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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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Cleavage and polyadenylation of the β-globin transcripts.(A) Detection of the downstream cleavage product (3′ fragment). The expression of Rat1 was silenced by RNAi in S2 cells expressing either mut or wt β-globin. Control cells were treated in parallel with GFP-dsRNA. Total RNA was purified and reverse-transcribed from dsRNA-treated cells, and the resulting cDNAs were quantified by qPCR with primers specific for the pre-mRNA, the mRNA (see Figure 4A) and the 3′ fragment (see the schematic picture above the histogram). The histogram shows the average values and standard deviations of β-globin levels, normalized to actin 5C RNA, from three independent experiments with two qPCR runs each (n = 6). The relative accumulation of downstream product was less pronounced in cells that expressed the mut β-globin gene than in cells that expressed the wt gene (p<0.01 using a paired, two-tailed Student's t-test, n = 6). (B) PCR-based detection of polyadenylated β-globin transcripts. Total RNA was purified from nuclear preparations of S2 cells expressing either mut or wt β-globin genes. The resulting cDNAs were used to amplify polyadenylated β-globin sequences by PCR with a 26 nt-long downstream primer complementary to the beginning of the poly(A) tail, as indicated in the figure (lanes 1–4). Control reactions with a 16 nt-long primer lacking the oligo(dT) extension were processed in parallel to rule out poly(A)-independent priming (lanes 5–8). Contamination with genomic DNA was assessed in parallel reactions without reverse transcriptase (RT-). (C) RT-qPCR quantification of polyadenylated β-globin transcripts. The samples described above were analyzed by RT-qPCR. Relative levels of polyadenylated β-globin transcripts, normalized to the total β-globin RNA, are shown. Values and standard deviations represent the average from two independent experiments with two qPCR runs each (n = 4). No significant differences were observed using a paired, two-tailed Student's t-test (p = 0.12, n = 4).
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pone-0011540-g003: Cleavage and polyadenylation of the β-globin transcripts.(A) Detection of the downstream cleavage product (3′ fragment). The expression of Rat1 was silenced by RNAi in S2 cells expressing either mut or wt β-globin. Control cells were treated in parallel with GFP-dsRNA. Total RNA was purified and reverse-transcribed from dsRNA-treated cells, and the resulting cDNAs were quantified by qPCR with primers specific for the pre-mRNA, the mRNA (see Figure 4A) and the 3′ fragment (see the schematic picture above the histogram). The histogram shows the average values and standard deviations of β-globin levels, normalized to actin 5C RNA, from three independent experiments with two qPCR runs each (n = 6). The relative accumulation of downstream product was less pronounced in cells that expressed the mut β-globin gene than in cells that expressed the wt gene (p<0.01 using a paired, two-tailed Student's t-test, n = 6). (B) PCR-based detection of polyadenylated β-globin transcripts. Total RNA was purified from nuclear preparations of S2 cells expressing either mut or wt β-globin genes. The resulting cDNAs were used to amplify polyadenylated β-globin sequences by PCR with a 26 nt-long downstream primer complementary to the beginning of the poly(A) tail, as indicated in the figure (lanes 1–4). Control reactions with a 16 nt-long primer lacking the oligo(dT) extension were processed in parallel to rule out poly(A)-independent priming (lanes 5–8). Contamination with genomic DNA was assessed in parallel reactions without reverse transcriptase (RT-). (C) RT-qPCR quantification of polyadenylated β-globin transcripts. The samples described above were analyzed by RT-qPCR. Relative levels of polyadenylated β-globin transcripts, normalized to the total β-globin RNA, are shown. Values and standard deviations represent the average from two independent experiments with two qPCR runs each (n = 4). No significant differences were observed using a paired, two-tailed Student's t-test (p = 0.12, n = 4).

Mentions: In a first series of experiments, we used RNA interference (RNAi) to knock down the expression of Rat1 in S2 cells that expressed either the wt or the mut β-globin genes, and we analyzed the levels of the downstream cleavage product by quantitative RT-PCR (RT-qPCR). The decreased expression of Rat1 was verified by RT-qPCR (Figure S3). The depletion of Rat1 increased the levels of the downstream product (3′ fragment in Figure 3A) for both wt and mut β-globin transcripts. However, the relative accumulation of downstream product was significantly less pronounced in cells that expressed the mut β-globin gene than in cells that expressed the wt gene (p<0.01). Altogether, these results indicated that both the wt and the mut β-globin transcripts are cleaved, but that the cleavage of the mut β-globin transcripts is less efficient.


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)

Cleavage and polyadenylation of the β-globin transcripts.(A) Detection of the downstream cleavage product (3′ fragment). The expression of Rat1 was silenced by RNAi in S2 cells expressing either mut or wt β-globin. Control cells were treated in parallel with GFP-dsRNA. Total RNA was purified and reverse-transcribed from dsRNA-treated cells, and the resulting cDNAs were quantified by qPCR with primers specific for the pre-mRNA, the mRNA (see Figure 4A) and the 3′ fragment (see the schematic picture above the histogram). The histogram shows the average values and standard deviations of β-globin levels, normalized to actin 5C RNA, from three independent experiments with two qPCR runs each (n = 6). The relative accumulation of downstream product was less pronounced in cells that expressed the mut β-globin gene than in cells that expressed the wt gene (p<0.01 using a paired, two-tailed Student's t-test, n = 6). (B) PCR-based detection of polyadenylated β-globin transcripts. Total RNA was purified from nuclear preparations of S2 cells expressing either mut or wt β-globin genes. The resulting cDNAs were used to amplify polyadenylated β-globin sequences by PCR with a 26 nt-long downstream primer complementary to the beginning of the poly(A) tail, as indicated in the figure (lanes 1–4). Control reactions with a 16 nt-long primer lacking the oligo(dT) extension were processed in parallel to rule out poly(A)-independent priming (lanes 5–8). Contamination with genomic DNA was assessed in parallel reactions without reverse transcriptase (RT-). (C) RT-qPCR quantification of polyadenylated β-globin transcripts. The samples described above were analyzed by RT-qPCR. Relative levels of polyadenylated β-globin transcripts, normalized to the total β-globin RNA, are shown. Values and standard deviations represent the average from two independent experiments with two qPCR runs each (n = 4). No significant differences were observed using a paired, two-tailed Student's t-test (p = 0.12, n = 4).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0011540-g003: Cleavage and polyadenylation of the β-globin transcripts.(A) Detection of the downstream cleavage product (3′ fragment). The expression of Rat1 was silenced by RNAi in S2 cells expressing either mut or wt β-globin. Control cells were treated in parallel with GFP-dsRNA. Total RNA was purified and reverse-transcribed from dsRNA-treated cells, and the resulting cDNAs were quantified by qPCR with primers specific for the pre-mRNA, the mRNA (see Figure 4A) and the 3′ fragment (see the schematic picture above the histogram). The histogram shows the average values and standard deviations of β-globin levels, normalized to actin 5C RNA, from three independent experiments with two qPCR runs each (n = 6). The relative accumulation of downstream product was less pronounced in cells that expressed the mut β-globin gene than in cells that expressed the wt gene (p<0.01 using a paired, two-tailed Student's t-test, n = 6). (B) PCR-based detection of polyadenylated β-globin transcripts. Total RNA was purified from nuclear preparations of S2 cells expressing either mut or wt β-globin genes. The resulting cDNAs were used to amplify polyadenylated β-globin sequences by PCR with a 26 nt-long downstream primer complementary to the beginning of the poly(A) tail, as indicated in the figure (lanes 1–4). Control reactions with a 16 nt-long primer lacking the oligo(dT) extension were processed in parallel to rule out poly(A)-independent priming (lanes 5–8). Contamination with genomic DNA was assessed in parallel reactions without reverse transcriptase (RT-). (C) RT-qPCR quantification of polyadenylated β-globin transcripts. The samples described above were analyzed by RT-qPCR. Relative levels of polyadenylated β-globin transcripts, normalized to the total β-globin RNA, are shown. Values and standard deviations represent the average from two independent experiments with two qPCR runs each (n = 4). No significant differences were observed using a paired, two-tailed Student's t-test (p = 0.12, n = 4).
Mentions: In a first series of experiments, we used RNA interference (RNAi) to knock down the expression of Rat1 in S2 cells that expressed either the wt or the mut β-globin genes, and we analyzed the levels of the downstream cleavage product by quantitative RT-PCR (RT-qPCR). The decreased expression of Rat1 was verified by RT-qPCR (Figure S3). The depletion of Rat1 increased the levels of the downstream product (3′ fragment in Figure 3A) for both wt and mut β-globin transcripts. However, the relative accumulation of downstream product was significantly less pronounced in cells that expressed the mut β-globin gene than in cells that expressed the wt gene (p<0.01). Altogether, these results indicated that both the wt and the mut β-globin transcripts are cleaved, but that the cleavage of the mut β-globin transcripts is less efficient.

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