Retron reverse transcriptase termination and phage defense are dependent on host RNase H1

Abstract

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.

An experimental census of retrons for DNA production and genome editing

“…This consists of conserved characteristics such as an inverted repeat (the a1/a2 region of a retron ncRNA) >8bp, a priming guanine near the beginning of the ncRNA, and multiple hairpins in close proximity to a retron RT. Retrons have been recently shown to engage in phage defense that requires one or more additional accessory proteins, multiple of which have been shown to be cellular toxins 8,9,12 . The accessory proteins and phage defense mechanisms are extremely diverse among retrons 10 .…”

“…Next, we prepared any resulting RT-DNA for sequencing. Because the retron RT-DNA sequences were unknown to us, we adapted an unbiased sequencing approach that we have previously used to examine Eco1 variant libraries 12 . Briefly, we added a single poly-nucleotide handle to the 5’ end of RT-DNA using TdT, used a complementary poly-nucleotide anchored primer and Klenow to synthesize a strand complementary to the RT-DNA, and then ligated adapters at the 3’ end of the now double-stranded RT-DNA ( Fig 1c ).…”

“…Either directly or indirectly, the phage modifies or degrades the RT-DNA, which releases an accessory retron protein that acts as a cellular toxin to remove the infected cell and spare the bacterial population 8,9,11 . Individual retrons have been shown to confer specific resistance to particular phages 8,12 . This specificity is due, at least in part, to the unique sequence of RT-DNA produced by the retron 8 .…”

“…All products were indexed and sequenced on an Illumina MiSeq instrument. Previous data from Palka et al 202212 was included in the sequencing analysis for retron-Sen2 (Mestre-116), retron-Eco6 (Mestre-488), retron-Eco2 (Mestre-1036), and retron-Eco4 (Mestre-64).…”

Continuous Multiplexed Phage Genome Editing Using Recombitrons

“…In bacteria, this RT-DNA is used in conjunction with retron accessory proteins to detect and respond to phage infection. The retron accessory proteins are necessary for the phage defense phenotype and are not included in the recombitron to avoid reconstituting an anti-phage system [21][22][23][24] . We modify the retron ncRNA by adding nucleotides to the region that is reverse transcribed that are homologous to a locus in the phage genome and carry the edit we aim to incorporate.…”