Manual Regulation of Alternative Splicing (Progress in Molecular and Subcellular Biology)

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As a control, staining with anti-Sm antibody against snRNPs and with anti-hPrp8 antibody U5-specific protein — p demonstrated that this effect was specific and not caused by the generation of nuclear leakage, because no cytoplasmic accumulation of Sm and hPrp8 proteins were observed at either dose level Fig. Similar experiments using only the second antibodies gave no background fluorescence data not shown. Addition of transcription and protein-synthesis inhibitors actinomycin-D and puromycin, respectively before the irradiation had no effect on hSlu7 cytoplasmic accumulation Fig.

This demonstrates that the accumulation of hSlu7 in the cytoplasm does not represent newly synthesized protein concomitant with nuclear import blockage. A time-scale analysis of hSlu7 export out of the nucleus following 0. Recovery is not seen even after 24 hours data not shown. Stress induces cytoplasmic hSlu7 accumulation. Following a 3 hour recovery period under optimal conditions, cells were fixed and hSlu7 cellular localization was observed by indirect immunofluorescence using anti-hSlu7 antibody.

B As described in A except that the cells were fixed and snRNP cellular localization was observed by indirect immunofluorescence using anti-Sm antibody. Treatments are indicated on the left. D, left Time-dependent export of hSlu7 out of the nucleus following irradiation at 0. The y -axis indicates the proportion of hSlu7 exported out of the nucleus. D, right The effects of various stress conditions on the export of hSlu7 out of the nucleus. HeLa cells were subjected to various stress conditions [UV-C 0. The error bars represent s.

The slightly different subcellular distribution observed after 0. This implies that the UV response is a unique effect of individual proteins to a stress-induced environment. The cytoplasmic accumulation is not cell-type specific, because UV-C irradiation caused similar subcellular distribution in three other cell lines T, HepG2 and U2OS; data not shown. The above results indicate that stress causes a shift in the cellular localization of hSlu7 — from nuclear to cytoplasmic accumulation. Other stress conditions, such as peroxidase, osmotic shock, cisplatin and neocarzinostatin, had no or very little effect on hSlu7 cytoplasmic accumulation Fig.

To test whether the cellular localization of hSlu7 has a functional significance in alternative splicing, we used two minigenes containing the genomic sequence of the gene encoding adenosine deaminase, ADAR2 , from exon 7 to exon 9, in which exon 8 is an alternatively spliced Alu exon Lev-Maor et al. The 4-nucleotide distance between the proximal and distal AGs also ensures the mode of alternative splicing: Increasing that distance leads to exon 8 skipping, and exon 8 inclusion can be restored by a high concentration of hSlu7 when the distance between the two AGs is over 8 nucleotides Lev-Maor et al.

RNA Trafficking and Nuclear Structure Dynamics Progress in Molecular and Subcellular Biology

We therefore reasoned that, if hSlu7 proteins partially shift from the nucleus to the cytoplasm, this might alter the alternative splicing pattern. Proximal left and distal right AGs are indicated in bold. Lower- and upper-case letters represent intron and exon sequences, respectively. Forty-eight hours after transfection, cells were either mock treated or UV-C irradiated and then incubated for 3 hours at optimal conditions. A schematic representation of PCR products is indicated to the right of the gel. The alternative Alu exon is colored black. GAPDH , a house-keeping gene, was amplified from each sample to confirm that approximately the same amount of cDNA was used for each reaction.

C A graph quantifying the spliced products, including exon 8 from B, expressed out of the total amount of spliced products in the same lane and experiment. The 55 kDa protein is a non-specific interaction of anti-hPrp18 antibody. To test whether the different alternative splicing patterns were caused by reduced nuclear hSlu7 levels we used RNAi for a review, see Novina and Sharp, to knockdown hSlu7 cellular concentrations. Cells with reduced levels of hSlu7 Fig. We note that a control plasmid and other unrelated RNAi plasmids had no or little effect on the alternative splicing pattern Fig.

In addition, reduced levels of hSlu7 had no effect on hPrp18 protein levels Fig. To test whether the effect on splicing acts on an endogenous gene as well, we assayed the alternative splicing of the D-aspartate-oxidase DDO gene. These results indicate that the reduced nuclear levels of hSlu7 are not restricted to the mutated minigene and might imply a broad effect of hSlu7 levels on alternative splicing of other exons as well.


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In fact, E1A spliced products were selected differently only as a response to UV-C irradiation and not following hSlu7 knockdown or transfection of a control plasmid Fig. C The relative amounts of 9S, 12S and 13S used in B expressed out of the total amount of spliced products in the same lane and experiment.

The signaling pathways activated by stress have been extensively studied. Stress stimuli such as UV-C irradiation activate two stress-response kinase cascades: Following a 1 hour incubation with the inhibitor, cells were UV-C irradiated and then allowed to recover for 3 hours, followed by fixation and staining with anti-hSlu7 Fig.

Blocking of both pathways was effective in reducing phosphorylated states of p38 and JNK kinases by ten- and sixfold after 1 hour, and nine- and fourfold after 3 hours, respectively Fig. Thus, the JNK pathway showed a stronger involvement than the p38 pathway in preventing the cytoplasmic accumulation of hSlu7 after UV-C irradiation even at the lowest concentration of nM; Fig. This suggests that the JNK signaling pathway might possibly be the significant player in the subcellular distribution of hSlu7. The JNK signaling pathway might be responsible for the subcellular distribution of hSlu7.

Cells were then UV-C irradiated 0. B Proportion of hSlu7 exported out of the nucleus. HeLa cells were UV-C irradiated 0. Cells were mock treated lane 2 or UV-C irradiated at a low dose 0. To test whether the alternative splicing activity of hSlu7 Fig. As expected, preventing hSlu7 shift from the nucleus to the cytoplasm abrogates the effect caused by reduced hSlu7 nuclear concentrations Fig. This supports the link between stress induced by UV-C, activation of the JNK pathway and hSlu7 nuclear levels, which cause an effect on alternative splicing.

To asses whether JNK1 phosphorylation is accountable for the induction of hSlu7 relocalization and, as a consequence, affect alternative splicing, we increased the cellular levels of active JNK1 protein by transfecting the cells with a JNK1 -cDNA-containing plasmid and then inducing its phosphorylation using mild UV-C irradiation 0. In this study, we identify physiological conditions that alter the nuclear concentration of second-step splicing protein hSlu7.

Our results strongly suggest that UV-C stress stimuli trigger changes in the alternative splicing patterns of cellular genes by decreasing the nuclear concentration of hSlu7 through the modulation of its nuclear to cytoplasmic transport. Therefore, this spatial and temporal regulatory mechanism might be the method by which hSlu7 protein levels are regulated within the nucleus upon exposure to environmental stimuli. Two known mechanisms control the levels of splicing proteins within the nucleus: All three proteins have been shown to affect alternative splicing of specific genes. The latter mechanism, which was also observed for hSlu7, seems to provide a finer regulation of nuclear protein levels that control splicing.

Several of the stress condition's effects might seem to be contradictory.

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In our experiments, we observed that UV-C irradiation and not osmotic shock resulted in cytoplasmic accumulation. However, the events upstream of these pathways are poorly defined and this inconsistent stress-induced localization effect has been reported elsewhere van der Houven van Oordt et al. It is also intriguing that UV-C irradiation and not cisplatin elicits a change in hSlu7 cellular localization, because they both exert similar physiological effects Hayakawa et al. UV-C light produces bulky adducts, pyrimidine dimers and other photoproducts in DNA, whereas cisplatin forms primarily 1,2-intrastrand cross-links between adjacent purines in DNA and also introduces DNA 1,3-intrastrand cross-links, interstrand cross-links and monoadducts Kartalou and Essigmann, These DNA obstructions must be removed or bypassed to prevent arrest of the DNA polymerase during replication and thus are expected to activate the same repair cascade.

However, a recent report showed that the mechanisms by which cells sensitize to UV and cisplatin are likely to be different Bowman et al.

Science , EMBO Journal 25, SR proteins as potential targets for therapy. Progress in Molecular and Subcellular Biology Invited review 44, Chromatin transcript elongation and alternative splicing. Nature Structural and Molecular Biology 13, Journal of Cell Science , Preparation of soluble extracts from adenovirus-infected cells for studies of RNA splicing. Methods in Molecular Medicine , LK, an adenovirus encoded alternative RNA splicing factor. Journal of Biological Chemistry , Genome Research 16, Cell Biology — a Laboratory Manual, Ed.

Cell Biology — a Laboratory Manual, ed. Experimental Cell Research , Abundance of the largest subunit of RNA polymerase II in the nucleus is regulated by nucleo-cytoplasmic shuttling. Molecular Biology of the Cell 17 10 , Molecular Cellular Biology 26 21 , Diversity of human U2AF splicing factors.

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This causes a longer version of exon 2 to be included in the processed transcript, including an early stop codon. The resulting mRNA encodes a truncated protein product that is inactive. Females produce the master sex determination protein Sex lethal Sxl. This prevents the use of this junction, shifting the spliceosome binding to the downstream acceptor site.

Splicing at this point bypasses the stop codon, which is excised as part of the intron. The resulting mRNA encodes an active Tra protein, which itself is a regulator of alternative splicing of other sex-related genes see dsx above. Multiple isoforms of the Fas receptor protein are produced by alternative splicing. Two normally occurring isoforms in humans are produced by an exon-skipping mechanism. An mRNA including exon 6 encodes the membrane-bound form of the Fas receptor, which promotes apoptosis , or programmed cell death. Increased expression of Fas receptor in skin cells chronically exposed to the sun, and absence of expression in skin cancer cells, suggests that this mechanism may be important in elimination of pre-cancerous cells in humans.

The inclusion or skipping of the exon depends on two antagonistic proteins, TIA-1 and polypyrimidine tract-binding protein PTB. This mechanism is an example of exon definition in splicing.

Alternative splicing - Wikipedia

A spliceosome assembles on an intron, and the snRNP subunits fold the RNA so that the 5' and 3' ends of the intron are joined. However, recently studied examples such as this one show that there are also interactions between the ends of the exon. In this particular case, these exon definition interactions are necessary to allow the binding of core splicing factors prior to assembly of the spliceosomes on the two flanking introns.

Competition between the activator and repressor ensures that both mRNA types with and without exon 2 are produced. Alternative splicing is one of several exceptions to the original idea that one DNA sequence codes for one polypeptide the One gene-one enzyme hypothesis. It might be more correct now to say "One gene — many polypeptides". Since the methods of regulation are inherited, this provides novel ways for mutations to affect gene expression.

It has been proposed that for eukaryotes alternative splicing was a very important step towards higher efficiency, because information can be stored much more economically. Several proteins can be encoded by a single gene, rather than requiring a separate gene for each, and thus allowing a more varied proteome from a genome of limited size. A single point mutation may cause a given exon to be occasionally excluded or included from a transcript during splicing, allowing production of a new protein isoform without loss of the original protein.

Such functional diversity achieved by isoforms is reflected by their expression patterns and can be predicted by machine learning approaches. This finding led to speculation that the perceived greater complexity of humans, or vertebrates generally, might be due to higher rates of alternative splicing in humans than are found in invertebrates.

When they compared alternative splicing frequencies in random subsets of genes from each organism, the authors concluded that vertebrates do have higher rates of alternative splicing than invertebrates. Changes in the RNA processing machinery may lead to mis-splicing of multiple transcripts, while single-nucleotide alterations in splice sites or cis-acting splicing regulatory sites may lead to differences in splicing of a single gene, and thus in the mRNA produced from a mutant gene's transcripts.

Abnormally spliced mRNAs are also found in a high proportion of cancerous cells.

Regulation of Alternative Splicing

For certain types of cancer, like in colorectal and prostate, the number of splicing errors per cancer has been shown to vary greatly between individual cancers, a phenomenon referred to as transcriptome instability. Mutation of DNMT3A has been demonstrated to contribute to hematologic malignancies , and that DNMT3A -mutated cell lines exhibit transcriptome instability as compared to their isogenic wildtype counterparts.

In fact, there is actually a reduction of alternative splicing in cancerous cells compared to normal ones, and the types of splicing differ; for instance, cancerous cells show higher levels of intron retention than normal cells, but lower levels of exon skipping. One example of a specific splicing variant associated with cancers is in one of the human DNMT genes. In two separate studies, expression of two of these abnormally spliced mRNAs in mammalian cells caused changes in the DNA methylation patterns in those cells.

Cells with one of the abnormal mRNAs also grew twice as fast as control cells, indicating a direct contribution to tumor development by this product. An important property of cancerous cells is their ability to move and invade normal tissue. The abnormal isoform of the Ron protein encoded by this mRNA leads to cell motility. Recent provocative studies point to a key function of chromatin structure and histone modifications in alternative splicing regulation. These insights suggest that epigenetic regulation determines not only what parts of the genome are expressed but also how they are spliced.

Genome-wide analysis of alternative splicing is a challenging task. Typically, alternatively spliced transcripts have been found by comparing EST sequences, but this requires sequencing of very large numbers of ESTs. Most EST libraries come from a very limited number of tissues, so tissue-specific splice variants are likely to be missed in any case. High-throughput approaches to investigate splicing have, however, been developed, such as: These methods can be used to screen for polymorphisms or mutations in or around splicing elements that affect protein binding. When combined with splicing assays, including in vivo reporter gene assays, the functional effects of polymorphisms or mutations on the splicing of pre-mRNA transcripts can then be analyzed.

In microarray analysis, arrays of DNA fragments representing individual exons e. The array is then probed with labeled cDNA from tissues of interest. This can reveal the presence of particular alternatively spliced mRNAs.