrates bearing the internal ribosome entry site of encephalomyocarditis virus in the RRL assay system. These data document that the vhs1 mutation does not eliminate catalytic activity and raise the possibility that the vhs-dependent endoribonuclease employs more than one mode of substrate recognition.

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18

Lee, Min Woo, Elizabeth E. Rogers, and Drake C. Stenger. "Xylella fastidiosa Plasmid-Encoded PemK Toxin Is an Endoribonuclease." Phytopathology® 102, no. 1 (January 2012): 32–40. http://dx.doi.org/10.1094/phyto-05-11-0150.

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Stable inheritance of pXF-RIV11 in Xylella fastidiosa is conferred by the pemI/pemK toxin-antitoxin (TA) system. PemK toxin inhibits bacterial growth; PemI is the corresponding antitoxin that blocks activity of PemK by direct binding. PemK and PemI were overexpressed in Escherichia coli and activities of each were assessed. Purified PemK toxin specifically degraded single-stranded RNA but not double-stranded RNA, double-stranded DNA, or single-stranded DNA. Addition of PemI antitoxin inhibited nuclease activity of PemK toxin. Purified complexes of PemI bound to PemK exhibited minimal nuclease activity; removal of PemI antitoxin from the complex restored nuclease activity of PemK toxin. Sequencing of 5′ rapid amplification of cDNA ends products of RNA targets digested with PemK revealed a preference for cleavage between U and A residues of the sequence UACU and UACG. Nine single amino-acid substitution mutants of PemK toxin were constructed and evaluated for growth inhibition, ribonuclease activity, and PemI binding. Three PemK point-substitution mutants (R3A, G16E, and D79V) that lacked nuclease activity did not inhibit growth. All nine PemK mutants retained the ability to bind PemI. Collectively, the results indicate that the mechanism of stable inheritance conferred by pXF-RIV11 pemI/pemK is similar to that of the R100 pemI/pemK TA system of E. coli.

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19

Kulichkova, Valentina A., Olga A. Fedorova, Anna S. Tsimokha, Tatiana N. Moiseeva, Andrew Bottril, Larissa Lezina, Larissa N. Gauze, Irina M. Konstantinova, Alexey G. Mittenberg, and Nick Barlev. "26S proteasome exhibits endoribonuclease activity controlled by extra-cellular stimuli." Cell Cycle 9, no. 4 (February 15, 2010): 840–49. http://dx.doi.org/10.4161/cc.9.4.10829.

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20

Kwon, A. R. "HP0315 fromHelicobacter pylorias a VapD protein with an endoribonuclease activity." Acta Crystallographica Section A Foundations of Crystallography 68, a1 (August 7, 2012): s163. http://dx.doi.org/10.1107/s0108767312096857.

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21

Wang, Zuoren, and Megerditch Kiledjian. "The Poly(A)-Binding Protein and an mRNA Stability Protein Jointly Regulate an Endoribonuclease Activity." Molecular and Cellular Biology 20, no. 17 (September 1, 2000): 6334–41. http://dx.doi.org/10.1128/mcb.20.17.6334-6341.2000.

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ABSTRACT We previously identified a sequence-specific erythroid cell-enriched endoribonuclease (ErEN) activity involved in the turnover of the stable α-globin mRNA. We now demonstrate that ErEN activity is regulated by the poly(A) tail. The unadenylated α-globin 3′ untranslated region (3′UTR) was an efficient substrate for ErEN cleavage, while the polyadenylated 3′UTR was inefficiently cleaved in an in vitro decay assay. The influence of the poly(A) tail was mediated through the poly(A)-binding protein (PABP) bound to the poly(A) tail, which can inhibit ErEN activity. ErEN cleavage of an adenylated α-globin 3′UTR was accentuated upon depletion of PABP from the cytosolic extract, while addition of recombinant PABP reestablished the inhibition of endoribonuclease cleavage. PABP inhibited ErEN activity indirectly through an interaction with the αCP mRNA stability protein. Sequestration of αCP resulted in an increase of ErEN cleavage activity, regardless of the polyadenylation state of the RNA. Using electrophoretic mobility shift assays, PABP was shown to enhance the binding efficiency of αCP to the α-globin 3′UTR, which in turn protected the ErEN target sequence. Conversely, the binding of PABP to the poly(A) tail was also augmented by αCP, implying that a stable higher-order structural network is involved in stabilization of the α-globin mRNA. Upon deadenylation, the interaction of PABP with αCP would be disrupted, rendering the α-globin 3′UTR more susceptible to endoribonuclease cleavage. The data demonstrated a specific role for PABP in protecting the body of an mRNA in addition to demonstrating PABP's well-characterized effect of stabilizing the poly(A) tail.

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22

Foster, Kawanda, Sabine Grüschow, Scott Bailey, Malcolm F. White, and Michael P. Terns. "Regulation of the RNA and DNA nuclease activities required for Pyrococcus furiosus Type III-B CRISPR–Cas immunity." Nucleic Acids Research 48, no. 8 (March 21, 2020): 4418–34. http://dx.doi.org/10.1093/nar/gkaa176.

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Abstract Type III CRISPR–Cas prokaryotic immune systems provide anti-viral and anti-plasmid immunity via a dual mechanism of RNA and DNA destruction. Upon target RNA interaction, Type III crRNP effector complexes become activated to cleave both target RNA (via Cas7) and target DNA (via Cas10). Moreover, trans-acting endoribonucleases, Csx1 or Csm6, can promote the Type III immune response by destroying both invader and host RNAs. Here, we characterize how the RNase and DNase activities associated with Type III-B immunity in Pyrococcus furiosus (Pfu) are regulated by target RNA features and second messenger signaling events. In vivo mutational analyses reveal that either the DNase activity of Cas10 or the RNase activity of Csx1 can effectively direct successful anti-plasmid immunity. Biochemical analyses confirmed that the Cas10 Palm domains convert ATP into cyclic oligoadenylate (cOA) compounds that activate the ribonuclease activity of Pfu Csx1. Furthermore, we show that the HEPN domain of the adenosine-specific endoribonuclease, Pfu Csx1, degrades cOA signaling molecules to provide an auto-inhibitory off-switch of Csx1 activation. Activation of both the DNase and cOA generation activities require target RNA binding and recognition of distinct target RNA 3′ protospacer flanking sequences. Our results highlight the complex regulatory mechanisms controlling Type III CRISPR immunity.

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23

Masuda, Hisako, and Masayori Inouye. "Toxins of Prokaryotic Toxin-Antitoxin Systems with Sequence-Specific Endoribonuclease Activity." Toxins 9, no. 4 (April 14, 2017): 140. http://dx.doi.org/10.3390/toxins9040140.

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24

Concha, Nestor O., Angela Smallwood, William Bonnette, Rachel Totoritis, Guofeng Zhang, Kelly Federowicz, Jingsong Yang та ін. "Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1α Endoribonuclease Activity". Molecular Pharmacology 88, № 6 (5 жовтня 2015): 1011–23. http://dx.doi.org/10.1124/mol.115.100917.

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25

Konstantinova, I. M., V. A. Kulichkova, I. N. Evteeva, A. G. Mittenberg, I. V. Volkova, J. B. Ermolaeva та L. N. Gause. "The specific endoribonuclease activity of small nuclear and cytoplasmic α-RNPs". FEBS Letters 462, № 3 (30 листопада 1999): 407–10. http://dx.doi.org/10.1016/s0014-5793(99)01565-3.

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26

Garg, Abhishek V., Nilesh Amatya, Kong Chen, J. Agustin Cruz, Prerna Grover, Natasha Whibley, Heather R. Conti, et al. "MCPIP1 Endoribonuclease Activity Negatively Regulates Interleukin-17-Mediated Signaling and Inflammation." Immunity 43, no. 3 (September 2015): 475–87. http://dx.doi.org/10.1016/j.immuni.2015.07.021.

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27

Bhattacharya, P. "An Endoribonuclease Activity Associated with Lettre Ehrlich Ascites Tumor Cell Mitochondria." Biochemical and Biophysical Research Communications 196, no. 1 (October 1993): 47–54. http://dx.doi.org/10.1006/bbrc.1993.2214.

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28

Lu, Patricia, Frank E. Jones, Holly A. Saffran, and James R. Smiley. "Herpes Simplex Virus Virion Host Shutoff Protein Requires a Mammalian Factor for Efficient In Vitro Endoribonuclease Activity." Journal of Virology 75, no. 3 (February 1, 2001): 1172–85. http://dx.doi.org/10.1128/jvi.75.3.1172-1185.2001.

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ABSTRACT The virion host shutoff protein (vhs) of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated mRNA turnover during virus infection and induces endoribonucleolytic cleavage of exogenous RNA substrates when it is produced in a rabbit reticulocyte (RRL) in vitro translation system. Although vhs induces RNA turnover in the absence of other HSV gene products, it is not yet known whether cellular factors are required for its activity. As one approach to addressing this question, we expressed vhs in the budding yeast Saccharomyces cerevisiae. Expression of vhs inhibited colony formation, and the severity of this effect varied with the carbon source. The biological relevance of this effect was assessed by examining the activity of five mutant forms of vhs bearing previously characterized in-frame linker insertions. The results indicated a complete concordance between the growth inhibition phenotype in yeast and mammalian host cell shutoff. Despite these results, expression of vhs did not trigger global mRNA turnover in vivo, and cell extracts of yeast expressing vhs displayed little if any vhs-dependent endoribonuclease activity. However, activity was readily detected when such extracts were mixed with RRL. These data suggest that the vhs-dependent endoribonuclease requires one or more mammalian macromolecular factors for efficient activity.

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Ingle, Shakti, Shivani Chhabra, Jiandong Chen, Michael B. Lazarus, Xing Luo, and David H. Bechhofer. "Discovery and initial characterization of YloC, a novel endoribonuclease in Bacillus subtilis." RNA 28, no. 2 (November 23, 2021): 227–38. http://dx.doi.org/10.1261/rna.078962.121.

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The Bacillus subtilis genome is predicted to encode numerous ribonucleases, including four 3′ exoribonucleases that have been characterized to some extent. A strain containing gene knockouts of all four known 3′ exoribonucleases is viable, suggesting that one or more additional RNases remain to be discovered. A protein extract from the quadruple RNase mutant strain was fractionated and RNase activity was followed, resulting in the identification of an enzyme activity catalyzed by the YloC protein. YloC is an endoribonuclease and is a member of the highly conserved “YicC family” of proteins that is widespread in bacteria. YloC is a metal-dependent enzyme that catalyzes the cleavage of single-stranded RNA, preferentially at U residues, and exists in an oligomeric form, most likely a hexamer. As such, YloC shares some characteristics with the SARS-CoV Nsp15 endoribonuclease. While the in vivo function of YloC in B. subtilis is yet to be determined, YloC was found to act similarly to YicC in an Escherichia coli in vivo assay that assesses decay of the small RNA, RyhB. Thus, YloC may play a role in small RNA regulation.

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Ro, Young-Tae, and Jean L. Patterson. "Identification of the Minimal Essential RNA Sequences Responsible for Site-Specific Targeting of theLeishmania RNA Virus 1-4 Capsid Endoribonuclease." Journal of Virology 74, no. 1 (January 1, 2000): 130–38. http://dx.doi.org/10.1128/jvi.74.1.130-138.2000.

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ABSTRACT The Leishmania RNA virus 1-4 capsid protein possesses an endoribonuclease activity responsible for single-site-specific cleavage within the 450-nucleotide 5′ untranslated region of its own viral RNA transcript. To characterize the minimal essential RNA determinants required for site-specific cleavage, mutated RNA transcripts were examined for susceptibility to cleavage by the virus capsid protein in an in vitro assay. Deletion analyses revealed that all determinants necessary for accurate cleavage are encoded in viral nucleotides 249 to 342. Nuclease mapping and site-specific mutagenesis of the minimal RNA sequence defined a stem-loop structure that is located 40 nucleotides upstream from the cleavage site (nucleotide 320) and that is essential for accurate RNA cleavage. Abrogation of cleavage by disruption of base pairing within the stem-loop was reversed through the introduction of complementary nucleotide substitutions that reestablished the structure. We also provide evidence that divalent cations, essential components of the cleavage reaction, stabilized the stem-loop structure in solution. That capsid-specific antiserum eliminated specific RNA cleavage provides further evidence that the virus capsid gene encodes the essential endoribonuclease activity.

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31

Hackbart, Matthew, Xufang Deng, and Susan C. Baker. "Coronavirus endoribonuclease targets viral polyuridine sequences to evade activating host sensors." Proceedings of the National Academy of Sciences 117, no. 14 (March 20, 2020): 8094–103. http://dx.doi.org/10.1073/pnas.1921485117.

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Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease designated EndoU that facilitates evasion of host pattern recognition receptor MDA5, but the target of EndoU activity was not known. Here, we report that EndoU cleaves the 5′-polyuridines from negative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNA synthesis. Using a virus containing an EndoU catalytic-inactive mutation, we detected a higher abundance of PUN RNA in the cytoplasm compared to wild-type−infected cells. Furthermore, we found that transfecting PUN RNA into cells stimulates a robust, MDA5-dependent interferon response, and that removal of the polyuridine extension on the RNA dampens the response. Overall, the results of this study reveal the PUN RNA to be a CoV MDA5-dependent pathogen-associated molecular pattern (PAMP). We also establish a mechanism for EndoU activity to cleave and limit the accumulation of this PAMP. Since EndoU activity is highly conserved in all CoVs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging CoV infections.

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Moszyńska, Adrianna, James F. Collawn та Rafal Bartoszewski. "IRE1 Endoribonuclease Activity Modulates Hypoxic HIF-1α Signaling in Human Endothelial Cells". Biomolecules 10, № 6 (11 червня 2020): 895. http://dx.doi.org/10.3390/biom10060895.

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While the role of hypoxia and the induction of the hypoxia inducible factors (HIFs) and the unfolded protein response (UPR) pathways in the cancer microenvironment are well characterized, their roles and relationship in normal human endothelium are less clear. Here, we examined the effects of IRE1 on HIF-1α protein levels during hypoxia in primary human umbilical vein endothelial cells (HUVECs). The results demonstrated that HIF-1α levels peaked at 6 h of hypoxia along with two of their target genes, GLUT1 and VEGFA, whereas at up to 12 h of hypoxia the mRNA levels of markers of the UPR, IRE1, XBP1s, BiP, and CHOP, did not increase, suggesting that the UPR was not activated. Interestingly, the siRNA knockdown of IRE1 or inhibition of IRE1 endonuclease activity with 4µ8C during hypoxia significantly reduced HIF-1α protein without affecting HIF1A mRNA expression. The inhibition of the endonuclease activity with 4µ8C in two other primary endothelial cells during hypoxia, human cardiac microvascular endothelial cells and human aortic endothelial cells showed the same reduction in the HIF-1α protein. Surprisingly, the siRNA knockdown of XBP1s during hypoxia did not decrease the HIF1α protein levels, indicating that the IRE1-mediated effect on stabilizing the HIF1α protein levels was XBP1s-independent. The studies presented here, therefore, provide evidence that IRE1 activity during hypoxia increases the protein levels of HIF1α in an XBP1s-independent manner.

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Liu, Xiaorong, Puxian Fang, Liurong Fang, Yingying Hong, Xinyu Zhu, Dang Wang, Guiqing Peng та Shaobo Xiao. "Porcine deltacoronavirus nsp15 antagonizes interferon-β production independently of its endoribonuclease activity". Molecular Immunology 114 (жовтень 2019): 100–107. http://dx.doi.org/10.1016/j.molimm.2019.07.003.

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34

Grosshans, Cheryl A., and Thomas R. Cech. "Metal ion requirements for sequence-specific endoribonuclease activity of the Tetrahymena ribozyme." Biochemistry 28, no. 17 (August 1989): 6888–94. http://dx.doi.org/10.1021/bi00443a017.

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35

Wang, Zuoren, and Megerditch Kiledjian. "Identification of an erythroid-enriched endoribonuclease activity involved in specific mRNA cleavage." EMBO Journal 19, no. 2 (January 17, 2000): 295–305. http://dx.doi.org/10.1093/emboj/19.2.295.

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36

Davletgildeeva, Anastasiia T., Alexandra A. Kuznetsova, Darya S. Novopashina, Alexander A. Ishchenko, Murat Saparbaev, Olga S. Fedorova, and Nikita A. Kuznetsov. "Comparative Analysis of Exo- and Endonuclease Activities of APE1-like Enzymes." International Journal of Molecular Sciences 23, no. 5 (March 6, 2022): 2869. http://dx.doi.org/10.3390/ijms23052869.

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Apurinic/apyrimidinic (AP)-endonucleases are multifunctional enzymes that are required for cell viability. AP-endonucleases incise DNA 5′ to an AP-site; can recognize and process some damaged nucleosides; and possess 3′-phosphodiesterase, 3′-phosphatase, and endoribonuclease activities. To elucidate the mechanism of substrate cleavage in detail, we analyzed the effect of mono- and divalent metal ions on the exo- and endonuclease activities of four homologous APE1-like endonucleases (from an insect (Rrp1), amphibian (xAPE1), fish (zAPE1), and from humans (hAPE1)). It was found that the enzymes had similar patterns of dependence on metal ions’ concentrations in terms of AP-endonuclease activity, suggesting that the main biological function (AP-site cleavage) was highly conserved among evolutionarily distant species. The efficiency of the 3′-5′ exonuclease activity was the highest in hAPE1 among these enzymes. In contrast, the endoribonuclease activity of the enzymes could be ranked as hAPE1 ≈ zAPE1 ≤ xAPE1 ≤ Rrp1. Taken together, the results revealed that the tested enzymes differed significantly in their capacity for substrate cleavage, even though the most important catalytic and substrate-binding amino acid residues were conserved. It can be concluded that substrate specificity and cleavage efficiency were controlled by factors external to the catalytic site, e.g., the N-terminal domain of these enzymes.

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Fu, Zhibiao, Niles P. Donegan, Guido Memmi, and Ambrose L. Cheung. "Characterization of MazFSa, an Endoribonuclease from Staphylococcus aureus." Journal of Bacteriology 189, no. 24 (October 12, 2007): 8871–79. http://dx.doi.org/10.1128/jb.01272-07.

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ABSTRACT The mazEF homologs of Staphylococcus aureus, designated mazEFsa , have been shown to cotranscribe with the sigB operon under stress conditions. In this study, we showed that MazEF Sa , as with their Escherichia coli counterparts, compose a toxin-antitoxin module wherein MazF Sa leads to rapid cell growth arrest and loss in viable CFU upon overexpression. MazF Sa is a novel sequence-specific endoribonuclease which cleaves mRNA to inhibit protein synthesis. Using ctpA mRNA as the model substrate both in vitro and in vivo, we demonstrated that MazF Sa cleaves single-strand RNA preferentially at the 5′ side of the first U or 3′ side of the second U residue within the consensus sequences VUUV′ (where V and V′ are A, C, or G and may or may not be identical). Binding studies confirmed that the antitoxin MazE Sa binds MazF Sa to form a complex to inhibit the endoribonuclease activity of MazF Sa . Contrary to the system in E. coli, exposure to selected antibiotics augmented mazEFsa transcription, akin to what one would anticipate from the environmental stress response of the sigB system. These data indicate that the mazEF system of S. aureus differs from the gram-negative counterparts with respect to mRNA cleavage specificity and antibiotic stresses.

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Deng, Zhongliang, Zizhong Liu, Yujing Bi, Xiaoyi Wang, Dongsheng Zhou, Ruifu Yang, and Yanping Han. "Rapid Degradation of Hfq-Free RyhB inYersinia pestisby PNPase Independent of Putative Ribonucleolytic Complexes." BioMed Research International 2014 (2014): 1–7. http://dx.doi.org/10.1155/2014/798918.

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The RNA chaperone Hfq in bacteria stabilizes sRNAs by protecting them from the attack of ribonucleases. Upon release from Hfq, sRNAs are preferably degraded by PNPase. PNPase usually forms multienzyme ribonucleolytic complexes with endoribonuclease E and/or RNA helicase RhlB to facilitate the degradation of the structured RNA. However, whether PNPase activity on Hfq-free sRNAs is associated with the assembly of RNase E or RhlB has yet to be determined. Here we examined the roles of the main endoribonucleases, exoribonucleases, and ancillary RNA-modifying enzymes in the degradation ofY. pestisRyhB in the absence of Hfq. Expectedly, the transcript levels of both RyhB1 and RyhB2 increase only after inactivating PNPase, which confirms the importance of PNPase in sRNA degradation. By contrast, the signal of RyhB becomes barely perceptible after inactivating of RNase III, which may be explained by the increase in PNPase levels resulting from the exemption ofpnpmRNA from RNase III processing. No significant changes are observed in RyhB stability after deletion of either the PNPase-binding domain of RNase E orrhlB. Therefore, PNPase acts as a major enzyme of RyhB degradation independent of PNPase-containing RNase E and RhlB assembly in the absence of Hfq.

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39

Shaku, Mao, Jung-Ho Park, Masayori Inouye, and Yoshihiro Yamaguchi. "Identification of MazF Homologue in Legionella pneumophila Which Cleaves RNA at the AACU Sequence." Journal of Molecular Microbiology and Biotechnology 28, no. 6 (2018): 269–80. http://dx.doi.org/10.1159/000497146.

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MazF is a sequence-specific endoribonuclease that is widely conserved in bacteria and archaea. Here, we found an MazF homologue (MazF-lp; LPO-p0114) in Legionella pneumophila. The mazF-lp gene overlaps 14 base pairs with the upstream gene mazE-lp (MazE-lp; LPO-p0115). The induction of mazF-lp caused cell growth arrest, while mazE-lp co-induction recovered cell growth in Escherichia coli. In vivo and in vitro primer extension experiments showed that MazF-lp is a sequence-specific endoribonuclease cleaving RNA at AACU. The endoribonuclease activity of purified MazF-lp was inhibited by purified MazE-lp. We found that MazE-lp and the MazEF-lp complex specifically bind to the palindromic sequence present in the 5′-untranslated region of the mazEF-lp operon. MazE-lp and MazEF-lp both likely function as a repressor for the mazEF-lp operon and for other genes, including icmR, whose gene product functions as a secretion chaperone for the IcmQ pore-forming protein, by specifically binding to the palindromic sequence in 5′-UTR of these genes.

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40

Yao, Shiyi, and David H. Bechhofer. "Initiation of Decay of Bacillus subtilis rpsO mRNA by Endoribonuclease RNase Y." Journal of Bacteriology 192, no. 13 (April 23, 2010): 3279–86. http://dx.doi.org/10.1128/jb.00230-10.

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ABSTRACT rpsO mRNA, a small monocistronic mRNA that encodes ribosomal protein S15, was used to study aspects of mRNA decay initiation in Bacillus subtilis. Decay of rpsO mRNA in a panel of 3′-to-5′ exoribonuclease mutants was analyzed using a 5′-proximal oligonucleotide probe and a series of oligonucleotide probes that were complementary to overlapping sequences starting at the 3′ end. The results provided strong evidence that endonuclease cleavage in the body of the message, rather than degradation from the native 3′ end, is the rate-determining step for mRNA decay. Subsequent to endonuclease cleavage, the upstream products were degraded by polynucleotide phosphorylase (PNPase), and the downstream products were degraded by the 5′ exonuclease activity of RNase J1. The rpsO mRNA half-life was unchanged in a strain that had decreased RNase J1 activity and no RNase J2 activity, but it was 2.3-fold higher in a strain with decreased activity of RNase Y, a recently discovered RNase of B. subtilis encoded by the ymdA gene. Accumulation of full-length rpsO mRNA and its decay intermediates was analyzed using a construct in which the rpsO transcription unit was under control of a bacitracin-inducible promoter. The results were consistent with RNase Y-mediated initiation of decay. This is the first report of a specific mRNA whose stability is determined by RNase Y.

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Itzhak, Daniel, Michael Bright, Peter McAndrew, Amin Mirza, Yvette Newbatt, Jade Strover, Marcella Widya та ін. "Multiple autophosphorylations significantly enhance the endoribonuclease activity of human inositol requiring enzyme 1α". BMC Biochemistry 15, № 1 (2014): 3. http://dx.doi.org/10.1186/1471-2091-15-3.

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MacBeth, K. J., Y. T. Ro, L. Gehrke, and J. L. Patterson. "Cleavage Site Mapping and Substrate-Specificity of Leishmaniavirus 2-1 Capsid Endoribonuclease Activity." Journal of Biochemistry 122, no. 1 (July 1, 1997): 193–200. http://dx.doi.org/10.1093/oxfordjournals.jbchem.a021728.

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43

Theis, Mirko, Maciej Paszkowski-Rogacz, Ina Weisswange, Debojyoti Chakraborty, and Frank Buchholz. "Targeting Human Long Noncoding Transcripts by Endoribonuclease-Prepared siRNAs." Journal of Biomolecular Screening 20, no. 8 (April 24, 2015): 1018–26. http://dx.doi.org/10.1177/1087057115583448.

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Broad sequencing enterprises such as the FANTOM or ENCODE projects have substantially extended our knowledge of the human transcriptome. They have revealed that a large portion of genomic DNA is actively transcribed and have identified a plethora of novel transcripts. Many newly identified transcripts belong to the class of long noncoding RNAs (lncRNAs), which range from a few hundred bases to multiple kilobases in length and harbor no protein-coding potential. Although the biological activity of some lncRNAs is understood, the functions of most lncRNAs remain elusive. Tools that allow rapid and cost-effective access to functional data of lncRNAs are therefore essential. Here, we describe the construction and validation of an endoribonuclease-prepared siRNA (esiRNA) library designed to target 1779 individual human lncRNAs by RNA interference. We present a compendium of lncRNA expression data for 11 human cancer cell lines. Furthermore, we show that the resource is suitable for combined knockdown and localization analysis. We discuss challenges in sequence annotation of lncRNAs with respect to their often low and cell type–specific expression and specify esiRNAs that are suitable for targeting lncRNAs in commonly used human cell lines.

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Gupta, Ankush, and Pramod C. Rath. "Curcumin, a Natural Antioxidant, Acts as a Noncompetitive Inhibitor of Human RNase L in Presence of Its Cofactor 2-5AIn Vitro." BioMed Research International 2014 (2014): 1–9. http://dx.doi.org/10.1155/2014/817024.

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Ribonuclease L (RNase L) is an antiviral endoribonuclease of the innate immune system, which is induced and activated by viral infections, interferons, and double stranded RNA (dsRNA) in mammalian cells. Although, RNase L is generally protective against viral infections, abnormal RNase L expression and activity have been associated with a number of diseases. Here, we show that curcumin, a natural plant-derived anti-inflammatory active principle, inhibits RNase L activity; hence, it may be exploited for therapeutic interventions in case of pathological situations associated with excess activation of RNase L.

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45

Schiaffini, Marlene, Clara Chicois, Aude Pouclet, Tiphaine Chartier, Elodie Ubrig, Anthony Gobert, Hélène Zuber, et al. "A NYN domain protein directly interacts with DECAPPING1 and is required for phyllotactic pattern." Plant Physiology 188, no. 2 (November 13, 2021): 1174–88. http://dx.doi.org/10.1093/plphys/kiab529.

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Abstract In eukaryotes, general mRNA decay requires the decapping complex. The activity of this complex depends on its catalytic subunit, DECAPPING2 (DCP2), and its interaction with decapping enhancers, including its main partner DECAPPING1 (DCP1). Here, we report that in Arabidopsis thaliana, DCP1 also interacts with a NYN domain endoribonuclease, hence named DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1). Interestingly, we found DNE1 predominantly associated with DCP1, but not with DCP2, and reciprocally, suggesting the existence of two distinct protein complexes. We also showed that the catalytic residues of DNE1 are required to repress the expression of mRNAs in planta upon transient expression. The overexpression of DNE1 in transgenic lines led to growth defects and a similar gene deregulation signature than inactivation of the decapping complex. Finally, the combination of dne1 and dcp2 mutations revealed a functional redundancy between DNE1 and DCP2 in controlling phyllotactic pattern formation. Our work identifies DNE1, a hitherto unknown DCP1 protein partner highly conserved in the plant kingdom and identifies its importance for developmental robustness.

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de Martin, Elena, and Matthias Schweizer. "Fifty Shades of Erns: Innate Immune Evasion by the Viral Endonucleases of All Pestivirus Species." Viruses 14, no. 2 (January 27, 2022): 265. http://dx.doi.org/10.3390/v14020265.

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The genus Pestivirus, family Flaviviridae, includes four historically accepted species, i.e., bovine viral diarrhea virus (BVDV)-1 and -2, classical swine fever virus (CSFV), and border disease virus (BDV). A large number of new pestivirus species were identified in recent years. A common feature of most members is the presence of two unique proteins, Npro and Erns, that pestiviruses evolved to regulate the host’s innate immune response. In addition to its function as a structural envelope glycoprotein, Erns is also released in the extracellular space, where it is endocytosed by neighboring cells. As an endoribonuclease, Erns is able to cleave viral ss- and dsRNAs, thus preventing the stimulation of the host’s interferon (IFN) response. Here, we characterize the basic features of soluble Erns of a large variety of classified and unassigned pestiviruses that have not yet been described. Its ability to form homodimers, its RNase activity, and the ability to inhibit dsRNA-induced IFN synthesis were investigated. Overall, we found large differences between the various Erns proteins that cannot be predicted solely based on their primary amino acid sequences, and that might be the consequence of different virus-host co-evolution histories. This provides valuable information to delineate the structure-function relationship of pestiviral endoribonucleases.

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Московцев, А. А., Т. С. Клементьева, Д. М. Зайченко, Д. В. Колесов, А. А. Соколовская, and А. А. Кубатиев. "Time-restricted IRE1 activity: impact on cell survival." ZHurnal «Patologicheskaia fiziologiia i eksperimental`naia terapiia», no. 4() (November 21, 2018): 21–27. http://dx.doi.org/10.25557/0031-2991.2018.04.21-27.

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Нарушения фолдинга белков в эндоплазматическом ретикулуме (ЭПР) являются причиной развития особой формы протеотоксического клеточного стресса - стресса ЭПР. При этом незрелые и неправильно свернутые белки накапливаются в просвете ЭПР и образуют цитотоксичные агрегаты. При стрессе ЭПР в клетке запускается неспецифический защитный механизм - система сигнальных каскадов ответа на белки с нарушенными конформациями (Unfolded Protein Response, UPR). Важным элементом UPR является сигнальный путь, опосредуемый трансмембранным белком ЭПР IRE1, эндорибонуклеазный домен которого при активации вырезает интрон в мРНК XBP1, что приводит к синтезу транскрипционного фактора sXBP1, индуцирующего экспрессию целого ряда проадаптивных генов. Кроме цитопротекторной функции, IRE1 является ключевым регулятором ЭПР-стресс-индуцированной клеточной гибели. Предполагается, что при продолжительной активации IRE1 переключается с проадаптивной на проапототическую регуляцию. Цель . Исследование зависимости выживаемости клеток от длительности активности сигнального пути, опосредуемого рибонуклеазной активностью IRE1, при стрессе эндоплазматического ретикулума. Методика . С применением RT-qPCR, ингибирования эндорибонуклеазного домена IRE1a соединением STF-083010 проведен анализ зависимости выживаемости клеток от периода активности IRE1a при стрессе ЭПР различной интенсивности. Результаты . Установлено, IRE1a оказывает преимущественно цитопротекторное действие при интенсивном стрессе - ингибирование соединением STF-083010 снижает жизнеспособность клеток. Характер зависимости выживаемости клеток от периода активности IRE1a при стрессе ЭПР клеточноспецифичен: выживаемость несекретирующих Т-лимфобластов Jurkat была выше при ингибировании IRE1a на ранних сроках интенсивного стресса, нежели на поздних; для секретирующих эндотелиоцитоподобных клеток EA.hy926 наблюдалось обратное отношение . Protein unfolding in the endoplasmic reticulum (ER) induces a particular form of proteotoxic cellular stress - ER stress: immature and incorrectly folded proteins can accumulate in the ER lumen and form cytotoxic aggregates. Under ER stress, the non-specific protective mechanism, Unfolded Protein Response (UPR), is activated. The key element of UPR is the signaling pathway mediated by transmembrane ER protein IRE1. The activated endoribonuclease domain IRE1a causes non-canonic XBP1 mRNA splicing, which leads to the synthesis of an active transcription factor sXBP1. It induces the expression of proadaptive genes. In addition to its cytoprotective function, IRE1 is also a key regulator of ER stress-induced cell death. It is assumed that with prolonged activation, IRE1 switches from proadaptive to proapototic regulation. Aim. This paper is devoted to studying possible IRE1a switching from proadaptive to proapoptotic regulation. Using the inhibition of the IRE1a endoribonuclease domain by the compound STF-083010, we analyzed the dependence of cell survival on the period of IRE1a activity under ER stress of varying intensity. We observed the cell specificity of this dependence: in non-secreting Jurkat cells, inhibition of IRE1a in the early stages of intense stress was less toxic than in the later ones; in secreting EA.hy926 cells, an inverse relationship was observed. Purpose of the study. The study of the dependence of cell survival on the duration of the activity of the signaling pathway, mediated by the ribonuclease activity of IRE1, during endoplasmic reticulum stress. Methods. Using RT-qPCR, inhibition of the IRE1a endoribonuclease domain by compound STF-083010, the dependence of cell survival on the period of IRE1a activity during ER stress of various intensities was analyzed. Results. IRE1a exerts a predominantly cytoprotective effect under intense stress - inhibition by the compound STF-083010 reduces cell viability. The character of the dependence of cell survival on the period of IRE1a activity under ER stress is cell-specific: the survival of non-secretive T-lymphoblasts Jurkat was higher when IRE1a was inhibited in the early stages of intense stress than in the latter; for secreting endotheliocyte-like cells EA.hy926, an inverse relationship was observed.

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Posthuma, Clara C., Danny D. Nedialkova, Jessika C. Zevenhoven-Dobbe, Jeroen H. Blokhuis, Alexander E. Gorbalenya, and Eric J. Snijder. "Site-Directed Mutagenesis of the Nidovirus Replicative Endoribonuclease NendoU Exerts Pleiotropic Effects on the Arterivirus Life Cycle." Journal of Virology 80, no. 4 (February 15, 2006): 1653–61. http://dx.doi.org/10.1128/jvi.80.4.1653-1661.2006.

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ABSTRACT The highly conserved NendoU replicative domain of nidoviruses (arteriviruses, coronaviruses, and roniviruses) belongs to a small protein family whose cellular branch is prototyped by XendoU, a Xenopus laevis endoribonuclease involved in nucleolar RNA processing. Recently, sequence-specific in vitro endoribonuclease activity was demonstrated for the NendoU-containing nonstructural protein (nsp) 15 of several coronaviruses. To investigate the biological role of this novel enzymatic activity, we have characterized a comprehensive set of arterivirus NendoU mutants. Deleting parts of the NendoU domain from nsp11 of equine arteritis virus was lethal. Site-directed mutagenesis of conserved residues exerted pleiotropic effects. In a first-cycle analysis, replacement of two conserved Asp residues in the C-terminal part of NendoU rendered viral RNA synthesis and virus production undetectable. In contrast, mutagenesis of other conserved residues, including two putative catalytic His residues that are absolutely conserved in NendoU and cellular homologs, produced viable mutants displaying reduced plaque sizes (20 to 80% reduction) and reduced yields of infectious progeny of up to 5 log units. A more detailed analysis of these mutants revealed a moderate reduction in RNA synthesis, with subgenomic RNA synthesis consistently being more strongly affected than genome replication. Our data suggest that the arterivirus nsp11 is a multifunctional protein with a key role in viral RNA synthesis and additional functions in the viral life cycle that are as yet poorly defined.

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Robinson, Sophie R., Sandra C. Viegas, Rute G. Matos, Susana Domingues, Marisa Bedir, Helen J. S. Stewart, Timothy J. Chevassut, Antony W. Oliver, Cecilia M. Arraiano, and Sarah F. Newbury. "DIS3 isoforms vary in their endoribonuclease activity and are differentially expressed within haematological cancers." Biochemical Journal 475, no. 12 (June 29, 2018): 2091–105. http://dx.doi.org/10.1042/bcj20170962.

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DIS3 (defective in sister chromatid joining) is the catalytic subunit of the exosome, a protein complex involved in the 3′–5′ degradation of RNAs. DIS3 is a highly conserved exoribonuclease, also known as Rrp44. Global sequencing studies have identified DIS3 as being mutated in a range of cancers, with a considerable incidence in multiple myeloma. In this work, we have identified two protein-coding isoforms of DIS3. Both isoforms are functionally relevant and result from alternative splicing. They differ from each other in the size of their N-terminal PIN (PilT N-terminal) domain, which has been shown to have endoribonuclease activity and tether DIS3 to the exosome. Isoform 1 encodes a full-length PIN domain, whereas the PIN domain of isoform 2 is shorter and is missing a segment with conserved amino acids. We have carried out biochemical activity assays on both isoforms of full-length DIS3 and the isolated PIN domains. We find that isoform 2, despite missing part of the PIN domain, has greater endonuclease activity compared with isoform 1. Examination of the available structural information allows us to provide a hypothesis to explain this altered behaviour. Our results also show that multiple myeloma patient cells and all cancer cell lines tested have higher levels of isoform 1 compared with isoform 2, whereas acute myeloid leukaemia and chronic myelomonocytic leukaemia patient cells and samples from healthy donors have similar levels of isoforms 1 and 2. Taken together, our data indicate that significant changes in the ratios of the two isoforms could be symptomatic of haematological cancers.

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Kim, Wan Cheol, Conan Ma, Wai-Ming Li, Manbir Chohan, David M. Wilson III, and Chow H. Lee. "Altered Endoribonuclease Activity of Apurinic/Apyrimidinic Endonuclease 1 Variants Identified in the Human Population." PLoS ONE 9, no. 3 (March 4, 2014): e90837. http://dx.doi.org/10.1371/journal.pone.0090837.

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