Three protein factors (SF1, SF3 and U2AF) function in pre-splicing complex formation in addition to snRNPs.

The splicing of nuclear messenger RNA precursors can be reproduced in vitro with fractions obtained after chromatography of HeLa cell nuclear extracts. Here we report the chromatographic separation of three protein factors: SF1, SF3 and U2AF. All factors function early in the splicing reaction, in the assembly of a pre-splicing complex. Likewise, all factors are essential for the production of spliced RNA. In addition to their distinct chromatographic properties, the splicing factors can be distinguished by their sensitivities to heat and N-ethylmaleimide. All activities can be detected in a cytoplasmic A-100 fraction from HeLa cells. The fact that SF1, SF3 and U2AF are essential factors in pre-splicing complex formation raises the possibility that SF1 and/or SF3 participate in the interaction of U2 snRNP with the branch point in addition to U2AF.     

Diversity of human U2AF splicing factors

U2 snRNP auxiliary factor (U2AF) is an essential heterodimeric splicing factor composed of two subunits, U2AF(65) and U2AF(35). During the past few years, a number of proteins related to both U2AF(65) and U2AF(35) have been discovered. Here, we review the conserved structural features that characterize the U2AF protein families and their evolutionary emergence. We perform a comprehensive database search designed to identify U2AF protein isoforms produced by alternative splicing, and we discuss the potential implications of U2AF protein diversity for splicing regulation.     

Myotonic dystrophy: the role of the CUG triplet repeats in splicing of a novel DMPK exon and altered cytoplasmic DMPK mRNA isoform ratios

The mechanism by which (CTG)n expansion in the 3' UTR of the DMPK gene causes myotonic dystrophy (DM) is unknown. We identified four RNA splicing factors--hnRNP C, U2AF (U2 auxiliary factor), PTB (polypyrimidine tract binding protein), and PSF (PTB associated splicing factor)--that bind to two short regions 3' of the (CUG)n, and found a novel 3' DMPK exon resulting in an mRNA lacking the repeats. We propose that the (CUG)n is an essential cis acting element for this splicing event. In contrast to (CUG)n containing mRNAs, the novel isoform is not retained in the nucleus in DM cells, resulting in imbalances in relative levels of cytoplasmic DMPK mRNA isoforms and a new dominant effect of the mutation on DMPK.     

SC35 autoregulates its expression by promoting splicing events that destabilize its mRNAs

SC35 belongs to the family of SR proteins that regulate alternative splicing in a concentration-dependent manner in vitro and in vivo. We previously reported that SC35 is expressed through alternatively spliced mRNAs with differing 3' untranslated sequences and stabilities. Here, we show that overexpression of SC35 in HeLa cells results in a significant decrease of endogenous SC35 mRNA levels along with changes in the relative abundance of SC35 alternatively spliced mRNAs. Remarkably, SC35 leads to both an exon inclusion and an intron excision in the 3' untranslated region of its mRNAs. In vitro splicing experiments performed with recombinant SR proteins demonstrate that SC35, but not ASF/SF2 or 9G8, specifically activates these alternative splicing events. Interestingly, the resulting mRNA is very unstable and we present evidence that mRNA surveillance is likely to be involved in this instability. SC35 therefore constitutes the first example of a splicing factor that controls its own expression through activation of splicing events leading to expression of unstable mRNA.     

Mitochondrial damage modulates alternative splicing in neuronal cells: implications for neurodegeneration

Mitochondrial damage is linked to many neurodegenerative conditions, such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. These diseases are associated with changes in the splicing pattern of individual mRNAs. Here, we tested the hypothesis that mitochondrial damage modulates alternative splicing, not only of a few mRNAs, but in a general manner. We incubated cultured human neuroblastoma cells with the chemical agent paraquat (a neurotoxin that interferes with mitochondrial function, causing energy deficit and oxidative stress) and analysed the splicing pattern of 13 genes by RT-PCR. For all mRNAs that are alternatively spliced, we observed a dose- and time-dependent increase of the smaller isoforms. In contrast, splicing of all constitutive splicing exons that we monitored did not change. Using other drugs, we show that the modulation of alternative splicing correlates with ATP depletion, not with oxidative stress. Such drastic changes in alternative splicing are not observed in cell lines of non-neuronal origin, suggesting a selective susceptibility of neuronal cells to modulation of splicing. As a significant percentage of all mammalian mRNAs undergo alternative splicing, we predict that mitochondrial failure will unbalance a vast number of isoform equilibriums, which would give an important contribution to neurodegeneration.     

Identification and characterization of three members of the human SR family of pre-mRNA splicing factors.

SR proteins have a characteristic C-terminal Ser/Arg-rich repeat (RS domain) of variable length and constitute a family of highly conserved nuclear phosphoproteins that can function as both essential and alternative pre-mRNA splicing factors. We have cloned a cDNA encoding a novel human SR protein designated SRp30c, which has an unusually short RS domain. We also cloned cDNAs encoding the human homologues of Drosophila SRp55/B52 and rat SRp40/HRS. Recombinant proteins expressed from these cDNAs are active in constitutive splicing, as shown by their ability to complement a HeLa cell S100 extract deficient in SR proteins. Additional cDNA clones reflect extensive alternative splicing of SRp40 and SRp55 pre-mRNAs. The predicted protein isoforms lack the C-terminal RS domain and might be involved in feedback regulatory loops. The ability of human SRp30c, SRp40 and SRp55 to modulate alternative splicing in vivo was compared with that of other SR proteins using a transient contransfection assay. The overexpression of individual SR proteins in HeLa cells affected the choice of alternative 5' splice sites of adenovirus E1A and/or human beta-thalassemia reporters. The resulting splicing patterns were characteristic for each SR protein. Consistent with the postulated importance of SR proteins in alternative splicing in vivo, we demonstrate complex changes in the levels of mRNAs encoding the above SR proteins upon T cell activation, concomitant with changes in the expression of alternatively spliced isoforms of CD44 and CD45.     

Genome-wide analysis reveals an unexpected function for the Drosophila splicing factor U2AF50 in the nuclear export of intronless mRNAs

The protein factor U2AF is an essential component required for pre-mRNA splicing. Mutations identified in the S. pombe large U2AF subunit were used to engineer transgenic Drosophila carrying temperature-sensitive U2AF large subunit alleles. Mutant recombinant U2AF heterodimers showed reduced polypyrimidine tract RNA binding at elevated temperatures. Genome-wide RNA profiling comparing wild-type and mutant strains identified more than 400 genes differentially expressed in the dU2AF50 mutant flies grown at the restrictive temperature. Surprisingly, almost 40% of the downregulated genes lack introns. Microarray analyses revealed that nuclear export of a large number of intronless mRNAs is impaired in Drosophila-cultured cells RNAi knocked down for dU2AF50. Immunopurification of nuclear RNP complexes showed that dU2AF50 associates with intronless mRNAs. These results reveal an unexpected role for the splicing factor dU2AF50 in the nuclear export of intronless mRNAs.     

High-throughput quantification of splicing isoforms

Most human messenger RNAs (mRNAs) are alternatively spliced and many exhibit disease-specific splicing patterns. However, the contribution of most splicing events to the development and maintenance of human diseases remains unclear. As the contribution of alternative splicing events to diagnosis and prognosis is becoming increasingly recognized, it becomes important to develop precise methods to quantify the abundance of these isoforms in clinical samples. Here we present a pipeline for real-time PCR annotation of splicing events (RASE) that allows accurate identification of a large number of splicing isoforms in human tissues. The RASE automatically designed specific primer pairs for 81% of all alternative splicing events in the NCBI build 36 database. Experimentally, the majority of the RASE designed primers resulted in isoform-specific amplification suitable for quantification in human cell lines or in formalin-fixed, paraffin-embedded (FFPE) RNA extract. Using this pipeline it is now possible to rapidly identify splicing isoform signatures in different types of human tissues or to validate complete sets of data generated by microarray expression profiling and deep sequencing techniques.