| 1. |
- Eberle, Andrea B., et al.
(författare)
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An Interaction between RRP6 and SU(VAR)3-9 Targets RRP6 to Heterochromatin and Contributes to Heterochromatin Maintenance in Drosophila melanogaster
- 2015
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Ingår i: PLOS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 11:9
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Tidskriftsartikel (refereegranskat)abstract
- RNA surveillance factors are involved in heterochromatin regulation in yeast and plants, but less is known about the possible roles of ribonucleases in the heterochromatin of animal cells. Here we show that RRP6, one of the catalytic subunits of the exosome, is necessary for silencing heterochromatic repeats in the genome of Drosophila melanogaster. We show that a fraction of RRP6 is associated with heterochromatin, and the analysis of the RRP6 interaction network revealed physical links between RRP6 and the heterochromatin factors HP1a, SU(VAR)3-9 and RPD3. Moreover, genome-wide studies of RRP6 occupancy in cells depleted of SU(VAR)3-9 demonstrated that SU(VAR)3-9 contributes to the tethering of RRP6 to a subset of heterochromatic loci. Depletion of the exosome ribonucleases RRP6 and DIS3 stabilizes heterochromatic transcripts derived from transposons and repetitive sequences, and renders the heterochromatin less compact, as shown by micrococcal nuclease and proximity-ligation assays. Such depletion also increases the amount of HP1a bound to heterochromatic transcripts. Taken together, our results suggest that SU(VAR)3-9 targets RRP6 to a subset of heterochromatic loci where RRP6 degrades chromatin-associated non-coding RNAs in a process that is necessary to maintain the packaging of the heterochromatin.
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| 2. |
- Eberle, Andrea B., et al.
(författare)
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Quality control of mRNP biogenesis : Networking at the transcription site
- 2014
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Ingår i: Seminars in Cell and Developmental Biology. - : Elsevier BV. - 1084-9521 .- 1096-3634. ; 32, s. 37-46
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Forskningsöversikt (refereegranskat)abstract
- Eukaryotic cells carry out quality control (QC) over the processes of RNA biogenesis to inactivate or eliminate defective transcripts, and to avoid their production. In the case of protein-coding transcripts, the quality controls can sense defects in the assembly of mRNA-protein complexes, in the processing of the precursor mRNAs, and in the sequence of open reading frames. Different types of defect are monitored by different specialized mechanisms. Some of them involve dedicated factors whose function is to identify faulty molecules and target them for degradation. Others are the result of a more subtle balance in the kinetics of opposing activities in the mRNA biogenesis pathway. One way or another, all such mechanisms hinder the expression of the defective mRNAs through processes as diverse as rapid degradation, nuclear retention and transcriptional silencing. Three major degradation systems are responsible for the destruction of the defective transcripts: the exosome, the 5'-3' exoribonucleases, and the nonsense-mediated mRNA decay (NMD) machinery. This review summarizes recent findings on the cotranscriptional quality control of mRNA biogenesis, and speculates that a protein-protein interaction network integrates multiple mRNA degradation systems with the transcription machinery.
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| 3. |
- Eberle, Andrea B., et al.
(författare)
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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
- 2010
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:7, s. e11540-
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Tidskriftsartikel (refereegranskat)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 β-globin gene with mutated splice sites in intron 2 (mut β-globin). The transcripts encoded by the mut β-globin gene are normally spliced at intron 1 but retain intron 2. The levels of the mut β-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 β-globin genes to investigate the mechanisms that down-regulate the production of defective mRNAs. Both wt and mut β-globin transcripts are processed at the 3′, but the mut β-globin transcripts are less efficiently cleaved than the wt transcripts. Moreover, the mut β-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 β-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 β-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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| 4. |
- Eberle, Andrea B., et al.
(författare)
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The use of a synthetic DNA-antibody complex as external reference for chromatin immunoprecipitation
- 2012
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 426:2, s. 147-152
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Tidskriftsartikel (refereegranskat)abstract
- Chromatin immunoprecipitation (ChIP) is an analytical method used to investigate the interactions between proteins and DNA in vivo. ChIP is often used as a quantitative tool, and proper quantification relies on the use of adequate references for data normalization. However, many ChIP experiments involve analyses of samples that have been submitted to experimental treatments with unknown effects, and this precludes the choice of suitable internal references. We have developed a normalization method based on the use of a synthetic DNA-antibody complex that can be used as an external reference instead. A fixed amount of this synthetic DNA-antibody complex is spiked into the chromatin extract at the beginning of the ChIP experiment. The DNA-antibody complex is isolated together with the sample of interest, and the amounts of synthetic DNA recovered in each tube are measured at the end of the process. The yield of synthetic DNA recovery in each sample is then used to normalize the results obtained with the antibodies of interest. Using this approach, we could compensate for losses of material, reduce the variability between ChIP replicates, and increase the accuracy and statistical resolution of the data.
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| 5. |
- Marin-Vicente, Consuelo, et al.
(författare)
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RRP6/EXOSC10 is required for the repair of DNA double-strand breaks by homologous recombination
- 2015
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 128:6, s. 1097-1107
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Tidskriftsartikel (refereegranskat)abstract
- The exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome factor RRP6 of Drosophila melanogaster and its human ortholog EXOSC10 play a role in DNA repair. Here, we show that RRP6 and EXOSC10 are recruited to DNA double-strand breaks (DSBs) in S2 cells and HeLa cells, respectively. Depletion of RRP6/ EXOSC10 does not interfere with the phosphorylation of the histone variant H2Av (Drosophila) or H2AX (humans), but impairs the recruitment of the homologous recombination factor RAD51 to the damaged sites, without affecting RAD51 levels. The recruitment of RAD51 to DSBs in S2 cells is also inhibited by overexpression of RRP6-Y361A-V5, a catalytically inactive RRP6 mutant. Furthermore, cells depleted of RRP6 or EXOSC10 are more sensitive to radiation, which is consistent with RRP6/EXOSC10 playing a role in DNA repair. RRP6/EXOSC10 can be co-immunoprecipitated with RAD51, which links RRP6/EXOSC10 to the homologous recombination pathway. Taken together, our results suggest that the ribonucleolytic activity of RRP6/EXOSC10 is required for the recruitment of RAD51 to DSBs.
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