Abstract
Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.
Highlights
Mobile group II introns are catalytic RNAs and mobile retroelements that were initially discovered in the mitochondrial and chloroplast genomes of lower eukaryotes and plants[1] and subsequently identified in bacteria and archaea[2,3,4]
We generated two control vectors: pEB-yellow fluorescent protein (YFP), encoding YFP only, and pEB-YFP DNMT3L, encoding YFP fused to the mouse de novo methyl transferase 3-like (DNMT3L) protein, a regulatory factor involved in DNA methylation[26]
With the pEB-YFP and pEB-YFP-DNMT3L controls, preferential nuclear localization was observed for both the proteins encoded, but, by contrast to the YFP-intron-encoded protein (IEP) fusion, these proteins were detected in the nucleolus (Fig. 1a)
Summary
Mobile group II introns are catalytic RNAs and mobile retroelements that were initially discovered in the mitochondrial and chloroplast genomes of lower eukaryotes and plants[1] and subsequently identified in bacteria and archaea[2,3,4]. We recently reported the expression and subcellular localization of the bacterial RmInt[1] group II IEP, which lacks the endonuclease domain[23], in Arabidopsis thaliana protoplasts[24] This IEP has been shown to accumulate in the nucleolus, whereas a mutant IEP with a modified maturase domain was localized in nuclear speckles. The localization of the RmInt[1] IEP in speckles in the absence of maturase activity is consistent with the hypothesis that group II introns are the ancestors of spliceosomal introns, because nuclear speckles are nuclear domains that accumulate high local concentrations of pre-mRNA splicing factors (snRNPs and SR proteins), located in the interchromatin nuclear space[25]
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