Selective mRNA degradation by antisense oligonucleotide-2,5A chimeras: Involvement of RNase H and RNase L

The increase in antibacterial resistance is a serious challenge for both the health and defence sectors and there is a need for both novel antibacterial targets and antibacterial strategies. RNA degradation and ribonucleases, such as the essential endoribonuclease RNase E, encoded by the rne gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As rne mRNA has not been previously targeted using an antisense approach, we decided to explore using antisense oligonucleotides to target the translation initiation region of the Escherichia coli rne mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of rne mRNA translation through two mechanisms. Either LNA gapmer binding could sterically block translation and/or LNA gapmer binding could facilitate RNase H-mediated cleavage of the rne mRNA. This may prove to be an advantage over the majority of previous antibacterial antisense oligonucleotide approaches which used oligonucleotide chemistries that restrict the mode-of-action of the antisense oligonucleotide to steric blocking of translation. Using an electrophoretic mobility shift assay, we demonstrate that the LNA gapmers bind to the translation initiation region of E. coli rne mRNA. We then use a cell-free transcription translation reporter assay to show that this binding is capable of inhibiting translation. Finally, in an in vitro RNase H cleavage assay, the LNA gapmers facilitate RNase H-mediated mRNA cleavage. Although the challenges of antisense oligonucleotide delivery remain to be addressed, overall, this work lays the foundations for the development of a novel antibacterial strategy targeting rne mRNA with antisense oligonucleotides.

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Maintenance of protein synthesis in spite of mRNA breakdown in interferon-treated HeLa cells infected with reovirus

Molecular and Cellular Biology ◽

10.1128/mcb.3.1.64-69.1983 ◽

1983 ◽

Vol 3 (1) ◽

pp. 64-69

Author(s):

T W Nilsen ◽

P A Maroney ◽

C Baglioni

Keyword(s):

Protein Synthesis ◽

Hela Cells ◽

Large Fraction ◽

Relative Concentration ◽

Mrna Degradation ◽

Rnase L ◽

Mrna Synthesis ◽

Double Stranded Rna ◽

Specific Mrnas ◽

Cloned Cdna

Interferon induces the synthesis of an enzyme which synthesizes 2',5'-oligoadenylate [2',5'-oligo(A)] when activated by double-stranded RNA. The 2',5'-oligo(A) in turn activates an endonuclease (RNase L). Concentrations of 2',5'-oligo(A) sufficient to activate RNase L are formed in interferon-treated HeLa cells infected with reovirus, and a large fraction of cellular mRNA is degraded (T. W. Nilsen, P. A. Maroney, and C. Baglioni, J. Virol. 42:1039-1045, 1982). We report here that in spite of this mRNA degradation, protein synthesis was not significantly inhibited in these cells. When mRNA synthesis was inhibited with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, protein synthesis was markedly decreased, as shown by reduced incorporation of labeled amino acids and a decrease in polyribosomes. This suggested that the turnover of mRNA could be compensated for by increased production of mRNA. The relative concentration of specific mRNAs was measured with cloned cDNA probes. The amount of these mRNAs present in control cells was comparable to that in interferon-treated cells infected with reovirus, whereas it was decreased in the latter cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

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RNase H sequence preferences influence antisense oligonucleotide efficiency

Nucleic Acids Research ◽

10.1093/nar/gkx1073 ◽

2017 ◽

Vol 45 (22) ◽

pp. 12932-12944 ◽

Cited By ~ 19

Author(s):

Łukasz J. Kiełpiński ◽

Peter H. Hagedorn ◽

Morten Lindow ◽

Jeppe Vinther

Keyword(s):

Antisense Oligonucleotide ◽

Rnase H

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RNase L reprograms translation by widespread mRNA turnover escaped by antiviral mRNAs

10.1101/486530 ◽

2018 ◽

Cited By ~ 1

Author(s):

James M Burke ◽

Stephanie L Moon ◽

Evan T Lester ◽

Tyler Matheny ◽

Roy Parker

Keyword(s):

Translation Initiation ◽

Mammalian Cells ◽

Mrna Degradation ◽

Stress Granule ◽

Mrna Turnover ◽

Rnase L ◽

Protein Kinase R ◽

Double Stranded Rna ◽

Translation Repression ◽

Eif2α Phosphorylation

SUMMARYIn response to foreign and endogenous double-stranded RNA (dsRNA), protein kinase R (PKR) and ribonuclease L (RNase L) reprogram translation in mammalian cells. PKR inhibits translation initiation through eIF2α phosphorylation, which triggers stress granule (SG) formation and promotes translation of stress responsive mRNAs. The mechanisms of RNase L-driven translation repression, its contribution to SG assembly, and its regulation of dsRNA stress-induced mRNAs are unknown. We demonstrate that RNase L drives translational shut-off in response to dsRNA by promoting widespread turnover of mRNAs. This alters stress granule assembly and reprograms translation by only allowing for the translation of mRNAs resistant to RNase L degradation, including numerous antiviral mRNAs such asIFN-β. Individual cells differentially activate dsRNA responses revealing variation that can affect cellular outcomes. This identifies bulk mRNA degradation and the resistance of antiviral mRNAs as the mechanism by which RNaseL reprograms translation in response to dsRNA.

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Conformation-specific cleavage of antisense oligonucleotide-RNA duplexes by RNase H

Journal of the Chemical Society Perkin Transactions 2 ◽

10.1039/b006476i ◽

2001 ◽

pp. 402-408 ◽

Cited By ~ 10

Author(s):

Pushpangadan I. Pradeepkumar ◽

Edouard Zamaratski ◽

András Földesi ◽

Jyoti Chattopadhyaya

Keyword(s):

Antisense Oligonucleotide ◽

Rnase H ◽

Rna Duplexes

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Inhibition of off-target cleavage by RNase H using an artificial cationic oligosaccharide

Organic & Biomolecular Chemistry ◽

10.1039/d1ob00983d ◽

2021 ◽

Author(s):

Rintaro Iwata Iwata Hara ◽

Takeshi Wada

Keyword(s):

Side Effects ◽

Antisense Oligonucleotide ◽

Rnase H ◽

Ribonuclease H ◽

Sequence Dependent ◽

Target Effects

Sequence-dependent off-target effects are a serious problem of antisense oligonucleotide-based drugs. Some of these side effects are induced by ribonuclease H (RNase H)-mediated cleavage of non-target RNAs with similar base...

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Maintenance of protein synthesis in spite of mRNA breakdown in interferon-treated HeLa cells infected with reovirus.

Molecular and Cellular Biology ◽

10.1128/mcb.3.1.64 ◽

1983 ◽

Vol 3 (1) ◽

pp. 64-69 ◽

Cited By ~ 21

Author(s):

T W Nilsen ◽

P A Maroney ◽

C Baglioni

Keyword(s):

Protein Synthesis ◽

Hela Cells ◽

Large Fraction ◽

Relative Concentration ◽

Mrna Degradation ◽

Rnase L ◽

Mrna Synthesis ◽

Double Stranded Rna ◽

Specific Mrnas ◽

Cloned Cdna

Interferon induces the synthesis of an enzyme which synthesizes 2',5'-oligoadenylate [2',5'-oligo(A)] when activated by double-stranded RNA. The 2',5'-oligo(A) in turn activates an endonuclease (RNase L). Concentrations of 2',5'-oligo(A) sufficient to activate RNase L are formed in interferon-treated HeLa cells infected with reovirus, and a large fraction of cellular mRNA is degraded (T. W. Nilsen, P. A. Maroney, and C. Baglioni, J. Virol. 42:1039-1045, 1982). We report here that in spite of this mRNA degradation, protein synthesis was not significantly inhibited in these cells. When mRNA synthesis was inhibited with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, protein synthesis was markedly decreased, as shown by reduced incorporation of labeled amino acids and a decrease in polyribosomes. This suggested that the turnover of mRNA could be compensated for by increased production of mRNA. The relative concentration of specific mRNAs was measured with cloned cDNA probes. The amount of these mRNAs present in control cells was comparable to that in interferon-treated cells infected with reovirus, whereas it was decreased in the latter cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

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