Koppstein David, Ashour Joseph, Bartel David P
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Whitehead Institute of Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA Howard Hughes Medical Institute, Whitehead Institute of Biomedical Research, Cambridge, MA 02142, USA.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Whitehead Institute of Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA.
Nucleic Acids Res. 2015 May 26;43(10):5052-64. doi: 10.1093/nar/gkv333. Epub 2015 Apr 21.
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.
流感病毒聚合酶在宿主RNA的5'端下游约10 - 13个核苷酸处切割宿主RNA，并利用这个带帽片段启动病毒mRNA的合成。为了更好地理解这种抢帽过程，我们使用高通量测序来确定A/WSN/33（H1N1）流感病毒mRNA的5'端。这些序列为转录起始过程中的新生链重排提供了明确证据，并揭示了病毒模板对重排频率的强烈影响。在考虑通过重排插入的额外核苷酸后，对带帽片段的分析表明，不同的病毒mRNA都预先加上了一组共同的序列，并且聚合酶通常在嘌呤之后切割宿主RNA，并且经常在病毒模板的末端或倒数第二个残基的单个碱基对上启动转录。尽管抢得的片段中信息含量有限，我们还开发了一种生物信息学方法来识别靶向的宿主转录本，发现小核RNA和小核仁RNA贡献了最丰富的带帽前导序列。这些结果为病毒转录起始机制提供了见解，并揭示了抢帽文库的多样性，表明非编码转录本以及mRNA都被用于制造流感病毒mRNA。
