Identification of RNase L-Dependent, 3'-End-Modified, Viral Small RNAs in Sindbis Virus-Infected Mammalian Cells

AbstractDouble-stranded RNA (dsRNA) is a danger signal that triggers endonucleolytic degradation of RNA inside infected and stressed mammalian cells. This mechanism inhibits growth and ultimately removes problematic cells via apoptosis. To elucidate the molecular functions of this program and understand the connection between RNA cleavage and programmed cell death, we visualized dsRNA-induced degradation of human small RNAs using RtcB ligase-assisted RNA sequencing (RtcB RNA-seq). RtcB RNA-seq revealed strong cleavage of select transfer RNAs (tRNAs) and autoantigenic Y-RNAs, and identified the innate immune receptor RNase L as the responsible endoribonuclease. RNase L cleaves the non-coding RNA (ncRNA) targets site-specifically, releasing abundant ncRNA fragments, and downregulating full-length tRNAs and Y-RNAs. The depletion of a single Y-RNA, RNY1, appears particularly important and the loss of this Y-RNA is sufficient to initiate apoptosis. Site-specific cleavage of small ncRNA by RNase L thus emerges as an important terminal step in dsRNA surveillance.

Download Full-text

Faculty Opinions recommendation of MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1089522.544775 ◽

2007 ◽

Author(s):

Mark Kay

Keyword(s):

Small Rnas ◽

Mammalian Cells ◽

Competitive Inhibitors ◽

Microrna Sponges

Download Full-text

m5U54 tRNA Hypomodification by Lack of TRMT2A Drives the Generation of tRNA-Derived Small RNAs

International Journal of Molecular Sciences ◽

10.3390/ijms22062941 ◽

2021 ◽

Vol 22 (6) ◽

pp. 2941

Author(s):

Marisa Pereira ◽

Diana R. Ribeiro ◽

Miguel M. Pinheiro ◽

Margarida Ferreira ◽

Stefanie Kellner ◽

...

Keyword(s):

Stress Response ◽

Small Rnas ◽

Mammalian Cells ◽

Cellular Stress ◽

Cellular Stress Response ◽

Translation Regulation ◽

Translation Efficiency ◽

Down Regulation ◽

Gene Expression Control ◽

The Impact

Transfer RNA (tRNA) molecules contain various post-transcriptional modifications that are crucial for tRNA stability, translation efficiency, and fidelity. Besides their canonical roles in translation, tRNAs also originate tRNA-derived small RNAs (tsRNAs), a class of small non-coding RNAs with regulatory functions ranging from translation regulation to gene expression control and cellular stress response. Recent evidence indicates that tsRNAs are also modified, however, the impact of tRNA epitranscriptome deregulation on tsRNAs generation is only now beginning to be uncovered. The 5-methyluridine (m5U) modification at position 54 of cytosolic tRNAs is one of the most common and conserved tRNA modifications among species. The tRNA methyltransferase TRMT2A catalyzes this modification, but its biological role remains mostly unexplored. Here, we show that TRMT2A knockdown in human cells induces m5U54 tRNA hypomodification and tsRNA formation. More specifically, m5U54 hypomodification is followed by overexpression of the ribonuclease angiogenin (ANG) that cleaves tRNAs near the anticodon, resulting in accumulation of 5′tRNA-derived stress-induced RNAs (5′tiRNAs), namely 5′tiRNA-GlyGCC and 5′tiRNA-GluCTC, among others. Additionally, transcriptomic analysis confirms that down-regulation of TRMT2A and consequently m5U54 hypomodification impacts the cellular stress response and RNA stability, which is often correlated with tiRNA generation. Accordingly, exposure to oxidative stress conditions induces TRMT2A down-regulation and tiRNA formation in mammalian cells. These results establish a link between tRNA hypomethylation and ANG-dependent tsRNAs formation and unravel m5U54 as a tRNA cleavage protective mark.

Download Full-text

Alpha/Beta Interferon Inhibits Cap-Dependent Translation of Viral but Not Cellular mRNA by a PKR-Independent Mechanism

Journal of Virology ◽

10.1128/jvi.01784-07 ◽

2007 ◽

Vol 82 (6) ◽

pp. 2620-2630 ◽

Cited By ~ 22

Author(s):

Mulu Z. Tesfay ◽

Jun Yin ◽

Christina L. Gardner ◽

Mikhail V. Khoretonenko ◽

Nadejda L. Korneeva ◽

...

Keyword(s):

Sindbis Virus ◽

Antiviral Effect ◽

Protein Accumulation ◽

Rnase L ◽

80S Ribosome ◽

Host Protection ◽

Beta Interferon ◽

Genes Encoding ◽

Alpha Beta ◽

Antiviral Proteins

ABSTRACT The alpha/beta interferon (IFN-α/β) response is critical for host protection against disseminated replication of many viruses, primarily due to the transcriptional upregulation of genes encoding antiviral proteins. Previously, we determined that infection of mice with Sindbis virus (SB) could be converted from asymptomatic to rapidly fatal by elimination of this response (K. D. Ryman et al., J. Virol. 74:3366-3378, 2000). Probing of the specific antiviral proteins important for IFN-mediated control of virus replication indicated that the double-stranded RNA-dependent protein kinase, PKR, exerted some early antiviral effects prior to IFN-α/β signaling; however, the ability of IFN-α/β to inhibit SB and protect mice from clinical disease was essentially undiminished in the absence of PKR, RNase L, and Mx proteins (K. D. Ryman et al., Viral Immunol. 15:53-76, 2002). One characteristic of the PKR/RNase L/Mx-independent antiviral effect was a blockage of viral protein accumulation early after infection (K. D. Ryman et al., J. Virol. 79:1487-1499, 2005). We show here that IFN-α/β priming induces a PKR-independent activity that inhibits m7G cap-dependent translation at a step after association of cap-binding factors and the small ribosome subunit but before formation of the 80S ribosome. Furthermore, the activity targets mRNAs that enter across the cytoplasmic membrane, but nucleus-transcribed RNAs are relatively unaffected. Therefore, this IFN-α/β-induced antiviral activity represents a mechanism through which IFN-α/β-exposed cells are defended against viruses that enter the cytoplasm, while preserving essential host activities, including the expression of antiviral and stress-responsive genes.

Download Full-text

Caenorhabditis elegans Deficient in DOT-1.1 Exhibit Increases in H3K9me2 at Enhancer and Certain RNAi-Regulated Regions

Cells ◽

10.3390/cells9081846 ◽

2020 ◽

Vol 9 (8) ◽

pp. 1846 ◽

Cited By ~ 1

Author(s):

Ruben Esse ◽

Alla Grishok

Keyword(s):

Small Rnas ◽

Mammalian Cells ◽

Leukemic Cells ◽

Rnai Pathway ◽

Open Chromatin ◽

Loss Of Function ◽

Promoter Regions ◽

Heterochromatin Formation ◽

C Elegans ◽

H3k79 Methylation

The methylation of histone H3 at lysine 79 is a feature of open chromatin. It is deposited by the conserved histone methyltransferase DOT1. Recently, DOT1 localization and H3K79 methylation (H3K79me) have been correlated with enhancers in C. elegans and mammalian cells. Since earlier research implicated H3K79me in preventing heterochromatin formation both in yeast and leukemic cells, we sought to inquire whether a H3K79me deficiency would lead to higher levels of heterochromatic histone modifications, specifically H3K9me2, at developmental enhancers in C. elegans. Therefore, we used H3K9me2 ChIP-seq to compare its abundance in control and dot-1.1 loss-of-function mutant worms, as well as in rde-4; dot-1.1 and rde-1; dot-1.1 double mutants. The rde-1 and rde-4 genes are components of the RNAi pathway in C. elegans, and RNAi is known to initiate H3K9 methylation in many organisms, including C. elegans. We have previously shown that dot-1.1(−) lethality is rescued by rde-1 and rde-4 loss-of-function. Here we found that H3K9me2 was elevated in enhancer, but not promoter, regions bound by the DOT-1.1/ZFP-1 complex in dot-1.1(−) worms. We also found increased H3K9me2 at genes targeted by the ALG-3/4-dependent small RNAs and repeat regions. Our results suggest that ectopic H3K9me2 in dot-1.1(−) could, in some cases, be induced by small RNAs.

Download Full-text

The Structure of Sindbis Virus Produced from Vertebrate and Invertebrate Hosts as Determined by Small-Angle Neutron Scattering

Journal of Virology ◽

10.1128/jvi.00044-10 ◽

2010 ◽

Vol 84 (10) ◽

pp. 5270-5276 ◽

Cited By ~ 19

Author(s):

Lilin He ◽

Amanda Piper ◽

Flora Meilleur ◽

Dean A. A. Myles ◽

Raquel Hernandez ◽

...

Keyword(s):

Neutron Scattering ◽

Small Angle ◽

Host Species ◽

Mammalian Cells ◽

Small Angle Neutron Scattering ◽

Sindbis Virus ◽

Insect Cells ◽

Lipid Membrane ◽

Scattering Data ◽

The Impact

ABSTRACT The complex natural cycle of vectored viruses that transition between host species, such as between insects and mammals, makes understanding the full life cycle of the virus an incredibly complex problem. Sindbis virus, an arbovirus and prototypic alphavirus having an inner protein shell and an outer glycoprotein coat separated by a lipid membrane, is one example of a vectored virus that transitions between vertebrate and insect hosts. While evidence of host-specific differences in Sindbis virus has been observed, no work has been performed to characterize the impact of the host species on the structure of the virus. Here, we report the first study of the structural differences between Sindbis viruses grown in mammalian and insect cells, which were determined by small-angle neutron scattering (SANS), a nondestructive technique that did not decrease the infectivity of the Sindbis virus particles studied. The scattering data and modeling showed that, while the radial position of the lipid bilayer did not change significantly, it was possible to conclude that it did have significantly more cholesterol when the virus was grown in mammalian cells. Additionally, the outer protein coat was found to be more extended in the mammalian Sindbis virus. The SANS data also demonstrated that the RNA and nucleocapsid protein share a closer interaction in the mammalian-cell-grown virus than in the virus from insect cells.

Download Full-text

Role of N-Linked Glycosylation for Sindbis Virus Infection and Replication in Vertebrate and Invertebrate Systems

Journal of Virology ◽

10.1128/jvi.02427-08 ◽

2009 ◽

Vol 83 (11) ◽

pp. 5640-5647 ◽

Cited By ~ 24

Author(s):

Ronald L. Knight ◽

Kimberly L. W. Schultz ◽

Rebekah J. Kent ◽

Meera Venkatesan ◽

Diane E. Griffin

Keyword(s):

Mammalian Cells ◽

Sindbis Virus ◽

Glycosylation Site ◽

Surface Glycoprotein ◽

Heparin Binding ◽

Virus Binding ◽

Bhk Cells ◽

Mosquito Cells ◽

Intrathoracic Inoculation

ABSTRACT Each Sindbis virus (SINV) surface glycoprotein has two sites for N-linked glycosylation (E1 positions 139 and 245 [E1-139 and E1-245] and E2 positions 196 and 318 [E2-196 and E2-318]). Studies of SINV strain TE12 mutants with each site eliminated identified the locations of carbohydrates by cryo-electron microscopy (S. V. Pletnev et al., Cell 105:127-136, 2001). In the current study, the effects of altered glycosylation on virion infectivity, growth in cells of vertebrates and invertebrates, heparin binding, virulence in mice, and replication in mosquitoes were assessed. Particle-to-PFU ratios for E1-139 and E2-196 mutant strains were similar to that for TE12, but this ratio for the E1-245 mutant was 100-fold lower than that for TE12. Elimination of either E2 glycosylation site increased virus binding to heparin and increased replication in BHK cells. Elimination of either E1 glycosylation site had no effect on heparin binding but resulted in an approximately 10-fold decrease in virus yield from BHK cells compared to the TE12 amount. No differences in pE2 processing were detected. E2-196 and E2-318 mutants were more virulent in mice after intracerebral inoculation, while E1-139 and E1-245 mutants were less virulent. The E1-245 mutant showed impaired replication in C7/10 mosquito cells and in Culex quinquefasciatus after intrathoracic inoculation. We conclude that the increased replication and virulence of E2-196 and E2-318 mutants are primarily due to increased efficiency of binding to heparan sulfate on mammalian cells. Lack of glycosylation at E1-139 or E1-245 impairs replication in vertebrate cells, while E1-245 also severely affects replication in invertebrate cells.

Download Full-text

Repertoire of virus-derived small RNAs produced by mosquito and mammalian cells in response to dengue virus infection

Virology ◽

10.1016/j.virol.2014.11.019 ◽

2015 ◽

Vol 476 ◽

pp. 54-60 ◽

Cited By ~ 8

Author(s):

Erin E. Schirtzinger ◽

Christy C. Andrade ◽

Nicholas Devitt ◽

Thiruvarangan Ramaraj ◽

Jennifer L. Jacobi ◽

...

Keyword(s):

Virus Infection ◽

Dengue Virus ◽

Small Rnas ◽

Mammalian Cells ◽

Dengue Virus Infection

Download Full-text

Protection against Fatal Sindbis Virus Encephalitis by Beclin, a Novel Bcl-2-Interacting Protein

Journal of Virology ◽

10.1128/jvi.72.11.8586-8596.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 8586-8596 ◽

Cited By ~ 731

Author(s):

Xiao Huan Liang ◽

Linda K. Kleeman ◽

Hui Hui Jiang ◽

Gerald Gordon ◽

James E. Goldman ◽

...

Keyword(s):

Mammalian Cells ◽

Sindbis Virus ◽

Stop Codon ◽

Coiled Coil ◽

Resonance Energy Transfer ◽

Premature Stop Codon ◽

Resonance Energy ◽

Cellular Protein ◽

Interacting Protein ◽

Induced Apoptosis

ABSTRACT bcl-2, the prototypic cellular antiapoptotic gene, decreases Sindbis virus replication and Sindbis virus-induced apoptosis in mouse brains, resulting in protection against lethal encephalitis. To investigate potential mechanisms by which Bcl-2 protects against central nervous system Sindbis virus infection, we performed a yeast two-hybrid screen to identify Bcl-2-interacting gene products in an adult mouse brain library. We identified a novel 60-kDa coiled-coil protein, Beclin, which we confirmed interacts with Bcl-2 in mammalian cells, using fluorescence resonance energy transfer microscopy. To examine the role of Beclin in Sindbis virus pathogenesis, we constructed recombinant Sindbis virus chimeras that express full-length human Beclin (SIN/beclin), Beclin lacking the putative Bcl-2-binding domain (SIN/beclinΔBcl-2BD), or Beclin containing a premature stop codon near the 5′ terminus (SIN/beclinstop). The survival of mice infected with SIN/beclin was significantly higher (71%) than the survival of mice infected with SIN/beclinΔBcl-2BD (9%) or SIN/beclinstop (7%) (P < 0.001). The brains of mice infected with SIN/beclin had fewer Sindbis virus RNA-positive cells, fewer apoptotic cells, and lower viral titers than the brains of mice infected with SIN/beclinΔBcl-2BD or SIN/beclinstop. These findings demonstrate that Beclin is a novel Bcl-2-interacting cellular protein that may play a role in antiviral host defense.

Download Full-text

Physiologically relevant miRNAs in mammalian oocytes are rare and highly abundant.

10.1101/2021.08.08.455568 ◽

2021 ◽

Author(s):

Shubhangini Kataruka ◽

Veronika Kinterova ◽

Filip Horvat ◽

Jiri Kanka ◽

Petr Svoboda

Keyword(s):

Gene Expression ◽

Small Rnas ◽

Mammalian Cells ◽

Negative Regulators ◽

Oocyte Growth ◽

Maternal To Zygotic Transition ◽

Porcine Oocyte ◽

Mirna Pathway ◽

Expression In Mammalian Cells ◽

Cognate Mrnas

miRNAs, ~22nt small RNAs associated with Argonaute (AGO) proteins, are important negative regulators of gene expression in mammalian cells. However, mammalian maternal miRNAs show negligible repressive activity and the miRNA pathway is dispensable for oocytes and maternal-to-zygotic transition. The stoichiometric hypothesis proposed that this is caused by dilution of maternal miRNAs during oocyte growth. As the dilution affects miRNAs but not mRNAs, it creates unfavorable miRNA:mRNA stoichiometry for efficient repression of cognate mRNAs. Here we report that porcine ssc-miR-205 and bovine bta-miR-10b are exceptional miRNAs, which resist the diluting effect of oocyte growth and can efficiently suppress gene expression. Additional analysis of ssc-miR-205 shows that it has higher stability, reduces expression of endogenous targets, and contributes to porcine oocyte-to-embryo transition. Consistent with the stoichiometric hypothesis, our results show that the endogenous miRNA pathway in mammalian oocytes is intact and that maternal miRNAs can efficiently suppress gene expression when a favorable miRNA:mRNA stoichiometry is established.

Download Full-text