A DNA damage signal activates and derepresses exon inclusion in Drosophila TAF1 alternative splicing

Signal-dependent alternative splicing is important for regulating gene expression in eukaryotes, yet our understanding of how signals impact splicing mechanisms is limited. A model to address this issue is alternative splicing of Drosophila TAF1 pre-mRNA in response to camptothecin (CPT)-induced DNA damage signals. CPT treatment of Drosophila S2 cells causes increased inclusion of TAF1 alternative cassette exons 12a and 13a through an ATR signaling pathway. To evaluate the role of TAF1 pre-mRNA sequences in the alternative splicing mechanism, we developed a TAF1 minigene (miniTAF1) and an S2 cell splicing assay that recapitulated key aspects of CPT-induced alternative splicing of endogenous TAF1. Analysis of miniTAF1 indicated that splice site strength underlies independent and distinct mechanisms that control exon 12a and 13a inclusion. Mutation of the exon 13a weak 5' splice site or weak 3' splice site to a consensus sequence was sufficient for constitutive exon 13a inclusion. In contrast, mutation of the exon 12a strong 5' splice site or moderate 3' splice site to a consensus sequence was only sufficient for constitutive exon 12a inclusion in the presence of CPT-induced signals. Analogous studies of the exon 13 3' splice site suggest that exon 12a inclusion involves signal-dependent pairing between constitutive and alternative splice sites. Finally, intronic elements identified by evolutionary conservation were necessary for full repression of exon 12a inclusion or full activation of exon 13a inclusion and may be targets of CPT-induced signals. In summary, this work defines the role of sequence elements in the regulation of TAF1 alternative splicing in response to a DNA damage signal.     

Myosin light-chain 1/3 gene alternative splicing: cis regulation is based upon a hierarchical compatibility between splice sites.

The mechanisms involved in the selective joining of appropriate 5' and 3' splice sites are still poorly understood in both constitutive and alternatively spliced genes. With two promoters associated with different exons, the myosin light-chain 1/3 gene generates two pre-mRNAs that also differ by the use of a pair of internal exons, 3 and 4, that are spliced in a mutually exclusive fashion. When the promoter upstream from exon 1 is used, only exon 4 is included. If the promoter upstream from exon 2 is used, only exon 3 is included. In an attempt to understand the molecular basis for the mutually exclusive behavior of these two exons and the basis of their specific selection, a number of minigene constructs containing exons 3 and 4 were tested in a variety of homologous or heterologous cis and trans environments. The results demonstrate that the mutually exclusive behavior of myosin light-chain exons 3 and 4 and selection between the two exons are cis regulated and are affected by the nature of the flanking sequences. Both exons competed for the common flanking 5' and 3' splice sites. Flanking exons were found that favored inclusion into mature mRNA of exon 3, exon 4, both, or neither, suggesting a specific cooperative interaction between certain 5' and 3' splice sites. Thus, alternative splicing of myosin light-chain 1/3 pre-mRNAs is regulated in cis by a hierarchy of compatibilities between pairs of 5' and 3' splice sites.     

Alternative splicing and gene structure of the transforming growth factor beta-activated kinase 1

We have identified a fourth splice variant of the TGF beta-activated kinase (TAK1), called TAK1-d, and identified an error in the previously published TAK1-c sequence. Our data shows that the c and d variants encode proteins whose carboxyl ends differ markedly from those of variants a and b. Analysis of the human TAK1 gene sequence, located at 6q16.1-q16.3, shows that the coding sequence is organised in 17 exons. The four splice variants result from alternative splicing of exons 12 and 16, the reading frame of exon 17 being determined by the presence or absence of exon 16. Study of the relative levels of expression of the four splice variants showed significant variations between tissues. Our evidence suggests that the alternative splicing of the TAK1 mRNA may have important functional implications.     

Combinatorial splicing of exon pairs by two-site binding of U1 small nuclear ribonucleoprotein particle.

A two-site model for the binding of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was tested in order to understand how exon partners are selected in complex pre-mRNAs containing alternative exons. In this model, it is proposed that two U1 snRNPs define a functional unit of splicing by base pairing to the 3' boundary of the downstream exon as well as the 5' boundary of the intron to be spliced. Three-exon substrates contained the alternatively spliced exon 4 (E4) region of the preprotachykinin gene. Combined 5' splice site mutations at neighboring exons demonstrate that weakened binding of U1 snRNP at the downstream site and improved U1 snRNP binding at the upstream site result in the failure to rescue splicing of the intron between the mutations. These results indicate the stringency of the requirement for binding a second U1 snRNP to the downstream 5' splice site for these substrates as opposed to an alternative model in which a certain threshold level of U1 snRNP can be provided at either site. Further support for the two-site model is provided by single-site mutations in the 5' splice site of the third exon, E5, that weaken base complementarity to U1 RNA. These mutations block E5 branchpoint formation and, surprisingly, generate novel branchpoints that are specified chiefly by their proximity to a cryptic 5' splice site located at the 3' terminus of the pre-mRNA. The experiments shown here demonstrate a true stimulation of 3' splice site activity by the downstream binding of U1 snRNP and suggest a possible mechanism by which combinatorial patterns of exon selection are achieved for alternatively spliced pre-mRNAs.