Localization of a bacterial group II intron-encoded protein in human cells

Toro

2016

Front. Mol. Biosci.

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The functional unit of mobile group II introns is a ribonucleoprotein particle (RNP) consisting of the intron-encoded protein (IEP) and the excised intron RNA. The IEP has reverse transcriptase activity but also promotes RNA splicing, and the RNA-protein complex triggers site-specific DNA insertion by reverse splicing, in a process called retrohoming. In vitro reconstituted ribonucleoprotein complexes from the Lactococcus lactis group II intron Ll.LtrB, which produce a double strand break, have recently been studied as a means of developing group II intron-based gene targeting methods for higher organisms. The Sinorhizobium meliloti group II intron RmInt1 is an efficient mobile retroelement, the dispersal of which appears to be linked to transient single-stranded DNA during replication. The RmInt1IEP lacks the endonuclease domain (En) and cannot cut the bottom strand to generate the 3′ end to initiate reverse transcription. We used an Escherichia coli expression system to produce soluble and active RmInt1 IEP and reconstituted RNPs with purified components in vitro. The RNPs generated were functional and reverse-spliced into a single-stranded DNA target. This work constitutes the starting point for the use of group II introns lacking DNA endonuclease domain-derived RNPs for highly specific gene targeting methods.

Section: Methodsmentioning

confidence: 99%

Functionality of In vitro Reconstituted Group II Intron RmInt1-Derived Ribonucleoprotein Particles

Molina-Sánchez

Toro

2016

Front. Mol. Biosci.

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“…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 IEP but lacking the ribozyme component of the intron; finally, as an IP control, pKGEMA4 encoded untagged functional RNPs (Nisa-Martínez et al, 2007). We constructed 18 libraries corresponding to the input and ChIP samples in triplicate, and we obtained a total of 508,880,560 high-quality reads, which we then mapped back onto the S. meliloti RMO17 genome (Supplementary Table S1), reaching around 300-fold genome coverage for each individual library.…”

Section: Resultsmentioning

confidence: 99%

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

Molina-Sánchez

Andrés-León

et al. 2022

Front. Mol. Biosci.

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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.

“…For the construction of pMalFlagIEP, a NotI fragment containing the IEP tagged with 3xFlag was obtained by the NotI digestion of pCEP4FlagIEP (35) and inserted into the pMAL-c5X vector (New England Biolabs) digested with NotI enzyme and dephosphorylated. We obtained pMALFlagIEPΔORF in a similar manner, but using a NotI fragment from pCEP4FlagIEPΔORF (35).…”

Section: Methodsmentioning

confidence: 99%

A group II intron-encoded protein interacts with the cellular replicative machinery through the β-sliding clamp

Neira

Marcia

et al. 2019

Nucleic Acids Research

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Group II introns are self-splicing mobile genetic retroelements. The spliced intron RNA and the intron-encoded protein (IEP) form ribonucleoprotein particles (RNPs) that recognize and invade specific DNA target sites. The IEP is a reverse transcriptase/maturase that may bear a C-terminal endonuclease domain enabling the RNP to cleave the target DNA strand to prime reverse transcription. However, some mobile introns, such as RmInt1, lack the En domain but nevertheless retrohome efficiently to transient single-stranded DNA target sites at a DNA replication fork. Their mobility is associated with host DNA replication, and they use the nascent lagging strand as a primer for reverse transcription. We searched for proteins that interact with RmInt1 RNPs and direct these RNPs to the DNA replication fork. Co-immunoprecipitation assays suggested that DnaN (the β-sliding clamp), a component of DNA polymerase III, interacts with the protein component of the RmInt1 RNP. Pulldown assays, far-western blots and biolayer interferometry supported this interaction. Peptide binding assays also identified a putative DnaN-interacting motif in the RmInt1 IEP structurally conserved in group II intron IEPs. Our results suggest that intron RNP interacts with the β-sliding clamp of the DNA replication machinery, favouring reverse splicing into the transient ssDNA at DNA replication forks.