RESUMEN
Influenza A virus (IAV) utilizes cap-snatching to obtain host capped small RNAs for priming viral mRNA synthesis, generating capped hybrid mRNAs for translation. Previous studies have been focusing on canonical cap-snatching, which occurs at the very 5' end of viral mRNAs. Here we discovered noncanonical cap-snatching, which generates capped hybrid mRNAs/noncoding RNAs mapped to the region â¼300 nucleotides (nt) upstream of each mRNA 3' end, and to the 5' region, primarily starting at the second nt, of each virion RNAs (vRNA). Like canonical cap-snatching, noncanonical cap-snatching utilizes a base-pairing between the last nt G of host capped RNAs and a nt C of template RNAs to prime RNA synthesis. However, the nt upstream of this template C is usually A/U rather than just U; prime-realignment occurs less frequently. We also demonstrate that IAV can snatch capped IAV RNAs in addition to host RNAs. Noncanonical cap-snatching likely generates novel mRNAs with start AUG encoded in viral or host RNAs. These findings expand our understanding of cap-snatching mechanisms and suggest that IAV may utilize noncanonical cap-snatching to diversify its mRNAs/ncRNAs.
Asunto(s)
Virus de la Influenza A/genética , Caperuzas de ARN/genética , ARN Mensajero/genética , ARN no Traducido/genética , Células A549 , Emparejamiento Base/genética , Línea Celular Tumoral , Humanos , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/genética , Transcripción Genética/genéticaRESUMEN
High-throughput sequencing has become a standard tool for analyzing RNA and DNA. This method usually needs a cDNA/DNA library ligated with specific 5' and 3' linkers. Unlike mRNA, small RNA often contains modifications including 5' cap or triphosphate and 2'-O-methyl, requiring additional processing steps before linker additions during cloning processes; due to low expression levels, it is difficult to clone small RNA with a small amount of total RNA. Here we present a new strategy to clone 5' modified or unmodified small RNA in an all-liquid-based reaction carried out in a single PCR tube with as little as 20 ng total RNA. The 7-h cloning process only needs â¼1 h of labor. Moreover, this method can also clone mRNA, simplifying the need to prepare two cloning systems for small RNA and mRNA; the barcoded PCR primers are also compatible with non-cDNA cloning applications, including the preparation of genomic libraries. Not only is our method more convenient for cloning modified RNA than available methods, but it is also more sensitive, versatile, and cost-effective. Moreover, the all-liquid-based reaction can be performed in an automated manner.
