Signaling pathways that stabilize interleukin-2 (IL-2) messenger RNA (mRNA) in activated T cells were examined. IL-2 mRNA contains at least two cis elements that mediated its stabilization in response to different signals, including activation of c-Jun amino-terminal kinase (JNK). This response was mediated through a cis element encompassing the 5' untranslated region (UTR) and the beginning of the coding region. IL-2 transcripts lacking this 5' element no longer responded to JNK activation but were still responsive to other signals generated during T cell activation, which were probably sensed through the 3' UTR. Thus, multiple elements within IL-2 mRNA modulate its stability in a combinatorial manner, and the JNK pathway controls turnover as well as synthesis of IL-2 mRNA.

Splicing of the K-SAM alternative exon of the fibroblast growth factor receptor 2 gene is heavily dependent on the U-rich sequence IAS1 lying immediately downstream from its 5 splice site. We show that IAS1 can activate the use of several heterologous 5 splice sites in vitro. Addition of the RNA-binding protein TIA-1 to splicing extracts preferentially enhances the use of 5 splice sites linked to IAS1. TIA-1 can provoke a switch to use of such sites on pre-mRNAs with competing 5 splice sites, only one of which is adjacent to IAS1. Using a combination of UV cross-linking and specific immunoprecipitation steps, we show that TIA-1 binds to IAS1 in cell extracts. This binding is stronger if IAS1 is adjacent to a 5 splice site and is U1 snRNP dependent. Overexpression of TIA-1 in cultured cells activates K-SAM exon splicing in an IAS1-dependent manner. If IAS1 is replaced with a bacteriophage MS2 operator, splicing of the K-SAM exon can no longer be activated by TIA-1. Splicing can, however, be activated by a TIA-1-MS2 coat protein fusion, provided that the operator is close to the 5 splice site. Our results identify TIA-1 as a novel splicing regulator, which acts by binding to intron sequences immediately downstream from a 5 splice site in a U1 snRNP-dependent fashion. TIA-1 is distantly related to the yeast U1 snRNP protein Nam8p, and the functional similarities between the two proteins are discussed.Many eucaryotic genes are made up of exons and introns (43). They are transcribed into pre-mRNAs, from which the intron sequences are removed by splicing. Exons to be included in mRNA must be identified as such. This involves interaction of short sequences at or close to the exon's 5Ј and 3Ј splice sites (5Јss and 3Јss, respectively) with spliceosome components such as snRNPs and associated proteins (for reviews, see references 4, 29, and 43). Exon splicing can be controlled, and several sequences which participate in the control of tissue-specific or developmentally controlled alternative splicing events have been described (for a review, see reference 32). These sequences are particularly interesting to study, as they may yield information on both splicing activation mechanisms and tissuespecific control mechanisms of gene expression. We have been studying fibroblast growth factor receptor 2 (FGFR-2) premRNA splicing for this reason.FGFR-2 alternative exons K-SAM and BEK are spliced in a tissue-specific, mutually exclusive manner, and the two types of FGFR-2 obtained bind different subsets of FGF family members (38). The K-SAM exon is under complex control. It has weak splice sites, and it contains an exon splicing silencer (ESS) which functions by recruiting hnRNP A1 (13). To overcome the activity of this silencer, at least three activating sequences in the downstream intron are required (6,10,12). One of these, IAS1, lies immediately downstream of the 5Јss and is a U-rich sequence (10). In the absence of IAS1 (10), or if IAS1 is moved further downstream from the 5Јss (F. Del GattoKonczak, unpublished data), the K-S...

Some exons contain exon splicing silencers. Their activity is frequently balanced by that of splicing enhancers, and this is important to ensure correct relative levels of alternatively spliced mRNAs. Using an immunoprecipitation and UV-cross-linking assay, we show that RNA molecules containing splicing silencers from the human immunodeficiency virus type 1 tat exon 2 or the human fibroblast growth factor receptor 2 K-SAM exon bind to hnRNP A1 in HeLa cell nuclear extracts better than the corresponding RNA molecule without a silencer. Two different point mutations which abolish the K-SAM exon splicing silencer's activity reduce hnRNP A1 binding twofold. Recruitment of hnRNP A1 in the form of a fusion with bacteriophage MS2 coat protein to a K-SAM exon whose exon splicing silencer has been replaced by a coat binding site efficiently represses splicing of the exon in vivo. Recruitment of only the glycine-rich C-terminal domain of hnRNP A1, which is capable of interactions with other proteins, is sufficient to repress exon splicing. Our results show that hnRNP A1 can function to repress splicing, and they suggest that at least some exon splicing silencers could work by recruiting hnRNP A1.Many eucaryotes make extensive use of alternative splicing to create more than one version of a protein from a single transcription unit. Alternative splicing can be controlled in a cell-type-specific fashion, allowing different cell types to make those versions of a protein best adapted to their particular needs. Such control acts on competing splice sites and can involve activation or repression.Two interesting cases of splicing activation involve construction of multiprotein complexes on the pre-mRNAs. In Drosophila, activation of splicing of a female-specific dsx exon requires assembly on the exon of a complex including the femalespecific protein tra, tra-2, and SR proteins (32, 33). Neuronspecific activation of splicing of the mouse c-src exon N1 is achieved by assembly on downstream intron sequences of a multiprotein complex including the protein KSRP (39). In vitro, KSRP induces the assembly of five other proteins, including hnRNP F, on the intronic splicing enhancer (38). Other exonic splicing enhancers have also been shown to interact with SR proteins (30,34,50,55). SR proteins are known to engage in protein-protein contacts important for splicing (34). Splicing activation thus often involves installation of multiprotein complexes on pre-mRNA sites in such a manner as to allow them to interact productively with spliceosome components.Intron sequences involved in splicing repression have been described for several systems. In Drosophila, the female-specific sxl protein represses use of a male-specific 3Ј splice site on the tra pre-mRNA by binding to the associated polypyrimidine sequence and blocking binding of U2AF (51). sxl blocks splicing of a male-specific sxl exon by binding to multiple pyrimidine-rich sites in the flanking introns (28). Splicing of some exons is repressed by binding of polypyrimidine tract binding pr...

TIA-1 has recently been shown to activate splicing of specific pre-mRNAs transcribed from transiently transfected minigenes, and of some 5 splice sites in vitro, but has not been shown to activate splicing of any endogenous pre-mRNA. We show here that overexpression of TIA-1 or the related protein TIAR has little effect on splicing of several endogenous pre-mRNAs containing alternative exons, but markedly activates splicing of some normally rarely used alternative exons on the TIA-1 and TIAR pre-mRNAs. These exons have weak 5 splice sites followed by U-rich stretches. When the Urich stretch following the 5 splice site of a TIA-1 alternative exon was deleted, TIAR overexpression induced use of a cryptic 5 splice site also followed by a U-rich stretch in place of the original splice site. Using in vitro splicing assays, we have shown that TIA-1 is directly involved in activating the 5 splice sites of the TIAR alternative exons. Activation requires a downstream Urich stretch of at least 10 residues. Our results confirm that TIA-1 activates 5 splice sites followed by U-rich sequences and show that TIAR exerts a similar activity. They suggest that both proteins may autoregulate their expression at the level of splicing.Many eucaryotic genes are transcribed to yield pre-mRNAs containing exon and intron sequences. The process of splicing then eliminates the introns and joins adjacent exons together (1). This requires precise recognition of exons and introns on the pre-mRNA, but it is not yet clear how this is achieved. Splice sites marking exon-intron junctions have specific sequence characteristics necessary for recruiting some spliceosome components: U1 snRNP at the 5Ј splice site (5Јss) 1 and U2AF and U2 snRNP close to the 3Ј splice site (3Јss), for example (for reviews, see Refs. 1-3). However, additional proteins acting through exon or intron sequences distinct from the splice sites are often required for correct splice site use, particularly, but not only, in alternative splicing (for reviews, see Refs. 4 -7). For example, splicing of the FGFR-2 gene alternative exon K-SAM (or IIIb) is repressed by an exon splicing silencer and by upstream intron sequences (8), but activated by three sequences in the downstream intron (9 -14). One of these sequences, IAS1, is U-rich and lies immediately downstream from the exon's 5Јss, suggesting that the protein that binds to it could interact directly with U1 snRNP bound to the adjacent 5Јss.Nam8p is one of several yeast U1 snRNP proteins that have no counterparts in mammalian U1 snRNP (15). Nam8p binds to nonconserved sequences downstream from 5Јss in yeast commitment complexes (16,17). This interaction can be important for use of some 5Јss and is most effective if the downstream sequences are U-rich (16). These observations led to the suggestion that a mammalian protein functionally equivalent to Nam8p might activate weak 5Јss followed by U-rich sequences, such as the 5Јss of the K-SAM exon. The closest mammalian relatives of Nam8p are a pair of related proteins, TIA-1 (18) a...

The CD44 gene alternative exons v8, v9, and v10 are frequently spliced as a block by epithelial cells. By transfecting minigenes containing only one of these alternative exons, we show that splicing of each of them is under cell type-specific control. By using minigenes carrying short block mutations within exons v8 and v9, we detected a candidate exon splicing enhancer in each of these exons. These candidates activated splicing in vitro of a heterologous transcript and are thus true exon splicing enhancers. We analyzed further a v9 exon splicing enhancer covering ϳ30 nucleotides. This enhancer can be UV cross-linked to SR proteins of 35 and 20 kDa in HeLa nuclear extract. By using individual recombinant SR proteins for UV cross-linking in S100 extract, these proteins were identified as 9G8, ASF/SF2, and SRp20. S100 complementation studies using recombinant 9G8, ASF/SF2, and SRp20 showed that all three proteins can activate splicing in vitro of a heterologous exon containing the v9 enhancer; the strongest activation was obtained with 9G8. Progressive truncation of the 30-nucleotide enhancer leads to a progressive decrease in splicing activation. We propose that 9G8, ASF/SF2, SRp20, and possibly other non-SR proteins cooperate in vivo to activate v9 exon splicing.Alternative splicing of pre-mRNA transcripts allows a gene to code for more than one protein (1, 2). Very often similar but distinct versions of the same protein are made from a gene in this way. When alternative splicing is controlled in a cell typespecific manner, cells benefit from the particular version of a protein best suited to their needs. The biological importance of this is underlined by numerous observations linking impaired control of alternative splicing to human diseases, including cancer (3). Understanding splicing control will thus shed light on important aspects of normal and pathological cell life.In mammalian systems, alternative splicing control is often very complex and is characterized by multiple combinatorial control (4). For example, splicing of the neuron-specific exon N1 of the mouse c-src gene is activated by a purine-rich exonic sequence and an intronic splicing enhancer; the latter contains binding sites for hnRNP 1 H, hnRNP F, and KH-type splicing regulatory protein (5). Polypyrimidine tract binding protein (PTB) binds to intron sites flanking the N1 exon to repress splicing, and one of these lies within the enhancer (6). A neurally enriched homologue of PTB binds more stably to this site than does PTB but is a weaker repressor of splicing in vitro. A neurally enriched homologue of PTB also greatly enhances the binding of hnRNP H and KH-type splicing-regulatory protein to the enhancer (5). The N1 exon is thus under control of both activators and repressors, and their relative influence determines its splicing efficiency (7).We and others have investigated another example of multiple combinatorial control: splicing of the FGFR-2 gene K-SAM alternative exon, an exon spliced by epithelial cells. Splicing requires the action of...