The activity of the SR protein family of splicing factors in constitutive or alternative splicing requires direct interactions with the pre-mRNA substrate. Thus it is important to define the high affinity targets of the various SR species and to evaluate their ability to discriminate between defined RNA targets. We have analyzed the binding specificity of the 30-kDa SR protein 9G8, which contains a zinc knuckle in addition to the RNA binding domain (RBD). Using a SELEX approach, we demonstrate that 9G8 selects RNA sequences formed by GAC triplets, whereas a mutated zinc knuckle variant selects different RNA sequences, centered around a (A/U)C(A/U)(A/U)C motif, indicating that the zinc knuckle is involved in the RNA recognition specificity of 9G8. In contrast, SC35 selects sequences composed of pyrimidine or purine-rich motifs. Analyses of RNA-protein interactions with purified recombinant 30-kDa SR proteins or in nuclear extracts, by means of UV crosslinking and immunoprecipitation, demonstrate that 9G8, SC35, and ASF/SF2 recognize their specific RNA targets with high specificity. Interestingly, the RNA sequences selected by the mutated zinc knuckle 9G8 variant are efficiently recognized by SRp20, in agreement with the fact that the RBD of 9G8 and SRp20 are similar. Finally, we demonstrate the ability of 9G8 and of its zinc knuckle variant, or SRp20, to act as efficient splicing transactivators through their specific RNA targets. Our results provide the first evidence for cooperation between an RBD and a zinc knuckle in defining the specificity of an RNA binding domain.

RNA helicases comprise the largest family of enzymes involved in the metabolism of mRNAs, the processing and fate of which rely on their packaging into messenger ribonucleoprotein particles (mRNPs). In this Review, we describe how the capacity of some RNA helicases to either remodel or lock the composition of mRNP complexes underlies their pleiotropic functions at different steps of the gene expression process. We illustrate the roles of RNA helicases in coordinating gene expression steps and programmes, and propose that RNA helicases function as molecular drivers and guides of the progression of their mRNA substrates from one RNA-processing factory to another, to a productive mRNA pool that leads to protein synthesis or to unproductive mRNA pools that are stored or degraded.

The RNA helicases DDX5 and DDX17 are members of a large family of highly conserved proteins that are involved in gene-expression regulation; however, their in vivo targets and activities in biological processes such as cell differentiation, which requires reprogramming of gene-expression programs at multiple levels, are not well characterized. Here, we uncovered a mechanism by which DDX5 and DDX17 cooperate with heterogeneous nuclear ribonucleoprotein (hnRNP) H/F splicing factors to define epithelial- and myoblast-specific splicing subprograms. We then observed that downregulation of DDX5 and DDX17 protein expression during myogenesis and epithelial-to-mesenchymal transdifferentiation contributes to the switching of splicing programs during these processes. Remarkably, this downregulation is mediated by the production of miRNAs induced upon differentiation in a DDX5/DDX17-dependent manner. Since DDX5 and DDX17 also function as coregulators of master transcriptional regulators of differentiation, we propose to name these proteins "master orchestrators" of differentiation that dynamically orchestrate several layers of gene expression.

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...