Abstract
The influenza polymerase cleaves host RNAs ∼10–13 nucleotides downstream of their 5′ ends and uses this capped fragment to prime viral mRNA synthesis. To better understand this process of cap snatching, we used high-throughput sequencing to determine the 5′ ends of A/WSN/33 (H1N1) influenza mRNAs. The sequences provided clear evidence for nascent-chain realignment during transcription initiation and revealed a strong influence of the viral template on the frequency of realignment. After accounting for the extra nucleotides inserted through realignment, analysis of the capped fragments indicated that the different viral mRNAs were each prepended with a common set of sequences and that the polymerase often cleaved host RNAs after a purine and often primed transcription on a single base pair to either the terminal or penultimate residue of the viral template. We also developed a bioinformatic approach to identify the targeted host transcripts despite limited information content within snatched fragments and found that small nuclear RNAs and small nucleolar RNAs contributed the most abundant capped leaders. These results provide insight into the mechanism of viral transcription initiation and reveal the diversity of the cap-snatched repertoire, showing that noncoding transcripts as well as mRNAs are used to make influenza mRNAs.
Highlights
In eukaryotic gene expression, a 7-methylguanosine (m7G) cap is added to the beginning of an mRNA by a 5 -5 triphosphate and is important for stability, export, and translation of that transcript [1,2,3]
At the 3 end, capped cellular fragments ending with a G that was used to prime transcription at the penultimate template nucleotide would have lost that G during our trimming step designed to remove the viral mRNA sequence
Our unanticipated finding that many of the cap-snatched leaders derived from small nuclear RNAs (snRNAs)/small nucleolar RNAs (snoRNAs) provided indirect evidence for cap snatching of nascent transcripts, presumably facilitated by the direct association between the viral polymerase and the C-terminal domain of polymerase II (Pol II) [57]
Summary
A 7-methylguanosine (m7G) cap is added to the beginning of an mRNA by a 5 -5 triphosphate and is important for stability, export, and translation of that transcript [1,2,3] This cap dependence poses a challenge for RNA viruses because many are unable to use the cellular RNA capping machinery, which is associated with DNA-dependent RNA polymerase II (Pol II) [4,5]. These viruses have developed diverse strategies to circumvent this problem. The polymerase is responsible for polyadenylation of viral mRNAs and replication of the negativestranded viral genome (vRNA), which consists of eight RNA segments, through complementary RNA (cRNA) intermediates [6,18,19,20]
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