Physiological state co-regulates thousands of mammalian mRNA splicing events at tandem splice sites and alternative exons

Thousands of tandem alternative splice sites (TASS) give rise to mRNA insertion/deletion variants with small size differences. Recent work has concentrated on the question of biological relevance in general, and the physiological regulation of TASS in particular. We have quantitatively studied 11 representative TASS cases in comparison to one mutually exclusive exon case and two cassette exons (CEs) using a panel of human and mouse tissues, as well as cultured cell lines. Tissues show small but significant differences in TASS isoform ratios, with a variance 4- to 20-fold lower than seen for CEs. Remarkably, in cultured cells, all studied alternative splicing (AS) cases showed a cell-density-dependent shift of isoform ratios with similar time series profiles. A respective genome-wide co-regulation of TASS splicing was shown by next-generation mRNA sequencing data. Moreover, data from human and mouse organs indicate that this co-regulation of TASS occurs in vivo, with brain showing the strongest difference to other organs. Together, the results indicate a physiological AS regulation mechanism that functions almost independently from the splice site context and sequence.     

Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene

In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branchpoints for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism by which alternative promoters can be coordinated with downstream alternative splicing.     

Conserved and species-specific alternative splicing in mammalian genomes

Background  

Alternative splicing has been shown to be one of the major evolutionary mechanisms for protein diversification and proteome expansion, since a considerable fraction of alternative splicing events appears to be species- or lineage-specific. However, most studies were restricted to the analysis of cassette exons in pairs of genomes and did not analyze functionality of the alternative variants.     

The relative strengths of SR protein-mediated associations of alternative and constitutive exons can influence alternative splicing.

We have characterized the functional role of SR protein-mediated exon/exon associations in the alternative splicing of exon 5 of chicken cardiac troponin T (cTnT). We have previously shown that SR proteins can promote the association of the alternative exon 5 with the flanking constitutive exon 6 of this pre-mRNA. In this study, we have shown that when exons 2, 3, and 4 of the cTnT pre-mRNA are spliced together, the composite exon 2/3/4 contains an additional SR protein binding site. Furthermore, we have found that SR proteins can also promote interactions between the pairs of exons 2/3/4-5 and 2/3/4-6. We then asked whether the SR protein binding sites in these exons play a role in cTnT alternative splicing in vivo. We found that the SR protein binding sites in exons 2/3/4 and 6 promote exon 5 skipping, and it has previously been shown that the SR protein binding site in exon 5 promotes exon 5 inclusion. Consistent with these results, we find that the SR protein-mediated association of exon 2/3/4 with 6 is preferred over associations involving exon 5, in that exons 2/3/4 and 6 are more efficient than exon 5 in competing an SR protein-mediated exon/exon association. We suggest that the relative strengths of SR protein-mediated associations of alternative and constitutive exons play a role in determining alternative splicing patterns.     

Adaptive evolution of multiple-variable exons and structural diversity of drug-metabolizing enzymes

Background  

The human genome contains a large number of gene clusters with multiple-variable-first exons, including the drug-metabolizing UDP glucuronosyltransferase (UGT1) and I-branching β-1,6-N-acetylglucosaminyltransferase (GCNT2, also known as IGNT) clusters, organized in a tandem array, similar to that of the protocadherin (PCDH), immunoglobulin (IG), and T-cell receptor (TCR) clusters. To gain insight into the evolutionary processes that may have shaped their diversity, we performed comprehensive comparative analyses for vertebrate multiple-variable-first-exon clusters.     

Human alpha 3(VI) collagen gene. Characterization of exons coding for the amino-terminal globular domain and alternative splicing in normal and tumor cells

We recently reported the isolation and sequencing of human cDNA clones corresponding to the alpha 3 chain of type VI collagen (Chu, M.-L., Zhang, R.-Z., Pan, T.-c., Stokes, D., Conway, D., Kuo, H.-J., Glanville, R., Mayer, U., Mann, K., Deutzmann, R., and Timpl, R. (1990) EMBO J. 9, 385-393). The study indicates that the amino-terminal globular domain of the alpha 3(VI) chain consists of nine repetitive subdomains of approximately 200 amino acid residues (N1-N9) and the gene appeared to undergo alternative splicing since some clones lacked regions encoding the N9 and part of the N3 subdomains. In the present study, we report the exon structure for the region encoding the amino-terminal globular domain of the human alpha 3(VI) chain. The nine repetitive subdomains are encoded by 10 exons spanning 26 kilobase pairs of genomic DNA. Eight of the repetitive subdomains (N2-N9) were found to be encoded by separate exons of approximately 600 base pairs each. The only exception is the N1 subdomain which is encoded by two exons of 417 and 146 base pairs. Characterization of the exon/intron structure showed that the cDNA variants were the result of splicing out of exon 9 (encoding the N9 subdomain) and part of exon 3 (encoding the N3 subdomain). Nuclease S1 analysis and the polymerase chain reaction demonstrated that exon 7 (N7 subdomain) was also subject to alternative splicing in normal skin fibroblasts. Examination of these splicing events by nuclease S1 analysis in normal fibroblasts, three different human tumor cell lines, and several human tissues showed that splicing out of exon 9 is much more efficient in normal as compared to tumor cells.     

Functional coordination of alternative splicing in the mammalian central nervous system

Background

Alternative splicing (AS) functions to expand proteomic complexity and plays numerous important roles in gene regulation. However, the extent to which AS coordinates functions in a cell and tissue type specific manner is not known. Moreover, the sequence code that underlies cell and tissue type specific regulation of AS is poorly understood.

Results

Using quantitative AS microarray profiling, we have identified a large number of widely expressed mouse genes that contain single or coordinated pairs of alternative exons that are spliced in a tissue regulated fashion. The majority of these AS events display differential regulation in central nervous system (CNS) tissues. Approximately half of the corresponding genes have neural specific functions and operate in common processes and interconnected pathways. Differential regulation of AS in the CNS tissues correlates strongly with a set of mostly new motifs that are predominantly located in the intron and constitutive exon sequences neighboring CNS-regulated alternative exons. Different subsets of these motifs are correlated with either increased inclusion or increased exclusion of alternative exons in CNS tissues, relative to the other profiled tissues.

Conclusion

Our findings provide new evidence that specific cellular processes in the mammalian CNS are coordinated at the level of AS, and that a complex splicing code underlies CNS specific AS regulation. This code appears to comprise many new motifs, some of which are located in the constitutive exons neighboring regulated alternative exons. These data provide a basis for understanding the molecular mechanisms by which the tissue specific functions of widely expressed genes are coordinated at the level of AS.     

Developmentally induced, muscle-specific trans factors control the differential splicing of alternative and constitutive troponin T exons

Alternative RNA splicing is a ubiquitous process permitting single genes to encode multiple protein isoforms. Here we report experiments in which a gene construct, containing combinatorial Troponin T (TnT) exons that manifest an exceptional diversity of alternative splicing in vivo, has been transfected into muscle and nonmuscle cells. Analyses of the spliced RNAs show that the alternative TnT exons retain their capacity for differential splicing in the modified minigene context when introduced into a variety of nonmuscle and muscle cells. The patterns of alternative splicing differ depending on cell type. Only in differentiated myotubes are the alternative exons normally incorporated during splicing, reproducing their behavior in the native gene; they are excluded in nonmuscle cells and myoblasts that do not express the endogenous TnT. These results provide proof that trans factors required for correct alternative splicing are induced during myogenesis. Surprisingly, such factors are also required for the correct splicing of constitutive TnT exons.