Alternative approach to a heavy weight problem

Obesity is reaching epidemic proportions in developed countries and represents a significant risk factor for hypertension, heart disease, diabetes, and dyslipidemia. Splicing mutations constitute at least 14% of disease-causing mutations, thus implicating polymorphisms that affect splicing as likely candidates for disease susceptibility. A recent study suggested that genes associated with obesity were significantly enriched for rare nucleotide variants. Here, we examined these variants and revealed that they are located near splice junctions and tend to affect exonic splicing regulatory sequences. We also show that the majority of the exons that harbor these SNPs are constitutively spliced, yet they exhibit weak splice sites, typical to alternatively spliced exons, and are hence suboptimal for recognition by the splicing machinery and prone to become alternatively spliced. Using ex vivo assays, we tested a few representative variants and show that they indeed affect splicing by causing a shift from a constitutive to an alternative pattern, suggesting a possible link between extreme body mass index and abnormal splicing patterns.     

Intronic sequences flanking alternatively spliced exons are conserved between human and mouse

Comparison of the sequences of mouse and human genomes revealed a surprising number of nonexonic, nonexpressed conserved sequences, for which no function could be assigned. To study the possible correlation between these conserved intronic sequences and alternative splicing regulation, we developed a method to identify exons that are alternatively spliced in both human and mouse. We compiled two exon sets: one of alternatively spliced conserved exons and another of constitutively spliced conserved exons. We found that 77% of the conserved alternatively spliced exons were flanked on both sides by long conserved intronic sequences. In comparison, only 17% of the conserved constitutively spliced exons were flanked by such conserved intronic sequences. The average length of the conserved intronic sequences was 103 bases in the upstream intron and 94 bases in the downstream intron. The average identity levels in the immediately flanking intronic sequences were 88% and 80% for the upstream and downstream introns, respectively, higher than the conservation levels of 77% that were measured in promoter regions. Our results suggest that the function of many of the intronic sequence blocks that are conserved between human and mouse is the regulation of alternative splicing.     

Uterine expression of alternatively spliced mRNAs of mouse splicing factor SC35 during early pregnancy

RNA differential display was applied to identify genes critical for the establishment of pregnancy in the mouse. One of the gene fragments identified was homologous to human SC35 splicing factor; the mouse counterpart had not then been cloned. To obtain the full cDNA sequence of the mouse gene, a cDNA library was screened and four positive clones were fully analysed. Sequencing analysis indicated that we had cloned alternatively spliced mRNA species of mouse SC35 splicing factor. A map of splicing structure for this gene's pre-mRNA was then proposed and region-specific mRNA species were tested on Northern blots. This analysis indicated that the overall expression level of SC35 mRNA was much higher in implantation sites than in inter-implantation sites in the mouse uterus during early pregnancy. The expression of alternatively spliced mRNAs for SC35 was differently regulated both during early pregnancy and by steroid hormones. Embryo-derived factors were also implicated in the up-regulation of SC35 mRNA at implantation sites. These results demonstrate, for the first time, that an essential splicing factor is regulated in a complex manner during implantation in the mouse uterus. Hence, its correct regulation could be important for the success of pregnancy.     

Evolution of exon-intron structure and alternative splicing in fruit flies and malarial mosquito genomes

Comparative analysis of alternative splicing of orthologous genes from fruit flies (Drosophila melanogaster and Drosophila pseudoobscura) and mosquito (Anopheles gambiae) demonstrated that both in the fruit fly genes and in fruit fly-mosquito comparisons, constitutive exons and splicing sites are more conserved than alternative ones. While >97% of constitutive D. melanogaster exons are conserved in D. pseudoobscura, only approximately 80% of alternative exons are conserved. Similarly, 77% of constitutive fruit fly exons are conserved in the mosquito genes, compared with <50% of alternative exons. Internal alternatives are more conserved than terminal ones. Retained introns are the least conserved, alternative acceptor sites are slightly more conserved than donor sites, and mutually exclusive exons are almost as conserved as constitutive exons. Cassette and mutually exclusive exons experience almost no intron insertions. We also observed cases of interconversion of various elementary alternatives, e.g., transformation of cassette exons into alternative sites. These results agree with the observations made earlier in human-mouse comparisons and demonstrate that the phenomenon of relatively low conservation of alternatively spliced regions may be universal, as it has been observed in different taxonomic groups (mammals and insects) and at various evolutionary distances.     

A systematic analysis of intronic sequences downstream of 5' splice sites reveals a widespread role for U-rich motifs and TIA1/TIAL1 proteins in alternative splicing regulation

To identify human intronic sequences associated with 5' splice site recognition, we performed a systematic search for motifs enriched in introns downstream of both constitutive and alternative cassette exons. Significant enrichment was observed for U-rich motifs within 100 nucleotides downstream of 5' splice sites of both classes of exons, with the highest enrichment between positions +6 and +30. Exons adjacent to U-rich intronic motifs contain lower frequencies of exonic splicing enhancers and higher frequencies of exonic splicing silencers, compared with exons not followed by U-rich intronic motifs. These findings motivated us to explore the possibility of a widespread role for U-rich motifs in promoting exon inclusion. Since cytotoxic granule-associated RNA binding protein (TIA1) and TIA1-like 1 (TIAL1; also known as TIAR) were previously shown in vitro to bind to U-rich motifs downstream of 5' splice sites, and to facilitate 5' splice site recognition in vitro and in vivo, we investigated whether these factors function more generally in the regulation of splicing of exons followed by U-rich intronic motifs. Simultaneous knockdown of TIA1 and TIAL1 resulted in increased skipping of 36/41 (88%) of alternatively spliced exons associated with U-rich motifs, but did not affect 32/33 (97%) alternatively spliced exons that are not associated with U-rich motifs. The increase in exon skipping correlated with the proximity of the first U-rich motif and the overall "U-richness" of the adjacent intronic region. The majority of the alternative splicing events regulated by TIA1/TIAL1 are conserved in mouse, and the corresponding genes are associated with diverse cellular functions. Based on our results, we estimate that approximately 15% of alternative cassette exons are regulated by TIA1/TIAL1 via U-rich intronic elements.     

Alternative RNA splicing in the nervous system

Tissue-specific alternative splicing profoundly effects animal physiology, development and disease, and this is nowhere more evident than in the nervous system. Alternative splicing is a versatile form of genetic control whereby a common pre-mRNA is processed into multiple mRNA isoforms differing in their precise combination of exon sequences. In the nervous system, thousands of alternatively spliced mRNAs are translated into their protein counterparts where specific isoforms play roles in learning and memory, neuronal cell recognition, neurotransmission, ion channel function, and receptor specificity. The essential nature of this process is underscored by the finding that its misregulation is a common characteristic of human disease. This review highlights the current views of the biological phenomenon of alternative splicing, and describes evidence for its intricate underlying biochemical mechanisms. The roles of RNA binding proteins and their tissue-specific properties are discussed. Why does alternative splicing occur in cosmic proportions in the nervous system? How does it affect integrated cellular functions? How are region-specific, cell-specific and developmental differences in splicing directed? How are the control mechanisms that operate in the nervous system distinct from those of other tissues? Although there are many unanswered questions, substantial progress has been made in showing that alternative splicing is of major importance in generating proteomic diversity, and in modulating protein activities in a temporal and spatial manner. The relevance of alternative splicing to diseases of the nervous system is also discussed.     

Differential subcellular localization of the two alternatively spliced isoforms of the Kv3.1 potassium channel subunit in brain

Voltage-gated K(+) channels containing pore-forming subunits of the Kv3 subfamily have specific roles in the fast repolarization of action potentials and enable neurons to fire repetitively at high frequencies. Each of the four known Kv3 genes encode multiple products by alternative splicing of 3' ends resulting in the expression of K(+) channel subunits differing only in their C-terminal sequence. The alternative splicing does not affect the electrophysiological properties of the channels, and its physiological role is unknown. It has been proposed that one of the functions of the alternative splicing of Kv3 genes is to produce subunit isoforms with differential subcellular membrane localizations in neurons and differential modulation by signaling pathways. We investigated the role of the alternative splicing of Kv3 subunits in subcellular localization by examining the brain distribution of the two alternatively spliced versions of the Kv3.1 gene (Kv3.1a and Kv3.1b) with antibodies specific for the alternative spliced C-termini. Kv3.1b proteins were prominently expressed in the somatic and proximal dendritic membrane of specific neuronal populations in the mouse brain. The axons of most of these neurons also expressed Kv3.1b protein. In contrast, Kv3.1a proteins were prominently expressed in the axons of some of the same neuronal populations, but there was little to no Kv3.1a protein expression in somatodendritic membrane. Exceptions to this pattern were seen in two neuronal populations with unusual targeting of axonal proteins, mitral cells of the olfactory bulb, and mesencephalic trigeminal neurons, which expressed Kv3.1a protein in dendritic and somatic membrane, respectively. The results support the hypothesis that the alternative spliced C-termini of Kv3 subunits regulate their subcellular targeting in neurons.     

atSRp30, one of two SF2/ASF-like proteins from Arabidopsis thaliana, regulates splicing of specific plant genes

SR proteins are nuclear phosphoproteins with a characteristic Ser/Arg-rich domain and one or two RNA recognition motifs. They are highly conserved in animals and plants and play important roles in spliceosome assembly and alternative splicing regulation. We have now isolated and partially sequenced a plant protein, which crossreacts with antibodies to human SR proteins. The sequence of the corresponding cDNA and genomic clones from Arabidopsis revealed a protein, atSRp30, with strong similarity to the human SR protein SF2/ASF and to atSRp34/SR1, a previously identified SR protein, indicating that plants possess two SF2/ASF-like proteins. atSRp30 expresses alternatively spliced mRNA isoforms that are expressed differentially in various organs and during development. Overexpression of atSRp30 via a strong constitutive promoter resulted in changes in alternative splicing of several endogenous plant genes, including atSRp30 itself. Interestingly, atSRp30 overexpression resulted in a pronounced down-regulation of endogenous mRNA encoding full-length atSRp34/SR1 protein. Transgenic plants overexpressing atSRp30 showed morphological and developmental changes affecting mostly developmental phase transitions. atSRp30- and atSRp34/SR1-promoter–GUS constructs exhibited complementary expression patterns during early seedling development and root formation, with overlapping expression in floral tissues. The results of the structural and expression analyses of both genes suggest that atSRp34/SR1 acts as a general splicing factor, whereas atSRp30 functions as a specific splicing modulator.     

Human-mouse comparative analysis reveals that branch-site plasticity contributes to splicing regulation

The formation of base-pairing between the branch-site (BS) sequence and the U2 snRNP is an important step in mRNA splicing. We developed a new algorithm to identify both the BS sequence and the polypyrimidine tract (PPT) and validated its predictions experimentally. To assess BS conservation between human and mouse, we assembled and analyzed 46 812 and 242 constitutively and alternatively spliced orthologs of human-mouse intron pairs, respectively. Combinations of BSs and PPTs can be found in most of the constitutive and alternative introns. The average distance between the BS and the 3' splice site (3'ss) is 33-34 nt. Acceptor-like AG dinucleotides that resided between the predicted BS and the 3'ss were found to appear mostly within 5 nt, but not more than 19 nt, downstream of the BS. However, although 32% of homologous alternatively spliced BS sequences were fully conserved between human and mouse, only a small fraction (3%) of homologous constitutive counterparts was fully conserved. This indicates that the full sequence of the BS is under weak purifying selection in constitutively spliced introns and further strengthens the view that the BS sequence is just one of several factors determining the ability of the splicing machinery to identify the BS location. Mutations in the putative BS revealed a shift from constitutive to alternative splicing, and it also controls the inclusion/skipping ratio in alternative splicing. This suggests a role for BS sequences in regulated splicing.