Fas exon 6 can be included or skipped to generate mRNAs encoding, respectively, a membrane bound form of the receptor that promotes apoptosis or a soluble isoform that prevents programmed cell death. We report that the apoptosis-inducing protein TIA-1 promotes U1 snRNP binding to the 5' splice site of intron 6, which in turn facilitates exon definition by enhancing U2AF binding to the 3' splice site of intron 5. The polypyrimidine tract binding protein (PTB) promotes exon skipping by binding to an exonic splicing silencer and inhibiting the association of U2AF and U2 snRNP with the upstream 3' splice site, without affecting recognition of the downstream 5' splice site by U1. Remarkably, U1 snRNP-mediated recognition of the 5' splice site is required both for efficient U2AF binding and for U2AF inhibition by PTB. We propose that TIA-1 and PTB regulate Fas splicing and possibly Fas-mediated apoptosis by targeting molecular events that lead to exon definition.

We report here that the apoptosis-promoting protein TIA-1 regulates alternative pre-mRNA splicing of the Drosophila melanogaster gene male-specific-lethal 2 and of the human apoptotic gene Fas. TIA-1 associates selectively with pre-mRNAs that contain 5' splice sites followed by U-rich sequences. TIA-1 binding to the U-rich stretches facilitates 5' splice site recognition by U1 snRNP. This activity is critical for activation of the weak 5' splice site of msl-2 and for modulating the choice of splice site partner in Fas. Structural and functional similarities with the Saccharomyces cerevisiae splicing factor Nam8 suggest striking evolutionary conservation of a mechanism of pre-mRNA splicing regulation that controls biological processes as diverse as meiosis in yeast, dosage compensation in fruit flies, or programmed cell death in humans.

The U1 small nuclear ribonucleoprotein (U1 snRNP) binds to the pre-mRNA 5¢ splice site (ss) at early stages of spliceosome assembly. Recruitment of U1 to a class of weak 5¢ ss is promoted by binding of the protein TIA-1 to uridine-rich sequences immediately downstream from the 5¢ ss. Here we describe a molecular dissection of the activities of TIA-1. RNA recognition motifs (RRMs) 2 and 3 are necessary and suf®cient for binding to the pre-mRNA. The nonconsensus RRM1 and the C-terminal glutamine-rich (Q) domain are required for association with U1 snRNP and to facilitate its recruitment to 5¢ ss. Coprecipitation experiments revealed a speci®c and direct interaction involving the N-terminal region of the U1 protein U1-C and the Q-rich domain of TIA-1, an interaction enhanced by RRM1. The results argue that binding of TIA-1 in the vicinity of a 5¢ ss helps to stabilize U1 snRNP recruitment, at least in part, via a direct interaction with U1-C, thus providing one molecular mechanism for the function of this splicing regulator. Keywords: TIA-1/U1-C/U1 snRNP IntroductionThe excision of introns from mRNA precursors is important to generate translatable mRNAs in higher eukaryotes. The process is often regulated to generate alternatively spliced transcripts able to encode distinct proteins (Hastings and Krainer, 2001;Will and Lu Èhrmann, 2001;Modrek and Lee, 2002). The chemical process of intron removal occurs within the spliceosome, a complex of >100 polypeptides and ®ve uridine-rich small nuclear ribonucleoproteins (U snRNPs) assembled on the premRNA (Nilsen, 2002;Zhou and Reed, 2002).U1 snRNP recognizes the 5¢ splice site (ss) and is among the ®rst factors to interact with the pre-mRNA to form complexes (complex E in mammalian extracts) that commit the pre-mRNA to the splicing pathway (Ruby and Abelson, 1988;Se Âraphin and Rosbash 1989; Reed, 1991, 1993). Human U1 snRNP is composed of a 165 nucleotide RNA (U1 snRNA), seven different Sm proteins common to other snRNPs and three U1-speci®c polypeptides: U1-70K, U1-A and U1-C . The sequence of the 5¢ end of U1 snRNA is complementary to the 5¢ ss region (Rinke et al., 1984), and stem±loops I and II are bound directly by U1-70K and U1-A, respectively. A uridine-rich motif, the Sm site, is bound by the Sm proteins, most probably forming a ringlike structure around the Sm site (Hamm et al., 1987;Patton and Pederson 1988;Scherly et al., 1989Scherly et al., , 1990Lutz-Freyermuth et al., 1990;Kambach et al., 1999). U1-C does not interact directly with naked U1 snRNA, but depends on other U1 protein components for association with the snRNP. Interactions have been detected between the N-terminal 45 amino acids of U1-C, which include a zinc ®nger-like motif, and U1-70K, as well as with the Sm proteins B¢/B (Nelissen et al., 1994). This region of U1-C has been shown to stimulate formation or stabilization of complex E . In contrast, reconstituted snRNPs lacking U1-A, or lacking the binding sites for U1-A or U1-70K, can support splicing and complex E formation . These results indica...

The protein Sex-lethal (SXL) controls dosage compensation in Drosophila by inhibiting the splicing and translation of male-specific-lethal-2 (msl-2) transcripts. Here we report that splicing inhibition of msl-2 requires a binding site for SXL at the polypyrimidine (poly(Y)) tract associated with the 3' splice site, and an unusually long distance between the poly(Y) tract and the conserved AG dinucleotide at the 3' end of the intron. Only this combination allows efficient blockage of U2 small nuclear ribonucleoprotein particle binding and displacement of the large subunit of the U2 auxiliary factor (U2AF65) from the poly(Y) tract by SXL. Crosslinking experiments with ultraviolet light indicate that the small subunit of U2AF (U2AF35) contacts the AG dinucleotide only when located in proximity to the poly(Y) tract. This interaction stabilizes U2AF65 binding such that SXL can no longer displace it from the poly(Y) tract. Our results reveal a novel function for U2AF35, a critical role for the 3' splice site AG at the earliest steps of spliceosome assembly and the need for a weakened U2AF35-AG interaction to regulate intron removal.

RBM5/Luca-15/H37 is a gene frequently inactivated in lung cancers and overexpressed in breast tumors. Its protein product has been detected in prespliceosomal complexes and modulates cell proliferation and Fas-mediated apoptosis. We report that RBM5 is a component of complexes involved in 3' splice site recognition and regulates alternative splicing of apoptosis-related genes, including the Fas receptor, switching between isoforms with antagonistic functions in programmed cell death. In contrast with classical mechanisms of splicing regulation, RBM5 does not affect early events of splice site recognition that lead to Fas exon 6 definition. Instead, RBM5 inhibits the transition between prespliceosomal complexes assembled around exon 6 to mature spliceosomes assembled on the flanking introns and promotes sequence-specific pairing of the distal splice sites. An OCRE domain important for RBM5 function contacts components of the U4/5/6 tri-snRNP, consistent with the idea that RBM5 modulates splice site pairing after prespliceosome assembly and exon definition.