Polypyrimdine tract binding protein (PTB) is a regulator of alternative splicing, mRNA 3' end formation, mRNA stability and localization, and IRES-mediated translation. Transient overexpression of PTB can influence alternative splicing, sometimes resulting in nonphysiological splicing patterns. Here, we show that alternative skipping of PTB exon 11 leads to an mRNA that is removed by NMD and that this pathway consumes at least 20% of the PTB mRNA in HeLa cells. We also show that exon 11 skipping is itself promoted by PTB in a negative feedback loop. This autoregulation may serve both to prevent disruptively high levels of PTB expression and to restore nuclear levels when PTB is mobilized to the cytoplasm. Our findings suggest that alternative splicing can act not only to generate protein isoform diversity but also to quantitatively control gene expression and complement recent bioinformatic analyses, indicating a high prevalence of human alternative splicing leading to NMD.

The poly(A) signal of the C2 complement gene is unusual in that it possesses an upstream sequence element (USE) required for full activity in vivo. We describe here in vitro experiments demonstrating that this USE enhances both the cleavage and poly(A) addition reactions. We also show that the C2 USE can be cross-linked efficiently to a 55-kD protein that we identify as the polypyrimidine tract-binding protein (PTB), implicated previously in modulation of pre-mRNA splicing. Mutation of the PTB-binding site significantly reduces the efficiency of the C2 poly(A) site both in vivo and in vitro. Furthermore, addition of PTB to reconstituted processing reactions enhances cleavage at the C2 poly(A) site, indicating that PTB has a direct role in recognition of this signal. The C2 USE, however, also increases the affinity of general polyadenylation factors independently for the C2 poly(A) signal as detected by enhanced binding of cleavage-stimulaton factor (CstF). Strikingly, this leads to a novel CstF-dependant enhancement of the poly(A) synthesis phase of the reaction. These studies both emphasize the interconnection between splicing and polyadenylation and indicate an unexpected flexibility in the organization of mammalian poly(A) sites.

Polypyrimidine tract binding protein (PTB) is an RNA-binding protein that regulates splicing by repressing specific splicing events. It also has roles in 39-end processing, internal initiation of translation, and RNA localization. PTB exists in three alternatively spliced isoforms, PTB1, PTB2, and PTB4, which differ by the insertion of 19 or 26 amino acids, respectively, between the second and third RNA recognition motif domains. Here we show that the PTB isoforms have distinct activities upon a-tropomyosin (TM) alternative splicing. PTB1 reduced the repression of TM exon 3 in transfected smooth muscle cells, whereas PTB4 enhanced TM exon 3 skipping in vivo and in vitro. PTB2 had an intermediate effect. The PTB4 . PTB2 . PTB1 repressive hierarchy was observed in all in vivo and in vitro assays with TM, but the isoforms were equally active in inducing skipping of a-actinin exons and showed the opposite hierarchy of activity when tested for activation of IRES-driven translation. These findings establish that the ratio of PTB isoforms could form part of a cellular code that in turn controls the splicing of various other pre-mRNAs.

Polypyrimidine tract binding protein (PTB) is a major hnRNP protein with multiple roles in mRNA metabolism, including regulation of alternative splicing and internal ribosome entry site-driven translation. We show here that a fourfold overexpression of PTB results in a 75% reduction of mRNA levels produced from transfected gene constructs with different polyadenylation signals (pA signals). This effect is due to the reduced efficiency of mRNA 3 end cleavage, and in vitro analysis reveals that PTB competes with CstF for recognition of the pA signal's pyrimidine-rich downstream sequence element. This may be analogous to its role in alternative splicing, where PTB competes with U2AF for binding to pyrimidine-rich intronic sequences. The pA signal of the C2 complement gene unusually possesses a PTB-dependent upstream sequence, so that knockdown of PTB expression by RNA interference reduces C2 mRNA expression even though PTB overexpression still inhibits polyadenylation. Consequently, we show that PTB can act as a regulator of mRNA expression through both its negative and positive effects on mRNA 3 end processing.Between the branch point and the 3Ј splice site (3ЈSS) of metazoan introns lies a pyrimidine-rich sequence which is critical for efficient splicing (49). Initial analysis of factors that recognize this sequence identified an hnRNP-like protein called polypyrimidine tract binding protein (PTB) or hnRNP I (20,22,23,40). PTB has strong RNA binding activity, since it possesses four tandem RNA recognition motif domains (42). In vitro RNA binding analysis (SELEX) revealed its preferred RNA binding site as UCUU flanked by pyrimidines rather than a nonspecific pyrimidine sequence (41). Subsequent studies indicated that far from being a positively acting splicing factor, PTB actually acts as a selective splicing repressor (55, 57). The splicing factor responsible for recognition of the 3ЈSS pyrimidine tract and AG dinucleotide is the dimeric U2 auxiliary factor protein or U2AF (7). The U2AF 65-kDa subunit interacts with the pyrimidine tract (63), while the smaller 35-kDa subunit directly contacts the 3ЈSS sequence (34). Recognition of the pyrimidine tract by U2AF has been identified as a major site of splicing regulation. This can be modulated in a positive fashion through interaction with splicing-regulatory proteins bound to adjacent exon enhancer sequences (4, 51) or in a negative fashion by competition for binding with PTB (57). In many cases the pyrimidine tract of PTB-regulated exons contains high-affinity binding sites for PTB (41), and direct competition between PTB and U2AF 65 for binding to the pyrimidine tract can lead to exon skipping (28, 50). However, regulation by PTB often requires additional PTB-binding elements remote from the 3ЈSS pyrimidine tract. These may mediate cooperative binding of PTB (11), which can interfere with binding of U2AF as well as other splicing factors (57). Furthermore, PTB exists in several different isoforms generated by alternative splicing (PTB1-referred to throughout the p...

PTB (polypyrimidine tract-binding protein) is a repressive regulator of alternative splicing. We have investigated the role of PTB in three model alternative splicing systems. In the alpha-actinin gene, PTB represses the SM (smooth muscle) exon by binding to key sites in the polypyrimidine tract. Repressive binding to these sites is assisted by co-operative binding to additional downstream sites. SM exon splicing can be activated by CELF proteins, which also bind co-operatively to interspersed sites and displace PTB from the pyrimidine tract. Exon 11 of PTB pre-mRNA is repressed by PTB in an autoregulatory feedback loop. Exon 11-skipped RNA gets degraded through nonsense-mediated decay. Less than 1% of steady-state PTB mRNA is represented by this isoform, but inhibition of nonsense-mediated decay by RNA interference against Upf1 shows that at least 20% of PTB RNA is consumed by this pathway. This represents a widespread but under-appreciated role of alternative splicing in the quantitative regulation of gene expression, an important addition to its role as a generator of protein isoform diversity. Repression of alpha-tropomyosin exon 3 is an exceptional example of PTB regulation, because repression only occurs at high levels in SM cells, despite the fact that PTB is widely expressed. In this case, a PTB-interacting cofactor, raver1, appears to play an important role. By the use of 'tethering' assays, we have identified discrete domains within both PTB and raver1 that mediate their repressive activities on this splicing event.

Poly(A) signals of mammalian pre‐mRNA have been defined as an AAUAAA sequence 10–30 nt upstream of the cleavage/poly(A) site followed by a GU/U‐rich element immediately downstream. However, a number of viral poly(A) signals have been shown to possess additional signals upstream of AAUAAA that increase poly(A) site efficiency. We describe the first non‐viral example of such an upstream sequence element (USE) for the poly(A) site of the human C2 complement gene. As this gene is very closely spaced to the related Factor B gene [the C2 poly(A) site is only 421 bp from the transcription start site of Factor B] we have isolated this same intergenic sequence from four other mammals (mouse, cat, rabbit and cow). We show that the USE of the C2 poly(A) site is highly conserved between these five different mammals. Furthermore, extensive mutagenesis of the human USE indicates that most of the 53 nt sequence is required for full activity. The human C2 poly(A) site does not possess any obvious downstream GU/U‐rich sequences, although sequences immediately 3′ to AAUAAA as well as 13 nt of sequence following the cleavage site are both required for full activity. Interestingly the other mammalian C2 poly(A) sites do possess significant downstream GU/U‐rich sequences. Finally we show that all five mammalian C2 poly(A) signals are immediately followed by conserved signals for transcriptional termination, consistent with the close proximity of the downstream Factor B gene.

Exons with distant branch points

Exons with predicted branch points were identified from a large dataset of human exons and the importance of these branch points for splicing was verified

35 b varies from 10-fold in the brain to 20-fold in skeletal muscle. We propose that post-transcriptional regulation of U2AF1 gene expression may provide a mechanism by which the relative cellular concentration and availability of U2AF 35 protein isoforms are modulated, thus contributing to the finely tuned control of splicing events in different tissues.In higher eukaryotes, most protein-coding genes contain sequences that are spliced from the nascent transcripts (pre-mRNAs) in the nucleus. Intron excision is carried out by an assembly of small nuclear RNAs and proteins that are collectively recruited to pre-mRNAs forming the spliceosome (reviewed in Ref. 1). Although introns are excised with a high degree of precision, for many pre-mRNAs there is flexibility in the choice of alternative splice sites, often in response to tissuespecific, physiologically or developmentally regulated states. Alternative splicing produces multiple mRNAs encoding distinct proteins, thus expanding the coding capacity of genes and contributing to the proteomic complexity of higher organisms (2-4). Moreover, alternative splicing may contribute to regulate protein expression by generating premature termination codons that target the transcript to nonsense-mediated mRNA decay (5).In metazoans, pre-mRNA sequences implicated in splicing are only weakly conserved. Multiple, relatively weak proteinprotein and protein-RNA interactions involving these sequences and additional regulatory sequence elements, which can positively or negatively affect spliceosome assembly at nearby splice sites, constitute the basis to control alternative splicing (for recent reviews see Refs. 4, 6, and 7). Proteins that bind to pre-mRNA and affect splicing regulation include SR proteins, hnRNPs, 1 and tissue-or developmental stage-specific factors. Individual SR proteins interact only weakly with enhancer elements, but their binding to pre-mRNA is highly cooperative and can lead to recognition of distinct RNA sequence motifs, thus contributing to the selection of regulated splice sites. A distinct role is played by hnRNP proteins, which in general antagonize the stimulatory activity of SR proteins, and cell type-specific proteins, which may either inhibit or promote splicing. Thus, control of alternative splicing is achieved through the combinatorial interplay of both regulatory sequence signals and trans-acting protein factors (4,8,9).The spliceosome is a multicomponent RNA-protein machine containing five uracil-rich small nuclear ribonucleoproteins (U snRNPs) and many non-snRNP protein splicing factors. In the late 1990s ϳ100 splicing factors were identified (10), and since then the number has nearly doubled (1). Initiation of spliceosome recruitment to a pre-mRNA involves recognition of the 5Ј splice site by the U1 snRNP, whereas the U2 snRNP associates with the 3Ј region of the intron. The establishment of a stable interaction between U2 snRNP and pre-mRNA requires an auxiliary factor, U2AF (11). The U2