Trans-acting factors required for inclusion of regulated exons in the Ultrabithorax mRNAs of Drosophila melanogaster

Alternatively spliced Ultrabithorax mRNAs differ by the presence of internal exons mI and mII. Two approaches were used to identify trans-acting factors required for inclusion of these cassette exons. First, mutations in a set of genes implicated in the control of other alternative splicing decisions were tested for dominant effects on the Ubx alternative splicing pattern. To identify additional genes involved in regulation of Ubx splicing, a large collection of deficiencies was tested first for dominant enhancement of the haploinsufficient Ubx haltere phenotype and second for effects on the splicing pattern. Inclusion of the cassette exons in Ubx mRNAs was reduced strongly in heterozygotes for hypomorphic alleles of hrp48, which encodes a member of the hnRNP A/B family and is implicated in control of P-element splicing. Significant reductions of mI and mII inclusion were also observed in heterozygotes for loss-of-function alleles of virilizer, fl(2)d, and crooked neck. The products of virilizer and fl(2)d are also required for Sxl autoregulation at the level of splicing; crooked neck encodes a protein with structural similarities to yeast-splicing factors Prp39p and Prp42p. Deletion of at least five other loci caused significant reductions in the inclusion of mI and/or mII. Possible roles of identified factors are discussed in the context of the resplicing strategy for generation of alternative Ubx mRNAs.     

Polypyrimidine tract sequences direct selection of alternative branch sites and influence protein binding.

IVS1, an intron derived from the rat fibronectin gene, is spliced inefficiently in vitro, involving the use of three alternative branch sites. Mutation of one branch point site, BP3, so as to increase complementarity to U2 snRNA resulted in exclusive use of that site and improved splicing efficiency, indicating that the wild type BP3 site is one determinant of poor IVS1 splicing. Deletions within the polypyrimidine tract had a variable effect on splicing efficiency and altered the pattern of branch site usage. Selection of each branch site was influenced negatively by purine substitutions ca. 20 nucleotides downstream. It is proposed that all three IVS1 branch sites are pyrimidine tract-dependent. Pyrimidine tract deletions also influenced the crosslinking of PTB (the polypyrimidine tract-binding protein), hnRNP C, and splicing factor U2AF65. All three proteins bound preferentially to distinct regions within the polypyrimidine tract and thus are candidates for mediating pyrimidine tract-dependent branch site selection. The findings indicate the complexity of the IVS1 polypyrimidine tract and suggest a crucial role for this region in modulating branch site selection and IVS1 splicing.     

Chironomus tentans-repressor splicing factor represses SR protein function locally on pre-mRNA exons and is displaced at correct splice sites

Chironomus tentans-repressor splicing factor (Ct-RSF) represses the activation of splicing by SR proteins in vitro. Ct-RSF colocalizes with the Ser-Arg-rich (SR) protein hrp45 in interchromatin granule clusters and coimmunoprecipitates with hrp45 in nuclear extracts. Ct-RSF and hrp45 can also interact directly in vitro. Ct-RSF and hrp45 are recruited together to transcribing genes and associate with growing pre-mRNAs. Ct-RSF and hrp45 colocalize at a large number of gene loci. Injection of anti-Ct-RSF antibodies into nuclei of living cells blocks association of both Ct-RSF and hrp45 with the growing pre-mRNA, whereas binding of U2 small nuclear ribonucleoprotein particle (snRNP) to the pre-mRNA is unaffected. On the intron-rich Balbiani ring (BR) 3 pre-mRNA, hrp45 as well as U1 and U2 snRNPs bind extensively, whereas relatively little Ct-RSF is present. In contrast, the BR1 and BR2 pre-mRNAs, dominated by exon sequences, bind relatively much Ct-RSF compared with hrp45 and snRNPs. Our data suggest that Ct-RSF represses SR protein function at exons and that the assembly of spliceosomes at authentic splice sites displaces Ct-RSF locally.     

Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping

Alternative splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9 is regulated by a combination of cis-acting elements distributed through the exon and both flanking introns (IVS8 and IVS9). Several studies have identified in the IVS8 intron 3' splice site a regulatory element that is composed of a polymorphic (TG)m(T)n repeated sequence. At present, no cellular factors have been identified that recognize this element. We have identified TDP-43, a nuclear protein not previously described to bind RNA, as the factor binding specifically to the (TG)m sequence. Transient TDP-43 overexpression in Hep3B cells results in an increase in exon 9 skipping. This effect is more pronounced with concomitant overexpression of SR proteins. Antisense inhibition of endogenous TDP-43 expression results in increased inclusion of exon 9, providing a new therapeutic target to correct aberrant splicing of exon 9 in CF patients. The clinical and biological relevance of this finding in vivo is demonstrated by our characterization of a CF patient carrying a TG10T9(DeltaF508)/TG13T3(wt) genotype leading to a disease-causing high proportion of exon 9 skipping.     

A regulator of Dscam mutually exclusive splicing fidelity

The Down syndrome cell adhesion molecule (Dscam) gene has essential roles in neural wiring and pathogen recognition in Drosophila melanogaster. Dscam encodes 38,016 distinct isoforms via extensive alternative splicing. The 95 alternative exons in Dscam are organized into clusters that are spliced in a mutually exclusive manner. The exon 6 cluster contains 48 variable exons and uses a complex system of competing RNA structures to ensure that only one variable exon is included. Here we show that the heterogeneous nuclear ribonucleoprotein hrp36 acts specifically within, and throughout, the exon 6 cluster to prevent the inclusion of multiple exons. Moreover, hrp36 prevents serine/arginine-rich proteins from promoting the ectopic inclusion of multiple exon 6 variants. Thus, the fidelity of mutually exclusive splicing in the exon 6 cluster is governed by an intricate combination of alternative RNA structures and a globally acting splicing repressor.     

Drosophila Hrp48 Is Required for Mushroom Body Axon Growth,Branching and Guidance

RNA binding proteins assemble on mRNAs to control every single step of their life cycle, from nuclear splicing to cytoplasmic localization, stabilization or translation. Consistent with an essential role of RNA binding proteins in neuronal maturation and function, mutations in this class of proteins, in particular in members of the hnRNP family, have been associated with neurological diseases. To date, however, the physiological function of hnRNPs during in vivo neuronal development has remained poorly explored. Here, we have investigated the role of Drosophila Hrp48, a fly homologue of mammalian hnRNP A2/B1, during central nervous system development. Using a combination of mutant conditions, we showed that hrp48 is required for the formation, growth and guidance of axonal branches in Mushroom Body neurons. Furthermore, our results revealed that hrp48 inactivation induces an overextension of Mushroom Body dorsal axonal branches, with a significantly higher penetrance in females than in males. Finally, as demonstrated by immunolocalization studies, Hrp48 is confined to Mushroom Body neuron cell bodies, where it accumulates in the cytoplasm from larval stages to adulthood. Altogether, our data provide evidence for a crucial in vivo role of the hnRNP Hrp48 in multiple aspects of axon guidance and branching during nervous system development. They also indicate cryptic sex differences in the development of sexually non-dimorphic neuronal structures.     

Synthesis of predominantly unspliced cytoplasmic RNAs by chimeric herpes simplex virus type 1 thymidine kinase-human beta-globin genes.

The herpes simplex virus type 1 thymidine kinase (tk) gene lacks introns and produces stable mRNA in the absence of splicing. We have prepared a hybrid gene by placing the first exon, first intron (first intervening sequence, designated IVS1), and most of the second exon of the normal human beta-globin gene into the 3' untranslated region of the tk gene. Although this hybrid gene contains all globin sequences presumed necessary for the splicing of IVS1, predominantly, unspliced stable cytoplasmic RNA is produced in both long- and short-term expression assays. Moreover, stable unspliced cytoplasmic RNA is detected whether the intron is situated in a sense or an antisense orientation. Efficient splicing of IVS1 is obtained either by deleting the majority of tk coding sequences or by relocating the globin sequences from the 3' to the 5' untranslated region of the tk gene.     

Sequence requirements for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA.

The sequence requirements for splicing of the Tetrahymena pre-rRNA have been examined by altering the rRNA gene to produce versions that contain insertions and deletions within the intervening sequence (IVS). The altered genes were transcribed and the RNA tested for self-splicing in vitro. A number of insertions (8-54 nucleotides) at three locations had no effect on self-splicing activity. Two of these insertions, located at a site 5 nucleotides preceding the 3'-end of the IVS, did not alter the choice of the 3' splice site. Thus the 3' splice site is not chosen by its distance from a fixed point within the IVS. Analysis of deletions constructed at two sites revealed two structures, a hairpin loop and a stem-loop, that are entirely dispensable for IVS excision in vitro. Three other regions were found to be necessary. The regions that are important for self-splicing are not restricted to the conserved sequence elements that define this class of intervening sequences. The requirement for structures within the IVS for pre-rRNA splicing is in sharp contrast to the very limited role of IVS structure in nuclear pre-mRNA splicing.