Wu, H.-Y. | Search bioRxiv

bioRxiv2026

N6-methyladenosine regulates Influenza A virus mRNA stability yet is rarely found on genomic RNA

Previous studies have found widespread N6-methyladenosine (m6A methylation) on all forms of Influenza A virus (IAV) RNA, with m6A found critical for viral replication, pathogenicity as well as viral RNA packaging. Here we applied the latest quantitative technologies to revisit the methylation landscape on the anti-sense genomic RNA of IAV. Unexpectedly, upon Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) analysis of IAV virion -extracted genomic RNA, we detected very little m6A regardless of production from human cells or chicken eggs. Concordantly, Nanopore direct RNA sequencing also detected an overall low occurrence and stoichiometry (generally <5%) of m6A across all viral genomic RNA segments, compared with abundant m6A sites on viral mRNAs at ~20-30% m6A. Cross validation with glyoxal- and nitrite-mediated deamination of unmethylated adenosines (GLORI) confirmed multiple m6A sites on viral mRNA yet very few m6A on the genomic RNA. This paucity of m6A on genomic RNA makes it unlikely that m6A contributes to viral RNA packaging. Knockdown or pharmacological inhibition of the m6A methyltransferase METTL3 as well as the reader protein YTHDF2 both reduced viral mRNA levels and infectious viral particle production, with YTHDF2 promoting viral mRNA stability. Thus, the presence of m6A on IAV transcripts is indeed proviral, yet it is the mRNAs instead of genomic RNAs that are methylated at functionally relevant levels. Lastly, we provide proof of concept that a METTL3 small molecule inhibitor can be antiviral, and propose that m6A-targeted antivirals would mainly impact the intracellular gene expression phase of IAV replication.

microbiology↗

bioRxiv2017

Dynein Pulling Forces On Ruptured Nuclei Counteract Lamin-Mediated Nuclear Envelope Repair Mechanisms In Vivo

Recent work done exclusively in tissue culture cells revealed that the nuclear envelope (NE) undergoes ruptures leading to transient mixing of nuclear and cytoplasmic components. The duration of transient NE ruptures depends on lamins, however the underlying mechanisms and the relevance to in vivo events is not known. Here, we use C. elegans embryos to show that dynein forces that position nuclei increase the severity of lamin-induced NE ruptures in vivo. In the absence of dynein forces, lamin prevents nuclear-cytoplasmic mixing caused by NE ruptures. By monitoring the dynamics of NE rupture events, we demonstrate that lamin is required for a distinct step in NE recovery that restricts nucleocytoplasmic mixing prior to the full restoration of NE rupture sites. We show that laser-induced puncture of the NE recapitulates phenotypes associated with NE recovery in wild type cells. Surprisingly, we find that embryonic lethality does not correlate with the incidence of NE rupture events suggesting that embryos survive transient losses of NE compartmentalization during early embryogenesis. In addition to presenting the first mechanistic analysis of transient NE ruptures in vivo, this work demonstrates that lamin controls the duration of NE ruptures by opposing dynein forces on ruptured nuclei to allow reestablishment of the NE permeability barrier and subsequent restoration of NE rupture sites.

developmental biology↗