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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.


Related in: MedlinePlus

Detection of RmInt1 intron RNA and IEP in HeLa cells.(a) Diagram of the plasmids used in this study: each plasmid is a derivative of the pCEP4 episomal vector. 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. The positions of the primers used for cDNA synthesis (PE1), and for PCR amplification (ε1 and PE1) are also shown. (b) Detection of ΔORF RmInt1 in vivo by reverse transcription and PCR. PCR was carried out with (+) and without (−) prior reverse transcription (RT), with RNA from HeLa cells harboring pCEP4flagIEPΔORF or pCEP4flagIEP plus pCEP4ΔORF. The RT-PCR products were subjected to electrophoresis in a 2% agarose gel. M; molecular weight marker. (c) IEP protein detection in HeLa cells by SDS-PAGE and western blotting. Whole-cell extract (input) or immunoprecipitated (elution) products from cells harboring pCEP4flagIEP.
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f3: Detection of RmInt1 intron RNA and IEP in HeLa cells.(a) Diagram of the plasmids used in this study: each plasmid is a derivative of the pCEP4 episomal vector. 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. The positions of the primers used for cDNA synthesis (PE1), and for PCR amplification (ε1 and PE1) are also shown. (b) Detection of ΔORF RmInt1 in vivo by reverse transcription and PCR. PCR was carried out with (+) and without (−) prior reverse transcription (RT), with RNA from HeLa cells harboring pCEP4flagIEPΔORF or pCEP4flagIEP plus pCEP4ΔORF. The RT-PCR products were subjected to electrophoresis in a 2% agarose gel. M; molecular weight marker. (c) IEP protein detection in HeLa cells by SDS-PAGE and western blotting. Whole-cell extract (input) or immunoprecipitated (elution) products from cells harboring pCEP4flagIEP.

Mentions: We studied the transcription of RmInt1 ΔORF (RmInt1 intron deleted from position 611 to 1,759), by transfecting HeLa cells with pCEP4flagIEPΔORF or cotransfecting them with pCEP4flagIEP and pCEP4ΔORF (Fig. 3a), and then subjecting the cells to selection on hygromycin for 10 days. RNA was then isolated and analyzed by RT-PCR (see Materials and Methods). The PCR products (Fig. 3b) were resolved by electrophoresis in 2% agarose gels. In both samples, amplification products of the expected size (~330 bp) were observed. These amplification products were not detected in samples to which no reverse transcriptase was added (RT-). For confirmation of the identity of the amplification products, the bands were excised from the gel, and the amplicons were cloned and analyzed by DNA sequencing. This analysis confirmed that the amplification products corresponded to the expected amplified fragments of the RmInt1 RNA. Thus, the RmInt1-derived intron ΔORF was expressed in the transfecting 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)

Detection of RmInt1 intron RNA and IEP in HeLa cells.(a) Diagram of the plasmids used in this study: each plasmid is a derivative of the pCEP4 episomal vector. 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. The positions of the primers used for cDNA synthesis (PE1), and for PCR amplification (ε1 and PE1) are also shown. (b) Detection of ΔORF RmInt1 in vivo by reverse transcription and PCR. PCR was carried out with (+) and without (−) prior reverse transcription (RT), with RNA from HeLa cells harboring pCEP4flagIEPΔORF or pCEP4flagIEP plus pCEP4ΔORF. The RT-PCR products were subjected to electrophoresis in a 2% agarose gel. M; molecular weight marker. (c) IEP protein detection in HeLa cells by SDS-PAGE and western blotting. Whole-cell extract (input) or immunoprecipitated (elution) products from cells harboring pCEP4flagIEP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Detection of RmInt1 intron RNA and IEP in HeLa cells.(a) Diagram of the plasmids used in this study: each plasmid is a derivative of the pCEP4 episomal vector. 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. The positions of the primers used for cDNA synthesis (PE1), and for PCR amplification (ε1 and PE1) are also shown. (b) Detection of ΔORF RmInt1 in vivo by reverse transcription and PCR. PCR was carried out with (+) and without (−) prior reverse transcription (RT), with RNA from HeLa cells harboring pCEP4flagIEPΔORF or pCEP4flagIEP plus pCEP4ΔORF. The RT-PCR products were subjected to electrophoresis in a 2% agarose gel. M; molecular weight marker. (c) IEP protein detection in HeLa cells by SDS-PAGE and western blotting. Whole-cell extract (input) or immunoprecipitated (elution) products from cells harboring pCEP4flagIEP.
Mentions: We studied the transcription of RmInt1 ΔORF (RmInt1 intron deleted from position 611 to 1,759), by transfecting HeLa cells with pCEP4flagIEPΔORF or cotransfecting them with pCEP4flagIEP and pCEP4ΔORF (Fig. 3a), and then subjecting the cells to selection on hygromycin for 10 days. RNA was then isolated and analyzed by RT-PCR (see Materials and Methods). The PCR products (Fig. 3b) were resolved by electrophoresis in 2% agarose gels. In both samples, amplification products of the expected size (~330 bp) were observed. These amplification products were not detected in samples to which no reverse transcriptase was added (RT-). For confirmation of the identity of the amplification products, the bands were excised from the gel, and the amplicons were cloned and analyzed by DNA sequencing. This analysis confirmed that the amplification products corresponded to the expected amplified fragments of the RmInt1 RNA. Thus, the RmInt1-derived intron ΔORF was expressed in the transfecting 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.


Related in: MedlinePlus