The Apoptosis-Promoting Factor TIA-1 Is a Regulator of Alternative Pre-mRNA Splicing

Abstract

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.

Evidence That Ternary Complex (eIF2-GTP-tRNAiMet)–Deficient Preinitiation Complexes Are Core Constituents of Mammalian Stress Granules

“…Recent reports indicate that TIA-1 regulates specific mRNA splicing events (Del Gatto-Konczak et al, 2000;Forch et al, 2000;Le Guiner et al, 2001), suggesting that an indirect function of SG assembly in the cytoplasm may be the altered splicing of specific nuclear transcripts, owing to the relocalization of TIA-1 from the nucleus to SGs. Indeed, the stressinduced relocalization of the shuttling protein hnRNP A1 from the nucleus to the cytoplasm appears to regulate premRNA splicing in vivo by such a mechanism (van der Houven van Oordt et al, 2000).…”

An in vitro-selected RNA-binding site for the KH domain protein PSI acts as a splicing inhibitor element

“…Antibody supershift assay+ Radiolabeled RNAs, wild-type 59 exon (lanes 1-4), L#456 (lanes 5-8), Up#6 (lanes 9-12), Up#2 (lanes 13-16), D#48 (lanes 17-20), and F1,F2-GT (lanes 20-24) were incubated with Kc nuclear extract for 30 min to allow complex assembly+ Affinity purified antibody was added after 30 min to the corresponding reactions as shown above each lane+ The free RNA (lanes 1, 5, 9, 13, 17, and 21) and RNA-protein complexes (A, B, and C) in the absence of antibody (lanes 2, 6, 10, 14, 18, and 22) are shown+ The supershifted complexes with anti- PSI (lanes 3, 7, 11, 15, 19, and 23) and anti-hrp48 (lanes 4, 8, 12, 16, 20, and 24) antibody are marked with arrows+ RNA-protein complex formed by PSI, hrp48, and several other unidentified proteins on the high affinity Up#6 SELEX RNA may inhibit splicing by sterically blocking U1 snRNP binding to the accurate 59 splice site and thereby preventing assembly of a functional spliceosome+ This idea of a steric block to U1 snRNP binding is consistent with previous results in which the F1/F2 sites were moved away from the IVS3 59 splice site resulting in a loss of inhibition (Siebel et al+, 1992)+ Because PSI can directly interact with U1 snRNP through its C-terminal A and B repeats, it is also possible that stably bound PSI protein on high affinity SELEX Up#6 RNA may bring U1 snRNP to the 59 exon RNA element in the absence of a strong U1 snRNA-pre-mRNA interaction and the presence of U1 snRNP and PSI would block the binding of U1 snRNP to the accurate 59 splice site+ Therefore, the presence of a strong PSI-binding site alone upstream of the IVS3 59 splice site appears to be sufficient to inhibit splicing of P element IVS3 in vitro+ There are several other reports of splicing factors that recruit U1 snRNP to the pre-mRNA and stabilize U1 snRNP-pre-mRNA interactions+ The yeast RNAbinding protein Mer1p, which has a single KH domain, is involved in mediating meiosis-specific alternative splicing of the MER2 pre-mRNA (Engebrecht et al+, 1991)+ Mer1p, which is expressed only in meiosis, binds to the region downstream of the weak 59 splice site of the MER2 transcript to facilitate meiosis-specific splicing of this intron+ Mer1p associates with U1 snRNP in the absence of MER2 target RNA (Spingola & Ares, 2000)+ By dividing the Mer1p protein into two fragments containing either the C-terminal KH domain or the N-terminal domain, it is known that only the N-terminal domain can coimmunoprecipitate U1 snRNA and that the KH domain has no direct interaction with U1 snRNP (Spingola & Ares, 2000)+ The yeast U1 snRNP-specific protein Nam8p, which is stably associated with U1 snRNA, has been shown to be required for efficient recognition of weak 59 splice sites (Puig et al+, 1999)+ It has been shown that Nam8p also binds to the region downstream of the weak 59 splice site of MER2 pre-mRNA and stabilizes the U1 snRNA-pre-mRNA base pairing interaction (Puig et al+, 1999)+ The mammalian homolog of Nam8p, the TIA-1 protein that was first identified as an effector of apoptosis (Tian et al+, 1991), also has been shown to function in U1 snRNP recruitment to a weak 59 splice site in the human apoptosis signaling receptor gene Fas premRNA (Forch et al+, 2000)+ TIA-1 interacts with the region downstream of the weak 59 splice site in the fas gene intron 5 to promote splicing and inclusion of the downstream exon 6 yields an mRNA encoding a membrane-bound active form of the fas receptor+ Another example of potential U1 snRNP...…”

“…Amarasinghe et al shows the preference of PSI for the pyrimidine-rich sequences in its natural target+ The recruitment of U1 snRNP to pseudo-59 splice sites, as seen for the P element IVS3 59 exon, or to weak 59 splice sites such as for MER2 and fas, could modulate the recognition of 59 splice sites to get either inhibition or activation, respectively+ Our data showed that a high affinity PSI SELEX RNA-binding sequence can function as a splicing inhibitor element and that the PSI protein is the major contributor to the inhibitory activity seen in the hybrid SELEX Up#6-P element IVS3 substrate splicing+ It is possible that similar sites are found near other 59 splice site-like sequences in PSI target cellular RNAs, in much the same way as the U-rich auxiliary regions are found near the msl-2 and fas 59 splice sites that function in TIA-1 binding (Forch et al+, 2000)+ Other examples of proteins with pyrimidinerich RNA-binding sites acting as splicing inhibitors include the Drosophila Sex-lethal protein (Singh et al+, 1995) and the hnRNPI/PTB protein (Singh et al+, 1995;Chou et al+, 2000)+ Thus, RNA-binding protein interaction with pre-mRNA sequences may act, both positively or negatively to control snRNP binding, splice site selection, and the early steps of intron recognition and spliceosome assembly+…”

High-affinity hnRNP A1 binding sites and duplex-forming inverted repeats have similar effects on 5??? splice site selection in support of a common looping out and repression mechanism

“…The fidelity of pre-messenger RNA splicing relies upon the appropriate selection of specific splicing partners amid a pool of sequences that resemble splice sites+ The process of splice site recognition and pairing, however, has to remain sufficiently flexible to accommodate the generation of alternatively spliced variants+ The control of splice site utilization can operate during the step-wise process of spliceosome assembly, and each step, in principle, can serve as a regulatory point+ Although recent progress has led to the identification of factors that either promote or inhibit the use of a splice site, their mechanisms of action remain poorly understood+ In one of the best-documented cases, the assembly of a soma-specific complex containing PSI, hrp48, and U1 snRNP prevents the binding of U1 snRNP to the authentic 59 splice site in the transposase premRNA of the P-element in Drosophila (Adams et al+, 1997)+ In contrast, the binding of TIA-1 to intron sequences facilitates 59 splice site recognition by U1 snRNP on some pre-mRNAs (Del Gatto-Konczak et al+, 2000;Forch et al+, 2000;Le Guiner et al+, 2001)+ In the case of the adenovirus L1 splicing unit, the binding of ASF/SF2 immediately upstream of the branch site flanking the IIIa exon sterically prevents U2 snRNP binding and hence represses splicing (Kanopka et al+, 1996)+ Likewise, hnRNP I/PTB has been found to antagonize the binding of U2AF to some 39 splice sites (Valcárcel & Gebauer, 1997)+ In other situations, exon enhancer elements bound by SR proteins can promote inclusion of an exon by stimulating the interaction of U2AF and/or U2 snRNP with the 39 splice site region (reviewed in Tacke & Manley, 1999;Graveley, 2000)+ Although some control elements do not affect the initial recognition of splicing signals (Gontarek et al+, 1993;Chou et al+, 2000), the exact mechanism by which many elements and regulatory factors affect splice site selection is currently unknown+…”