| 1. |
- Calado Botelho, Salomé, et al.
(författare)
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The association of Brahma with the Balbiani ring 1 gene of Chironomus tentans studied by immunoelectron microscopy and chromatin immunoprecipitation
- 2008
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Ingår i: Insect molecular biology (Print). - : Wiley. - 0962-1075 .- 1365-2583. ; 17:5, s. 505-13
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Tidskriftsartikel (refereegranskat)abstract
- Many steps of gene expression take place during transcription, and important functional information can thus be obtained by determining the distribution of specific factors along a transcribed gene. The Balbiani ring (BR) genes of the dipteran Chironomus tentans constitute a unique system for mapping the association of specific factors along a eukaryotic gene using immuno-electron microscopy (immuno-EM). The chromatin immunoprecipitation (ChIP) technique has provided an alternative, more general method for studying the association of proteins with specific genomic sequences. The immuno-EM and the ChIP methods suffer from different limitations, and thus a combination of both is advantageous. We have established optimal conditions for ChIP on chromatin extracted from the salivary glands of C. tentans, and we have analyzed the association of the SWI/SNF chromatin remodelling factor Brahma (Brm) with the BR1 gene by combined immuno-EM and ChIP. We show that Brm is not restricted to the promoter of the BR1 gene but is also associated with sequences in the middle and distal portions of the gene, which suggests that Brm has additional roles apart from regulating transcription initiation.
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| 2. |
- 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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| 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. |
- Hessle, Viktoria, 1970-
(författare)
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Characterization of RNA exosome in Insect Cells : Role in mRNA Surveillance
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The exosome, an evolutionarily conserved protein complex with exoribonucleolytic activity, is one of the key players in mRNA quality control. Little is known about the functions of the exosome in metazoans. We have studied the role of the exosome in nuclear mRNA surveillance using Chironomus tentans and Drosophila melanogaster as model systems. Studies of the exosome subunits Rrp4 and Rrp6 revealed that both proteins are associated with transcribed genes and nascent pre-mRNPs in C. tentans. We have shown that several exosome subunits interact in vivo with the mRNA-binding protein Hrp59/hnRNP M, and that depleting Hrp59 in D. melanogaster S2 cells by RNAi leads to reduced levels of Rrp4 at the transcription sites. Our results on Rrp4 suggest a model for cotranscriptional quality control in which the exosome is constantly recruited to nascent mRNAs through interactions with specific hnRNP proteins. Moreover, we show that Rrp6 interacts with mRNPs in transit from the gene to the nuclear pore complex, where it is released during early stages of nucleo-cytoplasmic translocation. Furthermore, we show that Rrp6 is enriched in discrete nuclear bodies in the salivary glands of C. tentans and D. melanogaster. In C. tentans, the Rrp6-rich nuclear bodies colocalize with SUMO. We have also constructed D. melanogaster S2 cells expressing human b-globin genes, with either wild type of mutated splice sites, and we have studied the mechanisms by which the cells react to pre-mRNA processing defects. Our results indicate that two surveillance responses operate co-transcriptionally in S2 cells. One requires Rrp6 and retains defective mRNAs at the transcription site. The other one reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications. These observations support the view that multiple mechanisms contribute to co-transcriptional surveillance in insects.
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| 5. |
- Hessle, Viktoria, et al.
(författare)
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Rrp6 is recruited to transcribed genes and accompanies the spliced mRNA to the nuclear pore
- 2012
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 18:8, s. 1466-1474
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Tidskriftsartikel (refereegranskat)abstract
- Rrp6 is an exoribonuclease involved in the quality control of mRNA biogenesis. We have analyzed the association of Rrp6 with the Balbiani ring pre-mRNPs of Chironomus tentans to obtain insight into the role of Rrp6 in splicing surveillance. Rrp6 is recruited to transcribed genes and its distribution along the genes does not correlate with the positions of exons and introns. In the nucleoplasm, Rrp6 is bound to both unspliced and spliced transcripts. Rrp6 is released from the mRNPs in the vicinity of the nuclear pore before nucleo-cytoplasmic translocation. We show that Rrp6 is associated with newly synthesized transcripts during all the nuclear steps of gene expression and is associated with the transcripts independently of their splicing status. These observations suggest that the quality control of pre-mRNA splicing is not based on the selective recruitment of the exoribonuclease Rrp6 to unprocessed mRNAs.
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| 6. |
- Hessle, Viktoria, et al.
(författare)
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The exosome Associates Cotranscriptionally with the Nascent Pre-mRNP through Interactions with Heterogeneous Nuclear Ribonucleoproteins
- 2009
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 20:15, s. 3459-3470
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Tidskriftsartikel (refereegranskat)abstract
- Eukaryotic cells have evolved quality control mechanisms to degrade aberrant mRNA molecules and prevent the synthesis of defective proteins that could be deleterious for the cell. The exosome, a protein complex with ribonuclease activity, is a key player in quality control. An early quality checkpoint takes place cotranscriptionally but little is known about the molecular mechanisms by which the exosome is recruited to the transcribed genes. Here we study the core exosome subunit Rrp4 in two insect model systems, Chironomus and Drosophila. We show that a significant fraction of Rrp4 is associated with the nascent pre-mRNPs and that a specific mRNA-binding protein, Hrp59/hnRNP M, interacts in vivo with multiple exosome subunits. Depletion of Hrp59 by RNA interference reduces the levels of Rrp4 at transcription sites, which suggests that Hrp59 is needed for the exosome to stably interact with nascent pre-mRNPs. Our results lead to a revised mechanistic model for cotranscriptional quality control in which the exosome is constantly recruited to newly synthesized RNAs through direct interactions with specific hnRNP proteins.
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| 7. |
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| 8. |
- Olsson, Viktoria, et al.
(författare)
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Sensory characteristics of meat from steers of various breeds and rearing intensities
- 2019
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Konferensbidrag (refereegranskat)abstract
- The most common category of young cattle slaughtered for beef in Sweden is bulls of dairy breed, most often reared indoors. There is however a potential in raising steers (castrated bulls) for slaughter on semi-natural pasture, which may have an impact on sensory properties of the meat. Furthermore, weight gain and carcass characteristics may be improved by crossing dairy breeds with specialised beef breeds. In combination with the new technique of sex-sorted dairy semen, beef breed semen can be used to the less superior cows in the herd without jeopardizing an adequate number of replacement heifers from the superior cows. The aim of the study was to investigate whether there are any differences in sensory meat quality between cross bred and purebred cattle and between two rearing intensities including semi-natural pasture. Sensory properties were evaluated by a trained, analytical panel consisting of six assessors by the use of descriptive analysis. The intensity of iron, acidic, tallow, milky and barny odour as well as metallic, barny and gamey flavour and basic tastes were assessed in triplicate along with attributes describing the appearance and texture of the meat. Differences were mainly found in appearance and texture attributes, but also gamey flavour and the intensity of umami were affected by the rearing and breeding regimes. The meat quality results from this study will be combined with results from other disciplines such as animal science, business administration and environmental science. It is important to be able to demonstrate various possible added values that comes from pasture-based beef production systems under Swedish conditions.
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| 9. |
- Olsson, Viktoria, et al.
(författare)
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Sensory characteristics of meat from steers of various breeds and rearing intensities
- 2019
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The most common category of young cattle slaughtered for beef in Sweden is bulls of dairy breed, most often reared indoors. There is however a potential in raising steers (castrated bulls) for slaughter on semi-natural pasture, which may have an impact on sensory properties of the meat. Furthermore, weight gain and carcass characteristics may be improved by crossing dairy breeds with specialised beef breeds. In combination with the new technique of sex-sorted dairy semen, beef breed semen can be used to the less superior cows in the herd without jeopardizing an adequate number of replacement heifers from the superior cows. The aim of the study was to investigate whether there are any differences in sensory meat quality between cross bred and purebred cattle and between two rearing intensities including semi-natural pasture. Sensory properties were evaluated by a trained, analytical panel consisting of six assessors by the use of descriptive analysis. The intensity of iron, acidic, tallow, milky and barny odour as well as metallic, barny and gamey flavour and basic tastes were assessed in triplicate along with attributes describing the appearance and texture of the meat. Differences were mainly found in appearance and texture attributes, but also gamey flavour and the intensity of umami were affected by the rearing and breeding regimes. The meat quality results from this study will be combined with results from other disciplines such as animal science, business administration and environmental science. It is important to be able to demonstrate various possible added values that comes from pasture-based beef production systems under Swedish conditions.
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| 10. |
- Söderberg, Emilia, et al.
(författare)
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Profilin is associated with transcriptionally active genes
- 2012
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Ingår i: Nucleus. - : Informa UK Limited. - 1949-1034 .- 1949-1042. ; 3:3, s. 290-299
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Tidskriftsartikel (refereegranskat)abstract
- We have raised antibodies against the profilin of Chironomus tentans to study the location of profilin relative to chromatin and to active genes in salivary gland polytene chromosomes. We show that a fraction of profilin is located in the nucleus, where profilin is highly concentrated in the nucleoplasm and at the nuclear periphery. Moreover, profilin is associated with multiple bands in the polytene chromosomes. By staining salivary glands with propidium iodide, we show that profilin does not co-localize with dense chromatin. profilin associates instead with protein-coding genes that are transcriptionally active, as revealed by co-localization with hnRNP and snRNP proteins. We have performed experiments of transcription inhibition with actinomycin D and we show that the association of profilin with the chromosomes requires ongoing transcription. however, the interaction of profilin with the gene loci does not depend on RNA. Our results are compatible with profilin regulating actin polymerization in the cell nucleus. however, the association of actin with the polytene chromosomes of C. tentans is sensitive to RNase, whereas the association of profilin is not, and we propose therefore that the chromosomal location of profilin is independent of actin.
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