Identification of Group II Intron RmInt1 Binding Sites in a Bacterial Genome.

María Dolores Molina-Sánchez,Fernando Manuel García-Rodríguez,Eduardo Andrés-León,Nicolás Toro

doi:10.3389/fmolb.2022.834020

Abstract

RmInt1 is a group II intron encoding a reverse transcriptase protein (IEP) lacking the C-terminal endonuclease domain. RmInt1 is an efficient mobile retroelement that predominantly reverse splices into the transient single-stranded DNA at the template for lagging strand DNA synthesis during host replication, a process facilitated by the interaction of the RmInt1 IEP with DnaN at the replication fork. It has been suggested that group II intron ribonucleoprotein particles bind DNA nonspecifically, and then scan for their correct target site. In this study, we investigated RmInt1 binding sites throughout the Sinorhizobium meliloti genome, by chromatin-immunoprecipitation coupled with next-generation sequencing. We found that RmInt1 binding sites cluster around the bidirectional replication origin of each of the three replicons comprising the S. meliloti genome. Our results provide new evidence linking group II intron mobility to host DNA replication.

Highlights

Group II introns are considered to be ancient genetic elements present in the genomes of Eubacteria, Archaebacteria, and the organelles of some eukaryotes (Ferat and Michel, 1993; Toro, 2003; Lambowitz and Zimmerly, 2004)
We aim to identify the regions of the S. meliloti genome binding RmInt1 RNPs in vivo using ChIP-Seq analyses, by introducing various plasmid constructs into RMO17, an intron-less S. meliloti strain (Figure 2). pKG4-FlagIEP encoded 3xFLAG-tagged active RNPs (Supplementary Figure S2; Reinoso-Colacio et al, 2015); pKG-FlagIEP expressed the FLAG-tagged intron encoding a reverse transcriptase protein (IEP) but lacking the ribozyme component of the intron; as an IP control, pKGEMA4 encoded untagged functional RNPs (Nisa-Martínez et al, 2007)
Further filtering was performed by removing any peaks found in non-tagged functional RNPs, which resulted in 321 distinct DNA fragments appearing only in the FLAGtagged, functional RNP output data (Figure 3)

Summary

Introduction

Group II introns are considered to be ancient genetic elements present in the genomes of Eubacteria, Archaebacteria, and the organelles of some eukaryotes (Ferat and Michel, 1993; Toro, 2003; Lambowitz and Zimmerly, 2004). The group II intron IEP is encoded by domain IV and typically consists of four functional domains: A reverse transcriptase (RT), a maturase (X), a DNA-binding domain (D) and an endonuclease (En) domain (San Filippo and Lambowitz, 2002). Under physiological conditions, both the RT and X domains form contacts with several intron RNA domains (DIV, DII, DIII and DVI) to promote intron folding and splicing (Wank et al, 1999; Zhao and Pyle, 2017). The IEP remains bound to the excised lariat RNA, forming the RNP complex, which performs the mobility reaction via an RNA intermediate (Cousineau et al, 1998; Martínez-Abarca et al, 2004)

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Conclusion