Retrons are genetic elements constituted of a non-coding RNA (ncRNA) associated with a reverse-transcriptase (RT). The RT reverse-transcribes part of the ncRNA to generate an RNA-DNA hybrid molecule. Although the existence of retrons have been known for decades, their biological functions were unknown. Recent studies revealed that most retrons could in fact be anti-phage systems (1,2).
Retrons are distinct genetic elements found in bacterial genomes that code for a reversetranscriptase (RT) and a non-coding RNA (ncRNA). These elements generate a unique satellite DNA/RNA hybrid in the cell termed multicopy single-stranded DNA (msDNA). Retrons were recently found to function as anti-phage defense systems protecting bacteria against phage infection :ref{doi=10.1016/j.cell.2020.09.065}. Their defensive unit is composed of three components: the reverse transcriptase, the non-coding RNA, and an effector protein.
### Discovery
Discovery
Retrons were originally discovered in 1984 in Myxococcus xanthus, when Yee et al. :ref{doi=10.1016/0092-8674(84)90541-5} identified a high copy, short, single-stranded linear ex-chromosomal DNA fragment in the gram-negative bacterium, Myxococcus xanthus. These multi-copy single-stranded DNA fragments were termed msDNA. Further studies showed that this single-stranded DNA (ssDNA) is covalently linked to an RNA molecule :ref{doi=10.1016/0092-8674(87)90596-4}. Although at the time reverse transcriptases were only known from Eukaryotes and viruses, Inouye and colleagues hypothesized that msDNA must be a product of a reverse transcription reaction :ref{doi=10.1016/j.gene.2016.10.031}. Five years later an RT was shown to be associated with the biosynthesis of msDNA :ref{doi=10.1016/0092-8674(89)90593-X,10.1016/0092-8674(89)90592-8}, this was the first discovery of an RT in bacteria.
_Fig 1. (Mestre et al., 2020) Structure and organisation of a retron_ . The two non-coding contiguous inverted sequences (named msr and msd) aretranscribed as a single RNA. The RT recognizes its specific structure and reverse-transcribes it, generating an RNA-DNA hybrid
Although retrons were biochemically well studied and characterized, it was only 36 years after msDNA discovery, when their biological function was discovered :ref{doi=10.1126/science.abf6127}. In a systematic screen for the discovery of novel anti-phage defense systems in bacterial genomes :ref{doi=10.1016/j.chom.2022.09.017}, Millman et al. discovered a new defense system that contained a retron element (Retron-Eco8), further analysis showed that retrons are enriched in bacterial defense islands and together with their accessory proteins many were shown to confer defense against phage infection :ref{doi=10.1016/j.cell.2020.09.065}. An independent screen for defense systems, later that same year, also reported similar conclusions showing retrons function in antiphage defense :ref{doi=10.1126/science.aba0372}.
Due to their ability to produce high copy of DNA within the cell, since their discovery retrons have served as a fertile ground for biotechnological applications :ref{doi=10.1073/pnas.2018181118,10.1038/s41589-021-00927-y,10.1038/s41596-023-00819-6}
The majority of retrons are encoded on a gene cassette that encodes the retron and one or two additional proteins, which act as the retrons effectors. Bioinformatic prediction reveals that these effectors are very diverse and include transmembrane proteins, proteases, Cold-shock proteins, TIR domains proteins, ATPase, endonucleases, etc. Interestingly, several of these effector domains have already been described in other defense systems, including CBASS and Septu. Most retrons appear to act through an Abortive infection strategy (1).
## Molecular mechanisms
The *E.coli* retron system Ec48 mediates growth arrest upon sensing the inactivation of the bacterial RecBCD complex, a key element of the bacterial DNA repair system and immunity (1). Another study demonstrates that several retrons are part of Toxin-Antitoxin systems, where the RT-msDNA complex acts as an antitoxin that binds to and inhibits its cognate toxin. The tempering of the RT-msDNA, possibly by phage-encoded anti-RM systems, abolishes the antitoxin properties of the retron element, resulting in cell death mediated by the toxin activity (2).
### General
When the retron ncRNA (msr-msd) is transcribed it folds into a typical structure that is recognized by the RT :ref{doi=10.1016/S0021-9258(18)83336-1}. The RT then reverse transcribes a portion of the ncRNA (msd), starting from the 2′-end of a conserved guanosine residue found immediately after a double-stranded RNA structure within the ncRNA :ref{doi=10.1016/0092-8674(89)90592-8}. During reverse transcription, cellular RNase H degrades the segment of the ncRNA that serves as template, but not other parts of the ncRNA (msr), yielding the mature RNA-DNA hybrid (msDNA) :ref{doi=10.1016/0092-8674(89)90592-8}. In some cases cellular nucleases have been shown to further process the msDNA :ref{doi=10.1111/j.1365-2958.1992.tb01788.x,10.1006/plas.1997.1298,10.1007/s12275-015-5304-0}.
### Retron-Eco6 (Ec48)
The Retron-Eco6 system encodes in addition to the retron an effector protein containing 2 transmembrane domains (2TM). Retron-Eco6 was shown to protect bacteria against phage through abortive infection (Abi) by guarding the integrity of the RecBCD complex in the cell. Many phages inhibit RecBCD in order to successfully infect the cell. Upon inhibition of RecBCD, the effector protein turns the membrane permeable and the cells lyse within 45 minutes post infection :ref{doi=10.1016/j.cell.2020.09.065}.
### Retron-Sen2 (St85), Retron-Eco9
The Retron-Sen2 system was shown to function as a three-partite toxin-antitoxin (TA) system. The accessory gene RcaT acts as a bona fide toxin and ectopically inhibits growth. The Retron-RT-msDNA complex acts as an antitoxin alleviating RcaT toxicity.
Several triggers were identified for the Sen2-TA system, including Dam that was shown to methylate the mature msDNA and thus likely disrupt the RcaT–RT–msDNA complex, and RecE that degrades mature msDNA and reduces the RT-msDNA antitoxin levels :ref{doi=10.1038/s41586-022-05091-4}
