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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.


Identification of the spliced form of RmInt1 in FLAG-immunoprecipitate from transfected HeLa cell.(a) Representation of the PCR products obtained from the spliced intron as a circle or lariat. The circled A is the bulged adenosine residue in domain VI. (b) RT-PCR products were subjected to electrophoresis in a 2% agarose gel. PCR was performed with (+) or without (−) prior reverse transcription (RT), with the immunoprecipitates indicated. The area in which the DNA fragments were isolated is indicated by a square bracket. Other unspecific PCR products likely derived from contaminant genomic DNA was also observed. M: molecular weight marker; the numbers on the right indicate the size of the marker band in base pairs.
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f4: Identification of the spliced form of RmInt1 in FLAG-immunoprecipitate from transfected HeLa cell.(a) Representation of the PCR products obtained from the spliced intron as a circle or lariat. The circled A is the bulged adenosine residue in domain VI. (b) RT-PCR products were subjected to electrophoresis in a 2% agarose gel. PCR was performed with (+) or without (−) prior reverse transcription (RT), with the immunoprecipitates indicated. The area in which the DNA fragments were isolated is indicated by a square bracket. Other unspecific PCR products likely derived from contaminant genomic DNA was also observed. M: molecular weight marker; the numbers on the right indicate the size of the marker band in base pairs.

Mentions: For confirmation of the physical association of the RmInt1 IEP with the spliced RNA of the RmInt1 intron, HeLa cells were transfected with pCEP4 or cotransfected with pCEP4flagIEP plus pCEP4ΔORF, or pCEP4flagIEP plus pCEP4dV (catalytic triad GTT is replaced by CGA in domain V of the RmInt1 ribozyme). Cell extracts were obtained and subjected to immunoprecipitation with the anti-FLAG M2 antibody fused to agarose beads, followed by western blotting, which demonstrated the presence of IEP in all samples, except those harboring pCEP4 alone (data not shown). The immunoprecipitated fractions were then used as a template for RT-PCR experiments, for detection of the spliced intron RNA. Amplification products were subjected to electrophoresis in 2% agarose gels (Fig. 4). Various bands were detected on the gel, and those of the expected size were isolated, cloned and sequenced. Only the immunoprecipitated material from samples cotransfected with pCEP4flagIEP plus pCEP4ΔORF contained the lariat (two of six clones) and circular (one of six clones) forms of the intron RNA. These data suggest that the IEP and intron RNA are associated and may be able to form a mature RNP.


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)

Identification of the spliced form of RmInt1 in FLAG-immunoprecipitate from transfected HeLa cell.(a) Representation of the PCR products obtained from the spliced intron as a circle or lariat. The circled A is the bulged adenosine residue in domain VI. (b) RT-PCR products were subjected to electrophoresis in a 2% agarose gel. PCR was performed with (+) or without (−) prior reverse transcription (RT), with the immunoprecipitates indicated. The area in which the DNA fragments were isolated is indicated by a square bracket. Other unspecific PCR products likely derived from contaminant genomic DNA was also observed. M: molecular weight marker; the numbers on the right indicate the size of the marker band in base pairs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Identification of the spliced form of RmInt1 in FLAG-immunoprecipitate from transfected HeLa cell.(a) Representation of the PCR products obtained from the spliced intron as a circle or lariat. The circled A is the bulged adenosine residue in domain VI. (b) RT-PCR products were subjected to electrophoresis in a 2% agarose gel. PCR was performed with (+) or without (−) prior reverse transcription (RT), with the immunoprecipitates indicated. The area in which the DNA fragments were isolated is indicated by a square bracket. Other unspecific PCR products likely derived from contaminant genomic DNA was also observed. M: molecular weight marker; the numbers on the right indicate the size of the marker band in base pairs.
Mentions: For confirmation of the physical association of the RmInt1 IEP with the spliced RNA of the RmInt1 intron, HeLa cells were transfected with pCEP4 or cotransfected with pCEP4flagIEP plus pCEP4ΔORF, or pCEP4flagIEP plus pCEP4dV (catalytic triad GTT is replaced by CGA in domain V of the RmInt1 ribozyme). Cell extracts were obtained and subjected to immunoprecipitation with the anti-FLAG M2 antibody fused to agarose beads, followed by western blotting, which demonstrated the presence of IEP in all samples, except those harboring pCEP4 alone (data not shown). The immunoprecipitated fractions were then used as a template for RT-PCR experiments, for detection of the spliced intron RNA. Amplification products were subjected to electrophoresis in 2% agarose gels (Fig. 4). Various bands were detected on the gel, and those of the expected size were isolated, cloned and sequenced. Only the immunoprecipitated material from samples cotransfected with pCEP4flagIEP plus pCEP4ΔORF contained the lariat (two of six clones) and circular (one of six clones) forms of the intron RNA. These data suggest that the IEP and intron RNA are associated and may be able to form a mature RNP.

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.