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

Reinoso-Colacio M, García-Rodríguez FM, García-Cañadas M, Amador-Cubero S, García Pérez JL, Toro N - Sci Rep (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Grupo de Ecología Genética, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Calle Profesor Albareda 1, 18008 Granada, Spain.

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.

No MeSH data available.


Subcellular distribution of the YFP-IEP RmInt1 fusion in HeLa cells.(a) Fluorescence and bright-field (BF) microscopy of transfected HeLa HA cells. Localization of the YFP-IEP fusion protein in yellow (YFP). DAPI (blue) was used to stain nuclear DNA, and merged images are shown in the right column. Schematic diagrams of the constructs are shown to the right of the micrographs. The domains of the IEP are indicated as follows: in pink, the reverse transcriptase; in green, the maturase and, in yellow, the C-terminal domain. (b) Colocalization experiments using an anti-fibrillarin antibody (red) as a nucleolar marker (Fibrillarin); The YFP-IEP fusion protein is shown in yellow (YFP); DAPI (blue) was used to stain nuclear DNA, and merged images are shown on the right. A diagram of the construct is shown to the right of the micrograph.
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f1: Subcellular distribution of the YFP-IEP RmInt1 fusion in HeLa cells.(a) Fluorescence and bright-field (BF) microscopy of transfected HeLa HA cells. Localization of the YFP-IEP fusion protein in yellow (YFP). DAPI (blue) was used to stain nuclear DNA, and merged images are shown in the right column. Schematic diagrams of the constructs are shown to the right of the micrographs. The domains of the IEP are indicated as follows: in pink, the reverse transcriptase; in green, the maturase and, in yellow, the C-terminal domain. (b) Colocalization experiments using an anti-fibrillarin antibody (red) as a nucleolar marker (Fibrillarin); The YFP-IEP fusion protein is shown in yellow (YFP); DAPI (blue) was used to stain nuclear DNA, and merged images are shown on the right. A diagram of the construct is shown to the right of the micrograph.

Mentions: We investigated the intracellular distribution of the RmInt1 protein (IEP), by fusing its coding sequence to that of the yellow fluorescent protein (YFP), under control of the human EF1 α promoter (Pol II), in the pEB-YFP vector. We also generated two control vectors: pEB-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 methylation26. In all constructs, the IEP and DNMT3L ORFs were fused to C-terminus of the YFP protein. Following the transfection of cultured human HeLa cells with these constructs, the YFP-IEP fusion protein was located in the nucleus but with some signal in the cytoplasm in 89.94% of the transfected cells. In 19.64% of the transfected cells, we also observed bright cytoplasmic foci (Fig. 1a). Remarkably, despite the localization of YFP-IEP to the nucleus, it appeared to be excluded from the nucleolus. This nucleolar exclusion was confirmed by colocalization experiments using the anti-fibrillarin antibody as a nucleolar marker (Fig. 1b). 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-IEP fusion, these proteins were also detected in the nucleolus (Fig. 1a). The cellular distribution of the IEP was unaffected in cells transfected with pEB-YFP-IEPΔORF, a construct that expresses the RmInt1 RNA together with the IEP, or with pEB-YFP-YYAAIEP, a construct encoding a mutated IEP with a modified maturase domain (residues Y354Y355 were replaced by alanine residues) (data not shown). Interestingly, the accumulation of the YFP-IEP fusion protein in the nucleus (74.5 kDa, thus exceeding the 40 kDa exclusion limit of the nuclear pore complex) suggests an active mechanism for translocation of the fusion protein through the nuclear membrane. Interestingly, LtrA, the protein encoded by the Ll.LtrB intron, requires the addition of a nuclear localization signal for translocation into the nucleus of human22 or yeast10 cells.


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

Reinoso-Colacio M, García-Rodríguez FM, García-Cañadas M, Amador-Cubero S, García Pérez JL, Toro N - Sci Rep (2015)

Subcellular distribution of the YFP-IEP RmInt1 fusion in HeLa cells.(a) Fluorescence and bright-field (BF) microscopy of transfected HeLa HA cells. Localization of the YFP-IEP fusion protein in yellow (YFP). DAPI (blue) was used to stain nuclear DNA, and merged images are shown in the right column. Schematic diagrams of the constructs are shown to the right of the micrographs. The domains of the IEP are indicated as follows: in pink, the reverse transcriptase; in green, the maturase and, in yellow, the C-terminal domain. (b) Colocalization experiments using an anti-fibrillarin antibody (red) as a nucleolar marker (Fibrillarin); The YFP-IEP fusion protein is shown in yellow (YFP); DAPI (blue) was used to stain nuclear DNA, and merged images are shown on the right. A diagram of the construct is shown to the right of the micrograph.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525487&req=5

f1: Subcellular distribution of the YFP-IEP RmInt1 fusion in HeLa cells.(a) Fluorescence and bright-field (BF) microscopy of transfected HeLa HA cells. Localization of the YFP-IEP fusion protein in yellow (YFP). DAPI (blue) was used to stain nuclear DNA, and merged images are shown in the right column. Schematic diagrams of the constructs are shown to the right of the micrographs. The domains of the IEP are indicated as follows: in pink, the reverse transcriptase; in green, the maturase and, in yellow, the C-terminal domain. (b) Colocalization experiments using an anti-fibrillarin antibody (red) as a nucleolar marker (Fibrillarin); The YFP-IEP fusion protein is shown in yellow (YFP); DAPI (blue) was used to stain nuclear DNA, and merged images are shown on the right. A diagram of the construct is shown to the right of the micrograph.
Mentions: We investigated the intracellular distribution of the RmInt1 protein (IEP), by fusing its coding sequence to that of the yellow fluorescent protein (YFP), under control of the human EF1 α promoter (Pol II), in the pEB-YFP vector. We also generated two control vectors: pEB-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 methylation26. In all constructs, the IEP and DNMT3L ORFs were fused to C-terminus of the YFP protein. Following the transfection of cultured human HeLa cells with these constructs, the YFP-IEP fusion protein was located in the nucleus but with some signal in the cytoplasm in 89.94% of the transfected cells. In 19.64% of the transfected cells, we also observed bright cytoplasmic foci (Fig. 1a). Remarkably, despite the localization of YFP-IEP to the nucleus, it appeared to be excluded from the nucleolus. This nucleolar exclusion was confirmed by colocalization experiments using the anti-fibrillarin antibody as a nucleolar marker (Fig. 1b). 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-IEP fusion, these proteins were also detected in the nucleolus (Fig. 1a). The cellular distribution of the IEP was unaffected in cells transfected with pEB-YFP-IEPΔORF, a construct that expresses the RmInt1 RNA together with the IEP, or with pEB-YFP-YYAAIEP, a construct encoding a mutated IEP with a modified maturase domain (residues Y354Y355 were replaced by alanine residues) (data not shown). Interestingly, the accumulation of the YFP-IEP fusion protein in the nucleus (74.5 kDa, thus exceeding the 40 kDa exclusion limit of the nuclear pore complex) suggests an active mechanism for translocation of the fusion protein through the nuclear membrane. Interestingly, LtrA, the protein encoded by the Ll.LtrB intron, requires the addition of a nuclear localization signal for translocation into the nucleus of human22 or yeast10 cells.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Grupo de Ecología Genética, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Calle Profesor Albareda 1, 18008 Granada, Spain.

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.

No MeSH data available.