Host Factors Influencing the Retrohoming Pathway of Group II Intron RmInt1, Which Has an Intron-Encoded Protein Naturally Devoid of Endonuclease Activity.

Rafael Nisa-Martínez,Nicolás Toro,María Dolores Molina-Sánchez,Akio Kanai

doi:10.1371/journal.pone.0162275

Rafael Nisa-Martínez, Nicolás Toro + Show 2 more

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https://doi.org/10.1371/journal.pone.0162275

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Abstract

Bacterial group II introns are self-splicing catalytic RNAs and mobile retroelements that have an open reading frame encoding an intron-encoded protein (IEP) with reverse transcriptase (RT) and RNA splicing or maturase activity. Some IEPs carry a DNA endonuclease (En) domain, which is required to cleave the bottom strand downstream from the intron-insertion site for target DNA-primed reverse transcription (TPRT) of the inserted intron RNA. Host factors complete the insertion of the intron. By contrast, the major retrohoming pathway of introns with IEPs naturally lacking endonuclease activity, like the Sinorhizobium meliloti intron RmInt1, is thought to involve insertion of the intron RNA into the template for lagging strand DNA synthesis ahead of the replication fork, with possible use of the nascent strand to prime reverse transcription of the intron RNA. The host factors influencing the retrohoming pathway of such introns have not yet been described. Here, we identify key candidates likely to be involved in early and late steps of RmInt1 retrohoming. Some of these host factors are common to En+ group II intron retrohoming, but some have different functions. Our results also suggest that the retrohoming process of RmInt1 may be less dependent on the intracellular free Mg2+ concentration than those of other group II introns.

Highlights

Retroelements, such as retroviruses and retrotransposons, are selfish genetic elements that use an RNA intermediate for mobility
The in vivo retrohoming efficiency of RmInt1 in its natural host S. meliloti was evaluated in a double-plasmid assay (Fig 1A), with an intron donor plasmid expressing the RmInt1 intron-encoded protein (IEP) followed by the ΔORF intron RNA, and a recipient plasmid containing the RmInt1 target site (-176/+466) inserted in the correct orientation to serve as the template for lagging-strand synthesis at the DNA replication fork
Some of these host factors are common to En+ group II intron retrohoming (e.g., RNase E), but some have different functions

Summary

Introduction

Retroelements, such as retroviruses and retrotransposons, are selfish genetic elements that use an RNA intermediate for mobility. Most bacterial group II introns have an open reading frame (ORF) in DIV encoding an intron-encoded protein (IEP) with an N-terminal reverse transcriptase (RT) domain, followed by an RNA-binding domain (X domain). In some of these IEPs, the X domain is followed by a C-terminal DNA-binding (D) region and a DNA endonuclease (En) domain. The mobility of group II introns, with and without D and En domains, is mediated by a ribonucleoprotein (RNP) complex consisting of the IEP and the spliced intron lariat RNA that recognizes intron targets through both RNP components [10,11,12]

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