Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA.
La Jolla Institute for Immunology, La Jolla, CA, USA.
Nucleic Acids Res. 2022 Apr 8;50(6):3490-3504. doi: 10.1093/nar/gkac177.
Retrons are bacterial retroelements that produce single-stranded, reverse-transcribed DNA (RT-DNA) that is a critical part of a newly discovered phage defense system. Short retron RT-DNAs are produced from larger, structured RNAs via a unique 2'-5' initiation and a mechanism for precise termination that is not yet understood. Interestingly, retron reverse transcriptases (RTs) typically lack an RNase H domain and, therefore, depend on endogenous RNase H1 to remove RNA templates from RT-DNA. We find evidence for an expanded role of RNase H1 in the mechanism of RT-DNA termination, beyond the mere removal of RNA from RT-DNA:RNA hybrids. We show that endogenous RNase H1 determines the termination point of the retron RT-DNA, with differing effects across retron subtypes, and that these effects can be recapitulated using a reduced, in vitro system. We exclude mechanisms of termination that rely on steric effects of RNase H1 or RNA secondary structure and, instead, propose a model in which the tertiary structure of the single-stranded RT-DNA and remaining RNA template results in termination. Finally, we show that this mechanism affects cellular function, as retron-based phage defense is weaker in the absence of RNase H1.
Retrons 是一种细菌反转录元件，可产生单链、反转录的 DNA（RT-DNA），这是新发现的噬菌体防御系统的关键部分。短的 retron RT-DNA 是通过独特的 2'-5'起始和尚未完全理解的精确终止机制从较大的、结构化的 RNA 产生的。有趣的是，retron 逆转录酶（RT）通常缺乏 RNase H 结构域，因此依赖内源性 RNase H1 从 RT-DNA 中去除 RNA 模板。我们发现 RNase H1 在 RT-DNA 终止机制中具有扩展的作用，超出了从 RT-DNA:RNA 杂种中去除 RNA 的简单作用：我们表明，内源性 RNase H1 决定了 retron RT-DNA 的终止点，在不同的 retron 亚型中具有不同的影响，并且可以使用简化的体外系统再现这些影响。我们排除了依赖 RNase H1 的空间效应或 RNA 二级结构的终止机制，而是提出了一种模型，其中单链 RT-DNA 和剩余的 RNA 模板的三级结构导致终止。最后，我们表明这种机制会影响细胞功能，因为在缺乏 RNase H1 的情况下，基于 retron 的噬菌体防御能力较弱。
