Regulation of Fas alternative splicing by antagonistic effects of TIA-1 and PTB on exon definition

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.     

The Pre-mRNA 5′ Cap Determines Whether U6 Small Nuclear RNA Succeeds U1 Small Nuclear Ribonucleoprotein Particle at 5′ Splice Sites

Efficient splicing of the 5′-most intron of pre-mRNA requires a 5′ m⁷G(5′)ppp(5′)N cap, which has been implicated in U1 snRNP binding to 5′ splice sites. We demonstrate that the cap alters the kinetic profile of U1 snRNP binding, but its major effect is on U6 snRNA binding. With two alternative wild-type splice sites in an adenovirus pre-mRNA, the cap selectively alters U1 snRNA binding at the site to which cap-independent U1 snRNP binding is stronger and that is used predominantly in splicing; with two consensus sites, the cap acts on both, even though one is substantially preferred for splicing. However, the most striking quantitative effect of the 5′ cap is neither on U1 snRNP binding nor on the assembly of large complexes but on the replacement of U1 snRNP by U6 snRNA at the 5′ splice site. Inhibition of splicing by a cap analogue is correlated with the loss of U6 interactions at the 5′ splice site and not with any loss of U1 snRNP binding.     

The RNA-binding protein TIA-1 is a novel mammalian splicing regulator acting through intron sequences adjacent to a 5' splice site

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.     

The splicing regulator TIA-1 interacts with U1-C to promote U1 snRNP recruitment to 5' splice sites

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 sufficient for binding to the pre-mRNA. The non- consensus RRM1 and the C-terminal glutamine-rich (Q) domain are required for association with U1 snRNP and to facilitate its recruitment to 5' ss. Co-precipitation experiments revealed a specific 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.     

The Conserved Intronic Cleavage and Polyadenylation Site of CstF-77 Gene Imparts Control of 3′ End Processing Activity through Feedback Autoregulation and by U1 snRNP

The human gene encoding the cleavage/polyadenylation (C/P) factor CstF-77 contains 21 exons. However, intron 3 (In3) accounts for nearly half of the gene region, and contains a C/P site (pA) with medium strength, leading to short mRNA isoforms with no apparent protein products. This intron contains a weak 5′ splice site (5′SS), opposite to the general trend for large introns in the human genome. Importantly, the intron size and strengths of 5′SS and pA are all highly conserved across vertebrates, and perturbation of these parameters drastically alters intronic C/P. We found that the usage of In3 pA is responsive to the expression level of CstF-77 as well as several other C/P factors, indicating it attenuates the expression of CstF-77 via a negative feedback mechanism. Significantly, intronic C/P of CstF-77 pre-mRNA correlates with global 3′UTR length across cells and tissues. In addition, inhibition of U1 snRNP also leads to regulation of the usage of In3 pA, suggesting that the C/P activity in the cell can be cross-regulated by splicing, leading to coordination between these two processes. Importantly, perturbation of CstF-77 expression leads to widespread alternative cleavage and polyadenylation (APA) and disturbance of cell proliferation and differentiation. Thus, the conserved intronic pA of the CstF-77 gene may function as a sensor for cellular C/P and splicing activities, controlling the homeostasis of CstF-77 and C/P activity and impacting cell proliferation and differentiation.     

Efficient polyadenylation of Rous sarcoma virus RNA requires the negative regulator of splicing element

Rous sarcoma virus pre-mRNA contains an element known as the negative regulator of splicing (NRS) that acts to inhibit viral RNA splicing. The NRS binds serine/arginine-rich (SR) proteins, hnRNP H and the U1/U11 snRNPs, and appears to inhibit splicing by acting as a decoy 5′ splice site. Deletions within the gag gene that encompass the NRS also lead to increased read-through past the viral polyadenylation site, suggesting a role for the NRS in promoting polyadenylation. Using NRS-specific deletions and mutations, we show here that a polyadenylation stimulatory activity maps directly to the NRS and is most likely dependent upon SR proteins and U1 and/or U11 snRNP. hnRNP H does not appear to mediate splicing control or stimulate RSV polyadenylation, since viral RNAs containing hnRNP H-specific mutations were spliced and polyadenylated normally. However, the ability of hnRNP H mutations to suppress the read-through caused by an SR protein mutation suggests the potential for hnRNP H to antagonize polyadenylation. Interestingly, disruption of splicing control closely correlated with increased read-through, indicating that a functional NRS is necessary for efficient RSV polyadenylation rather than binding of an individual factor. We propose a model in which the NRS serves to enhance polyadenylation of RSV unspliced RNA in a process analogous to the stimulation of cellular pre-mRNA polyadenylation by splicing complexes.     

Identification and characterization of two internal cleavage and polyadenylation sites of parvovirus B19 RNA

Polyadenylation of B19 pre-mRNAs at the major internal site, (pA)p1, is programmed by the nonconsensus core cleavage and polyadenylation specificity factor-binding hexanucleotide AUUAAA. Efficient use of this element requires both downstream and upstream cis-acting elements and is further influenced by an adjacent AAUAAC motif. The primary hexanucleotide element must be nonconsensus to allow efficient readthrough of P6-generated pre-mRNAs into the capsid-coding region. An additional cleavage and polyadenylation site, (pA)p2, 296 nucleotides downstream of (pA)p1 was shown to be used following both B19 infection and transfection of a genomic clone. RNAs polyadenylated at (pA)p2 comprise approximately 10% of B19 RNAs that are polyadenylated internally.