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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

Nuclear retention of mutant β-globin transcripts in S2 cells.(A) The location of the β-globin RNAs studied by FISH. Expression of the wt and mut β-globin genes was induced with 400 µM CuSO4 for 24 h and the location of the β-globin transcripts was analyzed by FISH. The β-globin sequence of the pβΔRS plasmid was labeled with digoxigenin and used as a probe (green). The preparations were counterstained with DAPI (blue). When indicated, the cells were treated with actinomycin D before fixation to study the presence of transcripts at the transcription site in the absence of ongoing RNA synthesis. The magnification bar represents 5 µm. (B) Quantitative analysis of the FISH experiments. The frequency of cells with an intensely fluorescent spot in the nucleus was counted in control cells (Act-, dark bars) and in cells treated with actinomycin D (Act+, light bars). For each treatment, 98 cells were analyzed. The experiment was carried out three independent times, with a total of 294 cells analyzed. To show the changes induced by actinomycin D, the results are expressed as average percentage relative to the frequencies obtained in non-treated cells. The error bars represent standard deviations of the mean (n = 3). Comparisons of wt to mut treated with actinomycin D using a paired, one-tailed Student's t-test gave p = 0.02, n = 3. (C) Depletion of Rrp6 by RNAi in S2 cells. S2 cells were treated with either Rrp6-dsRNA or control GFP-dsRNA. After 4 days or 7 days, as indicated, total RNA was purified and reverse-transcribed. The resulting cDNA was analyzed by PCR with primers specific for the rrp6 gene. The expressions of two unrelated genes, fur2 and tctp, were analyzed in parallel to assess the specificity of the treatment. Genomic DNA was analyzed in parallel. Equivalent amounts of cells expressing wt and mut transcripts were used for the analysis. (D) Depletion of Rrp6 restores the release of β-globin transcripts from the transcription site. The distribution of the β-globin transcripts was analyzed by FISH in cells treated with either Rrp6-dsRNA or GFP-dsRNA for 5 days. Induction, actinomycin treatment and FISH analysis were the same as those described for Figures 2A and 2B. The histogram shows the average proportion of cells with an intense fluorescent spot. Dark bars indicate data from non-treated control cells. Light bars correspond to cells treated with actinomycin D before fixation and FISH analysis. A total of 392 cells from two independent experiments, each in duplicate, was analyzed for each treatment. The error bars represent standard deviations (n = 4). Comparisons of cells expressing mut RNA and treated with Rrp6-dsRNA with those treated with GFP-dsRNA in the presence of actinomycin D using an unpaired, one-tailed Student's t-test gave p = 0.03, n = 4.
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pone-0011540-g002: Nuclear retention of mutant β-globin transcripts in S2 cells.(A) The location of the β-globin RNAs studied by FISH. Expression of the wt and mut β-globin genes was induced with 400 µM CuSO4 for 24 h and the location of the β-globin transcripts was analyzed by FISH. The β-globin sequence of the pβΔRS plasmid was labeled with digoxigenin and used as a probe (green). The preparations were counterstained with DAPI (blue). When indicated, the cells were treated with actinomycin D before fixation to study the presence of transcripts at the transcription site in the absence of ongoing RNA synthesis. The magnification bar represents 5 µm. (B) Quantitative analysis of the FISH experiments. The frequency of cells with an intensely fluorescent spot in the nucleus was counted in control cells (Act-, dark bars) and in cells treated with actinomycin D (Act+, light bars). For each treatment, 98 cells were analyzed. The experiment was carried out three independent times, with a total of 294 cells analyzed. To show the changes induced by actinomycin D, the results are expressed as average percentage relative to the frequencies obtained in non-treated cells. The error bars represent standard deviations of the mean (n = 3). Comparisons of wt to mut treated with actinomycin D using a paired, one-tailed Student's t-test gave p = 0.02, n = 3. (C) Depletion of Rrp6 by RNAi in S2 cells. S2 cells were treated with either Rrp6-dsRNA or control GFP-dsRNA. After 4 days or 7 days, as indicated, total RNA was purified and reverse-transcribed. The resulting cDNA was analyzed by PCR with primers specific for the rrp6 gene. The expressions of two unrelated genes, fur2 and tctp, were analyzed in parallel to assess the specificity of the treatment. Genomic DNA was analyzed in parallel. Equivalent amounts of cells expressing wt and mut transcripts were used for the analysis. (D) Depletion of Rrp6 restores the release of β-globin transcripts from the transcription site. The distribution of the β-globin transcripts was analyzed by FISH in cells treated with either Rrp6-dsRNA or GFP-dsRNA for 5 days. Induction, actinomycin treatment and FISH analysis were the same as those described for Figures 2A and 2B. The histogram shows the average proportion of cells with an intense fluorescent spot. Dark bars indicate data from non-treated control cells. Light bars correspond to cells treated with actinomycin D before fixation and FISH analysis. A total of 392 cells from two independent experiments, each in duplicate, was analyzed for each treatment. The error bars represent standard deviations (n = 4). Comparisons of cells expressing mut RNA and treated with Rrp6-dsRNA with those treated with GFP-dsRNA in the presence of actinomycin D using an unpaired, one-tailed Student's t-test gave p = 0.03, n = 4.

Mentions: We applied fluorescent in situ hybridization (FISH) to determine whether Drosophila cells have a mechanism for the retention of unspliced mRNAs at the transcription site. For this purpose, we induced the expression of the wt and mut β-globin genes and analyzed the cellular distribution of the β-globin RNAs by FISH (Figure 2). Intense fluorescence was observed in the cytoplasm of cells that expressed the wt gene, which indicated that the wt mRNA was exported to the cytoplasm, as expected. Moreover, a bright fluorescent spot was observed in the nucleus in more than 60% of the cells (Figure 2A, left panel). Previous FISH experiments carried out in mammalian cells have shown that the bright fluorescent spot coincides with the β-globin gene and is due to the presence of newly synthesized transcripts that have not left the transcription site [24]. Custodio and coworkers showed that the frequency of cells showing a bright fluorescent spot is drastically reduced when transcription is inhibited [24]. The interpretation of this reduction is that, in the absence of ongoing transcription, the already synthesized transcripts leave the gene and the transcription site becomes depleted of RNA. We observed the same effect in the S2 cells that expressed the wt β-globin RNA. The frequency of cells with a nuclear spot was reduced by a factor of almost 4 when transcription was inhibited by actinomycin D (Figure 2B). This result is consistent with the observations in mammalian cells [24] and with the rapid release and efficient export of the wt mRNA.


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)

Nuclear retention of mutant β-globin transcripts in S2 cells.(A) The location of the β-globin RNAs studied by FISH. Expression of the wt and mut β-globin genes was induced with 400 µM CuSO4 for 24 h and the location of the β-globin transcripts was analyzed by FISH. The β-globin sequence of the pβΔRS plasmid was labeled with digoxigenin and used as a probe (green). The preparations were counterstained with DAPI (blue). When indicated, the cells were treated with actinomycin D before fixation to study the presence of transcripts at the transcription site in the absence of ongoing RNA synthesis. The magnification bar represents 5 µm. (B) Quantitative analysis of the FISH experiments. The frequency of cells with an intensely fluorescent spot in the nucleus was counted in control cells (Act-, dark bars) and in cells treated with actinomycin D (Act+, light bars). For each treatment, 98 cells were analyzed. The experiment was carried out three independent times, with a total of 294 cells analyzed. To show the changes induced by actinomycin D, the results are expressed as average percentage relative to the frequencies obtained in non-treated cells. The error bars represent standard deviations of the mean (n = 3). Comparisons of wt to mut treated with actinomycin D using a paired, one-tailed Student's t-test gave p = 0.02, n = 3. (C) Depletion of Rrp6 by RNAi in S2 cells. S2 cells were treated with either Rrp6-dsRNA or control GFP-dsRNA. After 4 days or 7 days, as indicated, total RNA was purified and reverse-transcribed. The resulting cDNA was analyzed by PCR with primers specific for the rrp6 gene. The expressions of two unrelated genes, fur2 and tctp, were analyzed in parallel to assess the specificity of the treatment. Genomic DNA was analyzed in parallel. Equivalent amounts of cells expressing wt and mut transcripts were used for the analysis. (D) Depletion of Rrp6 restores the release of β-globin transcripts from the transcription site. The distribution of the β-globin transcripts was analyzed by FISH in cells treated with either Rrp6-dsRNA or GFP-dsRNA for 5 days. Induction, actinomycin treatment and FISH analysis were the same as those described for Figures 2A and 2B. The histogram shows the average proportion of cells with an intense fluorescent spot. Dark bars indicate data from non-treated control cells. Light bars correspond to cells treated with actinomycin D before fixation and FISH analysis. A total of 392 cells from two independent experiments, each in duplicate, was analyzed for each treatment. The error bars represent standard deviations (n = 4). Comparisons of cells expressing mut RNA and treated with Rrp6-dsRNA with those treated with GFP-dsRNA in the presence of actinomycin D using an unpaired, one-tailed Student's t-test gave p = 0.03, n = 4.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2902512&req=5

pone-0011540-g002: Nuclear retention of mutant β-globin transcripts in S2 cells.(A) The location of the β-globin RNAs studied by FISH. Expression of the wt and mut β-globin genes was induced with 400 µM CuSO4 for 24 h and the location of the β-globin transcripts was analyzed by FISH. The β-globin sequence of the pβΔRS plasmid was labeled with digoxigenin and used as a probe (green). The preparations were counterstained with DAPI (blue). When indicated, the cells were treated with actinomycin D before fixation to study the presence of transcripts at the transcription site in the absence of ongoing RNA synthesis. The magnification bar represents 5 µm. (B) Quantitative analysis of the FISH experiments. The frequency of cells with an intensely fluorescent spot in the nucleus was counted in control cells (Act-, dark bars) and in cells treated with actinomycin D (Act+, light bars). For each treatment, 98 cells were analyzed. The experiment was carried out three independent times, with a total of 294 cells analyzed. To show the changes induced by actinomycin D, the results are expressed as average percentage relative to the frequencies obtained in non-treated cells. The error bars represent standard deviations of the mean (n = 3). Comparisons of wt to mut treated with actinomycin D using a paired, one-tailed Student's t-test gave p = 0.02, n = 3. (C) Depletion of Rrp6 by RNAi in S2 cells. S2 cells were treated with either Rrp6-dsRNA or control GFP-dsRNA. After 4 days or 7 days, as indicated, total RNA was purified and reverse-transcribed. The resulting cDNA was analyzed by PCR with primers specific for the rrp6 gene. The expressions of two unrelated genes, fur2 and tctp, were analyzed in parallel to assess the specificity of the treatment. Genomic DNA was analyzed in parallel. Equivalent amounts of cells expressing wt and mut transcripts were used for the analysis. (D) Depletion of Rrp6 restores the release of β-globin transcripts from the transcription site. The distribution of the β-globin transcripts was analyzed by FISH in cells treated with either Rrp6-dsRNA or GFP-dsRNA for 5 days. Induction, actinomycin treatment and FISH analysis were the same as those described for Figures 2A and 2B. The histogram shows the average proportion of cells with an intense fluorescent spot. Dark bars indicate data from non-treated control cells. Light bars correspond to cells treated with actinomycin D before fixation and FISH analysis. A total of 392 cells from two independent experiments, each in duplicate, was analyzed for each treatment. The error bars represent standard deviations (n = 4). Comparisons of cells expressing mut RNA and treated with Rrp6-dsRNA with those treated with GFP-dsRNA in the presence of actinomycin D using an unpaired, one-tailed Student's t-test gave p = 0.03, n = 4.
Mentions: We applied fluorescent in situ hybridization (FISH) to determine whether Drosophila cells have a mechanism for the retention of unspliced mRNAs at the transcription site. For this purpose, we induced the expression of the wt and mut β-globin genes and analyzed the cellular distribution of the β-globin RNAs by FISH (Figure 2). Intense fluorescence was observed in the cytoplasm of cells that expressed the wt gene, which indicated that the wt mRNA was exported to the cytoplasm, as expected. Moreover, a bright fluorescent spot was observed in the nucleus in more than 60% of the cells (Figure 2A, left panel). Previous FISH experiments carried out in mammalian cells have shown that the bright fluorescent spot coincides with the β-globin gene and is due to the presence of newly synthesized transcripts that have not left the transcription site [24]. Custodio and coworkers showed that the frequency of cells showing a bright fluorescent spot is drastically reduced when transcription is inhibited [24]. The interpretation of this reduction is that, in the absence of ongoing transcription, the already synthesized transcripts leave the gene and the transcription site becomes depleted of RNA. We observed the same effect in the S2 cells that expressed the wt β-globin RNA. The frequency of cells with a nuclear spot was reduced by a factor of almost 4 when transcription was inhibited by actinomycin D (Figure 2B). This result is consistent with the observations in mammalian cells [24] and with the rapid release and efficient export of the wt mRNA.

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