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

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 b-globin gene with mutated splice sites in intron 2 (mut bglobin). The transcripts encoded by the mut b-globin gene are normally spliced at intron 1 but retain intron 2. The levels of the mut b-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 b-globin genes to investigate the mechanisms that downregulate the production of defective mRNAs. Both wt and mut b-globin transcripts are processed at the 39, but the mut bglobin transcripts are less efficiently cleaved than the wt transcripts. Moreover, the mut b-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 b-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 b-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

Characterization of RNA exosome in Insect Cells : Role in mRNA Surveillance

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.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript

Sensory characteristics of meat from steers of various breeds and rearing intensities

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

An Interaction between RRP6 and SU(VAR)3-9 Targets RRP6 to Heterochromatin and Contributes to Heterochromatin Maintenance in Drosophila melanogaster.

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

Genome-wide effects of RRP6 depletion on the transcriptome of S2 cells.

<p>The effects of RRP6 depletion on the steady-state expression levels were investigated by RNA-seq. Control experiments (GFP RNAi) were carried out in parallel and used as a reference. The expression levels in the control GFP cells and in RRP6-depleted cells expressed as reads per million (y-axis) are shown in green and orange, respectively. Genomic coordinates are indicated in the x-axis in B-C. (A) Pie diagram showing the effect of RRP6 depletion on the levels of different types of sequences, as indicated. (B) Examples of the effect of RRP6 depletion on the expression of repeat sequences. The upper and lower panels show subtelomeric regions of chromosome arms 2L and 3R, respectively, and the middle panel shows a region near the 2R centromere. (C) The effect of RRP6 depletion on the expression of selected transposon sequences. The genomic position of each sequence is indicated in the x-axis. (D) RNAi experiments were carried out to knock down RRP6 alone or RRP6 and DIS3 simultaneously. RNA was isolated and analysed by RT-qPCR using primer pairs designed to amplify selected sequences (the primer sequences are provided in the Supplementary Materials and Methods). The data was normalised to actin 5C RNA levels and expressed as a fold change compared to the levels observed in the GFP control. A protein-coding gene, <i>Pgk</i>, was analysed in parallel as a control. The histogram shows averages and standard deviations from three independent biological replicates. (E) ChIP experiments with antibodies against histone H3, H3K9ac, and H3K9me2 were carried out in untreated S2 cells to analyze the chromatin state of the selected genomic regions. Actin 5C was analyzed in parallel as a representative for euchromatin. The histogram shows averages and standard deviations from three independent biological replicates.</p

SU(VAR)3-9 depletion affects the association of RRP6 with chromatin.

<p>The association of selected proteins with the chromatin was analyzed by Western blotting using native chromatin preparations fractionated according to the scheme in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005523#pgen.1005523.g001" target="_blank">Fig 1F</a>. (A) Analysis of S2 cells that express the HA-tagged SU(VAR)3-9. The global levels of HP1a and SU(VAR)3-9 in the chromatin were analyzed in control cells (GFP) and in cells depleted of RRP6 and DIS3, or HP1a. The chromatin fractions were analysed using different antibodies, as indicated in the figure. An anti-HA antibody was used to detect SU(VAR)3-9. H3 and H3K9me2 served as controls. (B) Analysis of S2 cells that express the V5-tagged RRP6 depleted of HP1a. An anti-V5 antibody was used to detect RRP6 in the chromatin fractions. Depletion of HP1a does not affect the levels of RRP6 bound to the chromatin fraction. (C) Analysis of S2 cells that express the V5-tagged RRP6 depleted of SU(VAR)3-9. An anti-V5 antibody was used to detect RRP6. The quantification of the band intensities from three independent experiments is shown to the right. The standard deviations are given in parentheses. Histone H3 was used for normalization.</p

RRP6 is associated with heterochromatin <i>in vivo</i>.

<p>(A) Salivary gland polytene chromosomes immunostained with antibodies against RRP6 (green) and HP1a (red). The figure shows an overview micrograph. ch: chromocenter. (B) A detail showing a telomere stained with antibodies against RRP6 and HP1a, as in A. The fluorescence profile in the right part of the image shows the co-variation of both signals along the telomeric region. (C) A detail showing the chromocenter (ch) stained with antibodies against RRP6 and HP1a, as in A. (D) Co-localization of HP1a and RRP6 in dense chromatin in S2 cells analyzed by immuno-EM. An overview of a thin section through the nucleus of a cell is shown to the left. The bar represents 1 μm. A high-magnification micrograph shows co-localization of HP1a (12 nm gold) and RRP6 (6 nm gold) in the dense chromatin (<i>Dc</i>). The bar represents 100 nm. (E) The fractionation scheme used to isolate the different nuclear fractions in S2 cells: soluble (nucleoplasm), chromosomal RNP, and chromatin. (F) The distribution of HP1a, RPD3, and RRP6-V5 in the different nuclear fractions in S2 cells analyzed by Western blotting. The experiment was carried out in cells that expressed V5-tagged RRP6. Histone H3 was used as a control.</p

Depletion of SU(VAR)3-9 influences RRP6 genomic occupancy in S2 cells.

<p>ChIP-seq experiments were carried out using S2 cells that expressed V5-tagged RRP6 under low-induction conditions. Chromatin preparations from control GFP cells and from cells depleted of SU(VAR)3-9 were used for ChIP-seq using an anti-V5 antibody. (A) Pie diagram showing the association of RRP6-rich regions with different types of sequences in control cells. (B) Chromosome distribution of RRP6 expressed in control cells as percentage of RRP6-rich regions in each chromosome (green bars). Two different scales are shown due to the lower fraction of regions in the heterochromatic scaffolds compared to the rest of the chromosome arms. The grey bars indicate the fraction of the genome corresponding to each chromosome, for comparison. (C) Depletion of SU-VAR)3-9 affects RRP6 genomic occupancy. For each chromosome or scaffold, the number of RRP6-rich regions upregulated or downregulated is expressed as percentage of the number of changed regions relative to the number of regions in that same chromosome in control cells. The percentage of affected regions is much higher in heterochromatin. (D) RRP6 occupancy in the genomic regions analyzed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005523#pgen.1005523.g004" target="_blank">Fig 4</a>. The arrows indicate the regions amplified in the qPCR assays.</p

The catalytic activity of RRP6 is required for the silencing of transposon transcripts and for the maintenance of heterochromatin compaction.

<p>Wild-type RRP6-V5 or catalytically inactive mutants RRP6-Y361A-V5 and RRp6-D328A-V5 were expressed in S2 cells. Control cells that did not overexpress any protein were used in parallel for comparison. (A) Analysis of protein expression by Western blotting using an antibody against the V5 tag. Histone H3 served as loading control. (B) RT-qPCR analysis of transcript levels in cells that overexpress wither the wild-type RRP6-V5 or the catalytically inactive mutants. RNA was isolated and analyzed using primer pairs designed to amplify selected sequences, as indicated in the figure. The data was normalised to actin 5C mRNA levels and expressed as a fold change compared to the levels observed in the control cells (dark blue line). The histogram shows averages and standard deviations from three independent biological replicates. (C) PLA analysis of chromatin compaction using antibodies against HP1a and histone H3. The images show examples of PLA staining (magenta) in cells counterstained with DAPI (blue). The graph shows the number of PLA dots per cell in each condition. The mean number of dots per cell (magenta line) was 3,93 in the cells that overexpressed wild-type RRP6-V6 and 2,04 in cells that overexpressed RRP6-Y361A-V5. The difference was highly significant (P<0.0001; two-tailed Mann Whitney test; n = 150 cells analyzed in each condition, from two independent experiments).</p