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Splice-site mutations cause Rrp6-mediated nuclear retention of the unspliced RNAs and transcriptional down-regulation of the splicing-defective genes.

Eberle AB, Hessle V, Helbig R, Dantoft W, Gimber N, Visa N - PLoS ONE (2010)

Bottom Line: We have also shown that the mut beta-globin gene shows reduced levels of H3K4me3.One response requires Rrp6 and results in the inefficient release of defective mRNAs from the transcription site.The other response acts at the transcription level and reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Functional Genomics, Stockholm University, Stockholm, Sweden.

ABSTRACT

Background: Eukaryotic cells have developed surveillance mechanisms to prevent the expression of aberrant transcripts. An early surveillance checkpoint acts at the transcription site and prevents the release of mRNAs that carry processing defects. The exosome subunit Rrp6 is required for this checkpoint in Saccharomyces cerevisiae, but it is not known whether Rrp6 also plays a role in mRNA surveillance in higher eukaryotes.

Methodology/principal findings: We have developed an in vivo system to study nuclear mRNA surveillance in Drosophila melanogaster. We have produced S2 cells that express a human beta-globin gene with mutated splice sites in intron 2 (mut beta-globin). The transcripts encoded by the mut beta-globin gene are normally spliced at intron 1 but retain intron 2. The levels of the mut beta-globin transcripts are much lower than those of wild type (wt) ss-globin mRNAs transcribed from the same promoter. We have compared the expression of the mut and wt beta-globin genes to investigate the mechanisms that down-regulate the production of defective mRNAs. Both wt and mut beta-globin transcripts are processed at the 3', but the mut beta-globin transcripts are less efficiently cleaved than the wt transcripts. Moreover, the mut beta-globin transcripts are less efficiently released from the transcription site, as shown by FISH, and this defect is restored by depletion of Rrp6 by RNAi. Furthermore, transcription of the mut beta-globin gene is significantly impaired as revealed by ChIP experiments that measure the association of the RNA polymerase II with the transcribed genes. We have also shown that the mut beta-globin gene shows reduced levels of H3K4me3.

Conclusions/significance: Our results show that there are at least two surveillance responses that operate cotranscriptionally in insect cells and probably in all metazoans. One response requires Rrp6 and results in the inefficient release of defective mRNAs from the transcription site. The other response acts at the transcription level and reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications.

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Related in: MedlinePlus

The human β-globin genes expressed in Drosophila S2 cells.(A) Schematic representation of the β-globin transcripts expressed in S2 cells. The grey boxes represent the coding β-globin sequences. The white boxes indicate the 5′ UTR and 3′ UTR of the pMT vector (see Materials and Methods S1 for details). The box marked V5 indicates the position of the V5 tag. The mut RNA carries a shorter intron 2 and mutations in both the 5′ and 3′ splice sites of intron 2, as indicated in the figure. (B) Western blot analysis of the expression of the wt and mut β-globin genes. The expression of the β-globin genes was induced with 500 µM CuSO4 for 24 h and analyzed by Western blotting using the anti-V5 antibody. Protein expression was detected only from the wt construct. As a loading reference, a section of the PVDF filter containing proteins in the 50–90 kDa range was stained for total protein with Coomassie blue. (C) The expression of the β-globin genes analyzed by immunofluorescence. Expression of the wt and mut β-globin genes was induced as described above and the cells were stained with the anti-V5 antibody (red) to visualize β-globin expression. DAPI counterstaining was used to visualize the nuclei (blue). Exposure times were the same for all images. The magnification bar represents 5 µm. (D) The β-globin transcripts analyzed by RT-PCR. The expression of the wt and mut β-globin genes was induced as described above. Total RNA was purified and reverse-transcribed, and the β-globin sequences were amplified by PCR primers flanking intron 2, as indicated in the figure (lanes 2 and 4). The genomic DNA isolated from β-globin wt or mut S2 cells was used in parallel to check the splicing pattern (gDNA, lanes 1 and 3). Molecular mass standards are shown (M) and the length of the major bands is indicated in bp.
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pone-0011540-g001: The human β-globin genes expressed in Drosophila S2 cells.(A) Schematic representation of the β-globin transcripts expressed in S2 cells. The grey boxes represent the coding β-globin sequences. The white boxes indicate the 5′ UTR and 3′ UTR of the pMT vector (see Materials and Methods S1 for details). The box marked V5 indicates the position of the V5 tag. The mut RNA carries a shorter intron 2 and mutations in both the 5′ and 3′ splice sites of intron 2, as indicated in the figure. (B) Western blot analysis of the expression of the wt and mut β-globin genes. The expression of the β-globin genes was induced with 500 µM CuSO4 for 24 h and analyzed by Western blotting using the anti-V5 antibody. Protein expression was detected only from the wt construct. As a loading reference, a section of the PVDF filter containing proteins in the 50–90 kDa range was stained for total protein with Coomassie blue. (C) The expression of the β-globin genes analyzed by immunofluorescence. Expression of the wt and mut β-globin genes was induced as described above and the cells were stained with the anti-V5 antibody (red) to visualize β-globin expression. DAPI counterstaining was used to visualize the nuclei (blue). Exposure times were the same for all images. The magnification bar represents 5 µm. (D) The β-globin transcripts analyzed by RT-PCR. The expression of the wt and mut β-globin genes was induced as described above. Total RNA was purified and reverse-transcribed, and the β-globin sequences were amplified by PCR primers flanking intron 2, as indicated in the figure (lanes 2 and 4). The genomic DNA isolated from β-globin wt or mut S2 cells was used in parallel to check the splicing pattern (gDNA, lanes 1 and 3). Molecular mass standards are shown (M) and the length of the major bands is indicated in bp.

Mentions: Wild-type (wt) and mutant (mut) versions of the human β-globin gene have been used in previous studies as reporter genes to analyze the surveillance mechanisms that operate during transcription. These versions of the gene have been used, in particular, to study the retention of unprocessed pre-mRNAs at the transcription site in mammalian cells [24], [27]. We have applied the same approach to set up a Drosophila cell system to study nuclear retention of unspliced transcripts. For this purpose, we subcloned the wt β-globin gene and a mut version of it into a vector that was suitable for expression in Drosophila melanogaster S2 cells (see Materials and Methods S1). The wt and the mut sequences were tagged with a V5 epitope to facilitate expression analyses. The mut RNA carries a GT to AC mutation in the 5′ splice site and an AG to CT mutation in the 3′ splice site of the second intron (Figure 1A). These mutations impair removal of the second intron and, in human cells, cause retention of the RNA at the transcription site [24].


Splice-site mutations cause Rrp6-mediated nuclear retention of the unspliced RNAs and transcriptional down-regulation of the splicing-defective genes.

Eberle AB, Hessle V, Helbig R, Dantoft W, Gimber N, Visa N - PLoS ONE (2010)

The human β-globin genes expressed in Drosophila S2 cells.(A) Schematic representation of the β-globin transcripts expressed in S2 cells. The grey boxes represent the coding β-globin sequences. The white boxes indicate the 5′ UTR and 3′ UTR of the pMT vector (see Materials and Methods S1 for details). The box marked V5 indicates the position of the V5 tag. The mut RNA carries a shorter intron 2 and mutations in both the 5′ and 3′ splice sites of intron 2, as indicated in the figure. (B) Western blot analysis of the expression of the wt and mut β-globin genes. The expression of the β-globin genes was induced with 500 µM CuSO4 for 24 h and analyzed by Western blotting using the anti-V5 antibody. Protein expression was detected only from the wt construct. As a loading reference, a section of the PVDF filter containing proteins in the 50–90 kDa range was stained for total protein with Coomassie blue. (C) The expression of the β-globin genes analyzed by immunofluorescence. Expression of the wt and mut β-globin genes was induced as described above and the cells were stained with the anti-V5 antibody (red) to visualize β-globin expression. DAPI counterstaining was used to visualize the nuclei (blue). Exposure times were the same for all images. The magnification bar represents 5 µm. (D) The β-globin transcripts analyzed by RT-PCR. The expression of the wt and mut β-globin genes was induced as described above. Total RNA was purified and reverse-transcribed, and the β-globin sequences were amplified by PCR primers flanking intron 2, as indicated in the figure (lanes 2 and 4). The genomic DNA isolated from β-globin wt or mut S2 cells was used in parallel to check the splicing pattern (gDNA, lanes 1 and 3). Molecular mass standards are shown (M) and the length of the major bands is indicated in bp.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2902512&req=5

pone-0011540-g001: The human β-globin genes expressed in Drosophila S2 cells.(A) Schematic representation of the β-globin transcripts expressed in S2 cells. The grey boxes represent the coding β-globin sequences. The white boxes indicate the 5′ UTR and 3′ UTR of the pMT vector (see Materials and Methods S1 for details). The box marked V5 indicates the position of the V5 tag. The mut RNA carries a shorter intron 2 and mutations in both the 5′ and 3′ splice sites of intron 2, as indicated in the figure. (B) Western blot analysis of the expression of the wt and mut β-globin genes. The expression of the β-globin genes was induced with 500 µM CuSO4 for 24 h and analyzed by Western blotting using the anti-V5 antibody. Protein expression was detected only from the wt construct. As a loading reference, a section of the PVDF filter containing proteins in the 50–90 kDa range was stained for total protein with Coomassie blue. (C) The expression of the β-globin genes analyzed by immunofluorescence. Expression of the wt and mut β-globin genes was induced as described above and the cells were stained with the anti-V5 antibody (red) to visualize β-globin expression. DAPI counterstaining was used to visualize the nuclei (blue). Exposure times were the same for all images. The magnification bar represents 5 µm. (D) The β-globin transcripts analyzed by RT-PCR. The expression of the wt and mut β-globin genes was induced as described above. Total RNA was purified and reverse-transcribed, and the β-globin sequences were amplified by PCR primers flanking intron 2, as indicated in the figure (lanes 2 and 4). The genomic DNA isolated from β-globin wt or mut S2 cells was used in parallel to check the splicing pattern (gDNA, lanes 1 and 3). Molecular mass standards are shown (M) and the length of the major bands is indicated in bp.
Mentions: Wild-type (wt) and mutant (mut) versions of the human β-globin gene have been used in previous studies as reporter genes to analyze the surveillance mechanisms that operate during transcription. These versions of the gene have been used, in particular, to study the retention of unprocessed pre-mRNAs at the transcription site in mammalian cells [24], [27]. We have applied the same approach to set up a Drosophila cell system to study nuclear retention of unspliced transcripts. For this purpose, we subcloned the wt β-globin gene and a mut version of it into a vector that was suitable for expression in Drosophila melanogaster S2 cells (see Materials and Methods S1). The wt and the mut sequences were tagged with a V5 epitope to facilitate expression analyses. The mut RNA carries a GT to AC mutation in the 5′ splice site and an AG to CT mutation in the 3′ splice site of the second intron (Figure 1A). These mutations impair removal of the second intron and, in human cells, cause retention of the RNA at the transcription site [24].

Bottom Line: We have also shown that the mut beta-globin gene shows reduced levels of H3K4me3.One response requires Rrp6 and results in the inefficient release of defective mRNAs from the transcription site.The other response acts at the transcription level and reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Functional Genomics, Stockholm University, Stockholm, Sweden.

ABSTRACT

Background: Eukaryotic cells have developed surveillance mechanisms to prevent the expression of aberrant transcripts. An early surveillance checkpoint acts at the transcription site and prevents the release of mRNAs that carry processing defects. The exosome subunit Rrp6 is required for this checkpoint in Saccharomyces cerevisiae, but it is not known whether Rrp6 also plays a role in mRNA surveillance in higher eukaryotes.

Methodology/principal findings: We have developed an in vivo system to study nuclear mRNA surveillance in Drosophila melanogaster. We have produced S2 cells that express a human beta-globin gene with mutated splice sites in intron 2 (mut beta-globin). The transcripts encoded by the mut beta-globin gene are normally spliced at intron 1 but retain intron 2. The levels of the mut beta-globin transcripts are much lower than those of wild type (wt) ss-globin mRNAs transcribed from the same promoter. We have compared the expression of the mut and wt beta-globin genes to investigate the mechanisms that down-regulate the production of defective mRNAs. Both wt and mut beta-globin transcripts are processed at the 3', but the mut beta-globin transcripts are less efficiently cleaved than the wt transcripts. Moreover, the mut beta-globin transcripts are less efficiently released from the transcription site, as shown by FISH, and this defect is restored by depletion of Rrp6 by RNAi. Furthermore, transcription of the mut beta-globin gene is significantly impaired as revealed by ChIP experiments that measure the association of the RNA polymerase II with the transcribed genes. We have also shown that the mut beta-globin gene shows reduced levels of H3K4me3.

Conclusions/significance: Our results show that there are at least two surveillance responses that operate cotranscriptionally in insect cells and probably in all metazoans. One response requires Rrp6 and results in the inefficient release of defective mRNAs from the transcription site. The other response acts at the transcription level and reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications.

Show MeSH
Related in: MedlinePlus