Twenty years have passed since the discovery of premRNA sphcing (for review, see Sharp 1994). The studies leading to this discovery were carried out on the adeno virus late transcript, which undergoes complex alterna tive splicing, and, therefore, the concept of alternative splicing is also 20 years old. In the intervening time, intervening sequences, or introns, have been found in a vast majority of higher eukaryotic genes, and a large frac tion of intron-containing transcripts have been shown to be subject to alternative splicing. Furthermore, modula tion of pre-mRNA splicing is now known to be a wide spread mechanism of gene control. But despite consider able advances in our understanding of the splicing reac tion per se (for review, see Moore et al. 1993;Madhani and Guthrie 1994), insights into how alternative splicing is controlled have been slower to emerge.Several factors have been responsible for our some what limited understanding of how splicing can be reg ulated. First, the combined use of biochemical and ge netic methodologies available in yeast has greatly accel erated understanding of the basic splicing reaction. But true alternative splicing (i.e., selection of alternative splice sites) does not appear to occur in Saccharomyces ceievisiae, and studies on this process are therefore lim ited to higher eukaryotes. With the important exception of the Diosophila sex determination pathway (for re view, see Baker 1989), what we know about alternative splicing comes almost entirely from in vitro biochemical approaches without the aid of genetic approaches. Sec ond, again with a limited number of exceptions (for re view, see Rio 1993), the identification of cis-acting se quences responsible for modulating specific splicing events has been difficult, and, therefore, the isolation of sequence-specific RNA-binding splicing factors has been slow. Indeed, whether gene-specific regulators play a ma jor role in splicing control, as sequence-specific DNAbinding proteins do in transcription, constitutes an im portant, unresolved question in the field. Finally, the number of regulated splicing events that have proven amenable to in vitro analysis is small, adding to the dif ficulties in the identification of regulatory molecules and mechanisms. Despite these limitations, significant progress has been made in the identification and analysis of a family of proteins, the SR proteins, that likely play important roles in splicing control. (The name SR re flects the presence of a characteristic serine/argininerich domain; see Fig. I.) The purpose of this review is to summarize our knowledge of these proteins, concentrat ing on how they might participate in splicing regulation. A related review, including discussion of what appears to be an extended superfamily of SR-type proteins, has been published recently (Fu 1995). ASF/SF2, SC35, and the SR protein familyThe first two SR proteins were discovered as a result of biochemical studies of mammalian splicing. The proto typical SR protein, ASF/SF2 (also referred to as SF2/ AS...
ASF/SF2 and SC35 belong to a highly conserved family of nuclear proteins that are both essential for splicing of pre‐mRNA in vitro and are able to influence selection of alternative splice sites. An important question is whether these proteins display distinct RNA binding specificities and, if so, whether this influences their functional interactions with pre‐mRNA. To address these issues, we first performed selection/amplification from pools of random RNA sequences (SELEX) with portions of the two proteins comprising the RNA binding domains (RBDs). Although both molecules selected mainly purine‐rich sequences, comparison of individual sequences indicated that the motifs recognized are different. Binding assays performed with the full‐length proteins confirmed that ASF/SF2 and SC35 indeed have distinct specificities, and at the same time provided evidence that the highly charged arginine‐serine region of each protein is not a major determinant of specificity. In the case of ASF/SF2, evidence is presented that binding specificity involves cooperation between the protein's two RBDs. Finally, we demonstrate that an element containing three copies of a high‐affinity ASF/SF2 binding site constitutes a powerful splicing enhancer. In contrast, a similar element consisting of three SC35 sites was inactive. The ASF/SF2 enhancer can be activated specifically in splicing‐deficient S100 extracts by recombinant ASF/SF2 in conjunction with one or more additional protein factors. These and other results suggest a central role for ASF/SF2 in the function of purine‐rich splicing enhancers.
Diverse glycoproteins of cell surfaces and extracellular matrices operationally termed 'adhesion molecules' are important in the specification of cell interactions during development, maintenance and regeneration of the nervous system. These adhesion molecules have distinct functions involving different cells at different developmental stages, but may cooperate when expressed together. Families of adhesion molecules which share common carbohydrate domains do exist, despite the structural and functional diversity of these glycoproteins. These include the Ca2+-independent neural adhesion molecules: N-CAM, myelin associated glycoprotein (MAG) and L1. L1 is involved in neuron-neuron adhesion, neurite fasciculation, outgrowth of neurites, cerebellar granule cell migration, neurite outgrowth on Schwann cells and interactions among epithelial cells of intestinal crypts. We show here that in addition to sharing carbohydrate epitopes with N-CAM and MAG, L1 is also a member of the immunoglobulin superfamily. It contains six C2 domains and also shares three type III domains with the extracellular matrix adhesion molecule fibronectin.
RNA polymerase II (RNAP II) is responsible for transcription of mRNA precursors in eukaryotic cells. Recent studies, however, have suggested that RNAP II also participates in subsequent RNA processing reactions through interactions between the carboxy-terminal domain (CTD) of the RNAP II largest subunit and processing factors. Using reconstituted in vitro splicing assays, we show that RNAP II functions directly in pre-mRNA splicing by influencing very early steps in assembly of the spliceosome. We demonstrate that the phosphorylation status of the CTD dramatically affects activity: Hyperphosphorylated RNAP IIO strongly activates splicing, whereas hypophosphorylated RNAP IIA can inhibit the reaction.Received March 3, 1999; revised version accepted April 6, 1999.Splicing of mammalian pre-mRNA is a nuclear process in which introns are removed from primary transcripts synthesized by RNA polymerase II (RNAP II). Splicing takes place in a large macromolecular complex called the spliceosome, which is composed of small nuclear ribonucleoprotein (snRNP) particles and non-snRNP proteins including members of the serine/arginine-rich (SR) protein family (for review, see Moore et al. 1993;Kramer 1996). Although cytological studies have suggested that splicing can occur cotranscriptionally (e.g., Beyer et al. 1988;Bauren and Wieslander 1994) and factors required for splicing are found localized at sites of active transcription (e.g., Zhang et al. 1994), functional coupling between transcription and splicing does not seem obligatory because splicing can be reconstituted in vitro with pretranscribed RNA and splicing-competent cell extracts.Recent studies, however, have provided evidence indicating that the carboxy-terminal domain (CTD) of the largest subunit of RNAP II links transcription with premRNA processing (for reviews, see Corden and Patturajan 1997;Neugebauer and Roth 1997;Steinmetz 1997). The CTD is comprised of multiple repeats of the consensus sequence YSPTSPS, which is highly conserved throughout evolution (for review, see Corden 1990) and subject to reversible phosphorylation during the transcription cycle (for review, see Dahmus 1996). RNAP II with a hypophosphorylated CTD (RNAP IIA) is preferentially included in the preinitiation complex at the promoter, whereas RNAP II with a hyperphosphorylated CTD (RNAP IIO) is associated with elongation complexes.Biochemical studies have shown that RNAP II, via the CTD, can physically interact with capping enzymes McCracken et al. 1997a;Yue et al. 1997), polyadenylation factors (McCracken et al. 1997b), and splicing factors, including both snRNPs and SR-like proteins (Chabot et al. 1995;Yuryev et al. 1996;Mortillaro et al. 1996;Kim et al. 1997). Notably, only RNAP IIO has been found to associate with capping and splicing factors, and this isoform has also been detected in active spliceosomes (Mortillaro et al. 1996). In addition, in vivo studies using mammalian cultured cells have demonstrated that RNAs transcribed by RNAP II with a shortened CTD undergo inefficient capping, ...
The RNA-binding protein Tra2 is an important regulator of sex determination in Drosophila. Recently, two mammalian Tra2 homologs of unknown function have been described. Here, we show that human Tra2 proteins are present in HeLa cell nuclear extracts and that they bind efficiently and specifically to a previously characterized pre-mRNA splicing enhancer element. Indeed, both purified proteins bound preferentially to RNA sequences containing GAA repeats, characteristic of many enhancer elements. Neither Tra2 protein functioned in constitutive splicing in vitro, but both activated enhancer-dependent splicing in a sequence-specific manner and restored it after inhibition with competitor RNA. Our findings indicate that mammalian Tra2 proteins are sequence-specific splicing activators that likely participate in the control of cell-specific splicing patterns.
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