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METTL3 alters capping enzyme expression and its activity on ribosomal proteins.

ABSTRACT: The 5' cap, catalyzed by RNA guanylyltransferase and 5'-phosphatase (RNGTT), is a vital mRNA modification for the functionality of mRNAs. mRNA capping occurs in the nucleus for the maturation of the functional mRNA and in the cytoplasm for fine-tuning gene expression. Given the fundamental importance of RNGTT in mRNA maturation and expression there is a need to further investigate the regulation of RNGTT. N6-methyladenosine (m⁶A) is one of the most abundant RNA modifications involved in the regulation of protein translation, mRNA stability, splicing, and export. We sought to investigate whether m⁶A could regulate the expression and activity of RNGTT. A motif for the m⁶A writer methyltransferase 3 (METTL3) in the 3'UTR of RNGTT mRNA was identified. Knockdown of METTL3 resulted in destabilizing RNGTT mRNA, and reduced protein expression. Sequencing of capped mRNAs identified an underrepresentation of ribosomal protein mRNA overlapping with 5' terminal oligopyrimidine (TOP) mRNAs and genes are dysregulated when cytoplasmic capping is inhibited. Pathway analysis identified disruptions in the mTOR and p70S6K pathways. A reduction in RPS6 mRNA capping, protein expression, and phosphorylation was detected with METTL3 knockdown.

SUBMITTER: Del Valle-Morales D

PROVIDER: S-EPMC10690260 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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METTL3 alters capping enzyme expression and its activity on ribosomal proteins.

Del Valle-Morales Daniel D Romano Giulia G Le Patricia P Saviana Michela M Brown Rachel R Micalo Lavender L Li Howard H La Ferlita Alessandro A Nigita Giovanni G Nana-Sinkam Patrick P Acunzo Mario M

bioRxiv : the preprint server for biology 20231122

The 5' cap, catalyzed by RNA guanylyltransferase and 5'-phosphatase (RNGTT), is a vital mRNA modification for the functionality of mRNAs. mRNA capping occurs in the nucleus for the maturation of the functional mRNA and in the cytoplasm for fine-tuning gene expression. Given the fundamental importance of RNGTT in mRNA maturation and expression there is a need to further investigate the regulation of RNGTT. N6-methyladenosine (m<sup>6</sup>A) is one of the most abundant RNA modifications involved ...[more]

PMID: 38045284

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Project description:The binding of viral RNA to RIG-I-like receptors triggers the formation of mitochondrial antiviral signaling (MAVS) protein aggregates critical for interferon (IFN) expression. Several rotavirus strains have been shown to suppress IFN expression by inducing MAVS degradation. Relying on transient expression assays, previous studies reached different conclusions regarding the identity of the rotavirus protein responsible for MAVS degradation, suggesting it was an activity of the rotavirus capping enzyme VP3 or the interferon antagonist NSP1. Here, we have used recombinant SA11 rotaviruses to identify the endogenous viral protein responsible for MAVS degradation and to analyze how the attack on MAVS impacts IFN expression. The recombinant viruses included those expressing modified VP3 or NSP1 proteins deficient in the ability to induce the degradation of MAVS or interferon regulatory factor-3 (IRF3), or both. With these viruses, we determined that VP3 directs the proteasomal degradation of MAVS but plays no role in IRF3 degradation. Moreover, NSP1 was determined to induce IRF3 degradation but to have no impact on MAVS degradation. Analysis of rotavirus-infected cells indicated that IRF3 degradation was more efficient than MAVS degradation and that NSP1 was primarily responsible for suppressing IFN expression in infected cells. However, VP3-mediated MAVS degradation contributed to IFN suppression in cells that failed to produce functional NSP1, pointing to a subsidiary role for VP3 in the IFN antagonist activity of NSP1. Thus, VP3 is a multifunctional protein with several activities that counter anti-rotavirus innate immune responses, including capping of viral (+)RNAs, hydrolysis of the RNase L 2-5A (2'-5' oligoadenylate) signaling molecule, and proteasomal degradation of MAVS. IMPORTANCE Rotavirus is an enteric RNA virus that causes severe dehydrating gastroenteritis in infants and young children through infection of enterocytes in the small intestine. Timely clearance of the virus demands a robust innate immune response by cells associated with the small intestine, including the expression of interferon (IFN). Previous studies have shown that some rotavirus strains suppress the production of interferon, by inducing the degradation of mitochondrial antiviral signaling (MAVS) protein and interferon regulatory factor-3 (IRF3). In this study, we have used reverse genetics to generate recombinant rotaviruses expressing compromised forms of VP3 or NSP1, or both, to explore the function of these viral proteins in the degradation of MAVS and IRF3. Our results demonstrate that VP3 is responsible for MAVS depletion in rotavirus-infected cells, and through this activity, helps to suppress IFN production. Thus, VP3 functions to support the activity of rotavirus NSP1, the major interferon antagonist of the virus.

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METTL3 alters capping enzyme expression and its activity on ribosomal proteins.

Publications

METTL3 alters capping enzyme expression and its activity on ribosomal proteins.

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