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PubMed2006

Role for Fox-1/Fox-2 in mediating the neuronal pathway of calcitonin/calcitonin gene-related peptide alternative RNA processing.

Although multiple regulatory elements and protein factors are known to regulate the non-neuronal pathway of alternative processing of the calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA, the mechanisms controlling the neuron-specific pathway have remained elusive. Here we report the identification of Fox-1 and Fox-2 proteins as novel regulators that mediate the neuron-specific splicing pattern. Fox-1 and Fox-2 proteins function to repress exon 4 inclusion, and this effect depends on two UGCAUG elements surrounding the 3' splice site of the calcitonin-specific exon 4. In neuron-like cells, mutation of a subset of UGCAUG elements promotes the non-neuronal pattern in which exon 4 is included. In HeLa cells, overexpression of Fox-1 or Fox-2 protein decreases exon 4 inclusion. Fox-1 and Fox-2 proteins interact with the UGCAUG elements specifically and regulate splicing by blocking U2AF(65) binding to the 3' splice site upstream of exon 4. We further investigated the inter-relationship between the UGCAUG silencer elements and the previously identified intronic and exonic splicing regulatory elements and found that exon 4 is regulated by an intricate balance of positive and negative regulation. These results define a critical role for Fox-1 and Fox-2 proteins in exon 4 inclusion of calcitonin/CGRP pre-mRNA and establish a regulatory network that controls the fate of exon 4.

Alternative Splicing↗

PubMed1995

Reconstitution of exon-bridging activity with purified U2AF and U1 snRNP components.

For pre-mRNAs containing multiple introns, the exon definition hypothesis has been proposed to account for the interactions that specify relatively short exons and prevent inappropriate exon-skipping [1]. Support for this hypothesis includes the finding that naturally occurring, or engineered mutations in 5' splice sites that weaken base complementary to U1 snRNA result in exon skipping due to a decrease in upstream 3' splice site activity. The reciprocal effect is also observed. For example, we found previously that the selection of the alternatively spliced rat preprotachykinin exon 4 is improved under conditions in which the adjacent 5' splice site is converted to a site with strengthened base pairing to U1 snRNA [2]. In the latter study, 3' splice site activity is improved in parallel with strengthened U1 snRNP binding to the downstream 5' splice site. Subsequent RNA-protein crosslinking experiments have provided evidence for exon bridging interactions between U2AF bound to the 3' splice site and U1 snRNP bound to the downstream 5' splice site in the preprotachykinin substrates [3]; see Figure 1. U2AF, a polypyrimidine tract binding protein composed of 65 and 35 kD subunits, is required for U2 snRNP binding to the adjacent branch site [4], [5]. In this work we have reconstituted exon bridging activity with purified components. These results show that U1 snRNP in addition to U2AF are the two components required to reconstitute full activity in vitro. The purified system has been used to test variants of U2AF and U1 snRNP. Our results show that the U1-A and U1-C proteins are dispensable for exon bridging activity. In addition, the 35 kD subunit of U2AF appears to be dispensable, at least under certain conditions.

Animals↗

PubMed1993

RNA annealing activity is intrinsically associated with U2AF.

U2AF is a protein that is essential for the formation of the prespliceosome complex during pre-mRNA splicing. It contains two subunits, 65 and 35 kDa, although only the 65-kDa subunit has been shown to be essential for its splicing activity. Here, we show that the 65-kDa subunit mediates the annealing of complementary single-stranded RNAs or single-stranded DNAs. This activity was shown to reverse the action of RNA helicase A, an enzyme that catalyzes the displacement of duplex RNAs. The NH2-terminal region of the 65-kDa subunit of U2AF, containing arginine-serine (RS) dipeptides and basic amino acid sequences, was shown to be essential for the annealing of complementary sequences, RNA binding, and the inhibition of RNA helicase A activity. Thus, through the combined action of U2AF and RNA helicases, duplex RNA regions can be reversibly formed and displaced. Such reactions appear to be critical for pre-mRNA splicing, translation, and transcription.

Base Sequence↗

PubMed2006

Structural and functional characterization of the TgDRE multidomain protein, a DNA repair enzyme from Toxoplasma gondii.

The parasite Toxoplasma gondii expresses a 55 kDa protein or TgDRE that belongs to a novel family of proteins characterized by the presence of three domains, a human splicing factor 45-like motif (SF), a glycine-rich motif (G-patch), and a RNA recognition motif (RRM). The two latter domains are mainly known as RNA-binding domains, and their presence in TgDRE, whose partial DNA repair function was demonstrated, suggests that the protein could also be involved in the RNA metabolism. In this work, we characterized the structure and function of the different domains by using single or multidomain proteins to define their putative role. The SF45-like domain has a helical conformation and is involved in the oligomerization of the protein. The G-patch domain, mainly unstructured on its own as well as in the presence of the SF upstream and RRM downstream domains, is able to bind small RNA oligonucleotides. We also report the structure determination of the RRM domain from the NMR data. It adopts a classical betaalphabetabetaalphabeta topology consisting of a four-stranded beta sheet packed against two alpha helices but does not present the key residues for the RNA interaction. In contrast, our analysis shows that the RRM of TgDRE is not only unable to bind small RNA oligonucleotides but it also shares the protein-protein interaction characteristics with two unusual RRMs of the U2AF heterodimeric splicing factor. The presence of both RNA- and protein-binding domains seems to indicate that TgDRE could also be involved in RNA metabolism.

Amino Acid Sequence↗

PubMed1995

Accumulation of a novel spliceosomal complex on pre-mRNAs containing branch site mutations.

Pre-mRNA assembles into spliceosomal complexes in the stepwise pathway E-->A-->B-->C. We show that mutations in the metazoan branchpoint sequence (BPS) have no apparent effect on E complex formation but block the assembly of the A complex and the UV cross-linking of U2 small nuclear ribonucleoprotein particle (snRNP) proteins. Unexpectedly, a novel complex, designated E*, assembles on pre-mRNAs containing BPS mutations. Unlike the E complex, the E* complex accumulates in the presence of ATP. U1 snRNP and U2AF, which are tightly bound to pre-mRNA in the E complex, are not tightly bound in the E* complex. Significantly, previous work showed that U1 snRNP and U2AF become destabilized from pre-mRNA after E complex assembly on normal pre-mRNAs. Thus, our data are consistent with a model in which there are two steps in the transition from the E complex to the A complex (E-->E*-->A). In the first step, U1 snRNP and U2AF are destabilized in an ATP-dependent, BPS-independent reaction. In the second step, the stable binding of U2 snRNP occurs in a BPS-dependent reaction.

Base Sequence↗

PubMed2005

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.

Alternative Splicing↗

PubMed1995

Isolation and mapping of human homologues of an imprinted mouse gene U2af1-rs1.

We have isolated human homologues of the imprinted mouse gene, U2af1-rs1. Two different types of cDNAs and three distinct genomic DNAs belonging to different groups were isolated. We have identified chromosomal genes corresponding to each cDNA by restriction mapping and sequencing. Using both a panel of rodent/human somatic cell hybrids and fluorescence in situ hybridization, group 1 and group 2 genes were mapped to chromosome 5q22 and chromosome Xp22.1, respectively. We designated group 1 and group 2 genes as human U2AF1-RS1 and U2AF1-RS2, respectively, because these genes corresponded to mouse U2af1-rs1 (chromosome 11) and U2af1-rs2 (chromosome X), which we also isolated and mapped. Amino acid sequences of human U2AF1-RS1 and U2AF-RS2 showed significant homology to U2AF small subunit. The group 3 gene, designated as U2AF1-RS3, of which the cDNA has not yet been isolated, was mapped to chromosome 19p13.2.

Amino Acid Sequence↗

PubMed2000

A novel type of splicing enhancer regulating adenovirus pre-mRNA splicing.

Splicing of the adenovirus IIIa pre-mRNA is subjected to a temporal regulation, such that efficient IIIa 3' splice site usage is confined to the late phase of the infectious cycle. Here we show that IIIa pre-mRNA splicing is activated more than 200-fold in nuclear extracts prepared from late adenovirus-infected cells (Ad-NE) compared to uninfected HeLa cell nuclear extracts (HeLa-NE). In contrast, splicing of the beta-globin pre-mRNA is repressed in Ad-NE. We constructed hybrid pre-mRNAs between IIIa and beta-globin in order to identify the minimal IIIa sequence element conferring enhanced splicing in Ad-NE. Using this approach, we show that the IIIa branch site/pyrimidine tract functions as a Janus element: it blocks splicing in HeLa-NE and functions as a splicing enhancer in Ad-NE. Therefore, we named this sequence the IIIa virus infection-dependent splicing enhancer (3VDE). This element is essential for regulated IIIa pre-mRNA splicing in Ad-NE and sufficient to confer an enhanced splicing phenotype to the beta-globin pre-mRNA in Ad-NE. We further show that the increase in IIIa splicing observed in Ad-NE is not accompanied by a similar increase in U2AF binding to the IIIa pyrimidine tract. This finding suggests that splicing activation by the 3VDE may operate without efficient U2AF interaction with the pre-mRNA. Importantly, this report represents the first description of a splicing enhancer that has evolved to function selectively in the context of a virus infection, a finding that adds a new level at which viruses may subvert the host cell RNA biosynthetic machinery to facilitate their own replication.

Adenoviridae↗

PubMed2002

The human papillomavirus type 31 late 3' untranslated region contains a complex bipartite negative regulatory element.

The papillomavirus life cycle is tightly linked to epithelial cell differentiation. Production of virus capsid proteins is restricted to the most terminally differentiated keratinocytes in the upper layers of the epithelium. However, mRNAs encoding the capsid proteins can be detected in less-differentiated cells, suggesting that late gene expression is controlled posttranscriptionally. Short sequence elements (less than 80 nucleotides in length) that inhibit gene expression in undifferentiated epithelial cells have been identified in the late 3' untranslated regions (UTRs) of several papillomaviruses, including the high-risk mucosal type human papillomavirus type 16 (HPV-16). Here we show that closely related high-risk mucosal type HPV-31 also contains elements that can act to repress gene expression in undifferentiated epithelial cells. However, the HPV-31 negative regulatory element is surprisingly complex, comprising a major inhibitory element of approximately 130 nucleotides upstream of the late polyadenylation site and a minor element of approximately 110 nucleotides mapping downstream. The first 60 nucleotides of the major element have 68% identity to the negative regulatory element of HPV-16, and these elements bind the same cellular proteins, CstF-64, U2AF(65), and HuR. The minor inhibitory element binds some cellular proteins in common with the major inhibitory element, though it also binds certain proteins that do not bind the upstream element.

3' Untranslated Regions↗

PubMed2000

Serologic responses in patients with malignant mesothelioma: evidence for both public and private specificities.

Malignant mesothelioma (MM) is a pulmonary malignancy that appears to be immunogenic based on a large number of studies in both animals and humans. This notion is supported by our recent demonstration using Western blot analysis of immunoglobulin G antibodies reactive with a variety of autoantigens in many patients with MM. In view of the enormous potential of such antigens in early diagnosis, immunotherapy, and vaccination of at-risk individuals, it was essential to identify these antigens. We therefore applied the SEREX technique (serologic identification by recombinant expression cloning), using a serum pool from six patients as the probe against an expressed complementary DNA library derived from a cloned MM cell line. We screened over one million recombinants and obtained sequence information on eight antigens that had provoked immunoglobulin heavy chain class switching, presumably as a consequence of T-cell recognition. Six of these antigens were identifiable (U2AF[65], Siah binding protein, topoisomerase IIbeta, ZFM1, mIre1, and pendulin), and of the others, one was found as a single EST from a myotube library (Jemm-1); the other (Jemm-2) was not represented in any EST database even as a weak homolog. Consistent with our previous findings, each of the characterizable antigens would be expected to be associated with the cell nucleus. Each of the autoantibody specificities was uniquely associated with a single patient with the exception of antibodies to TOPIIbeta and U2AF(65). We found 13 of 14 (93%) patients with MM had antibodies to TOPIIbeta and two of 14 (14%) patients had antibodies to U2AF(65). The number of serum reactivities, taken as a measure of the complexity of the immune response, correlates with patient survival and with an index of systemic inflammation. These data suggest that a broader range of serologic reactivities reflects a more active host response to the presence of tumor.

Animals↗

PubMed1997

Three-dimensional image reconstruction of large nuclear RNP (lnRNP) particles by automated electron tomography.

Nuclear RNA transcripts of split genes and their splicing products, as well as the general population of nuclear polyadenylated RNA are packaged in multi-component large nuclear ribonucleoprotein (lnRNP) particles. These lnRNP particles, which sediment at the 200 S region in sucrose gradients, contain all U small nuclear RNPs required for precursor messenger RNA (pre-mRNA) splicing and several protein splicing factors, including U2AF and the SR proteins. Electron microscopy of lnRNP particles revealed a large compact structure of 50 nm in diameter. In this study we employed automated computed tomography from electron micrographs for the three-dimensional (3D) image reconstruction of individual lnRNP particles isolated from mammalian cells nuclei and negatively stained. For each particle, a tilt series of 71 images was collected by direct digital recording of the images on a CCD camera attached to a computer controlled TEM facility. The 3D image was reconstructed according to the back projection principle. For rendering, real time display and comparison of the reconstructed particles, interactive computer graphics was employed. The reconstructed 3D images show a compact structure composed of four major subunits connected to each other. Comparison of the reconstructed lnRNP particles revealed morphological similarity of the individual particles, as well as similarity among the sub-structures. Based on these observations we propose a model for the packaging of nuclear pre-mRNAs in lnRNP particles where each substructure represents a functional unit. This model is compatible with the requirements for alternative splicing in multi-intronic pre-mRNAs, and with the fact that the splicing of multi-intronic pre-mRNAs does not occur in a sequential manner.

Animals↗

PubMed1994

Identification of an imprinted U2af binding protein related sequence on mouse chromosome 11 using the RLGS method.

A new imprinted gene has been discovered in mice using the technique of restriction landmark genomic scanning (RLGS) with methylation sensitive enzymes. Eight out of 3,100 strain-specific NotI and BssHII spots were identified as imprinted in reciprocal F1 hybrids. Subsequently, we isolated a genomic clone for one locus on proximal chromosome 11 near the Glns locus, an imprinted region in uniparental disomic mice, and its corresponding cDNA clone. Expression of this transcript from the paternal allele was established using RT-PCR of reciprocal F1-hybrid mice. The amino-acid sequence deduced from the cDNA showed significant homology to the U2 small nuclear ribonucleoprotein auxiliary factor 35 kDa subunit.

Amino Acid Sequence↗

PubMed2001

The hnRNP A1 protein regulates HIV-1 tat splicing via a novel intron silencer element.

The generation of >30 different HIV-1 mRNAs is achieved by alternative splicing of one primary transcript. The removal of the second tat intron is regulated by a combination of a suboptimal 3' splice site and cis-acting splicing enhancers and silencers. Here we show that hnRNP A1 inhibits splicing of this intron via a novel heterogeneous nuclear ribonucleoprotein (hnRNP) A1-responsive intron splicing silencer (ISS) that can function independently of the previously characterized exon splicing silencer (ESS3). Surprisingly, depletion of hnRNP A1 from the nuclear extract (NE) enables splicing to proceed in NE that contains 100-fold reduced concentrations of U2AF and normal levels of SR proteins, conditions that do not support processing of other efficiently spliced pre-mRNAs. Reconstituting the extract with recombinant hnRNP A1 protein restores splicing inhibition at a step subsequent to U2AF binding, mainly at the time of U2 snRNP association. hnRNP A1 interacts specifically with the ISS sequence, which overlaps with one of three alternative branch point sequences, pointing to a model where the entry of U2 snRNP is physically blocked by hnRNP A1 binding.

Alternative Splicing↗

PubMed2001

Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions.

It has been previously observed that the intrinsically weak variant GC donor sites, in order to be recognized by the U2-type spliceosome, possess strong consensus sequences maximized for base pair formation with U1 and U5/U6 snRNAs. However, variability in signal strength is a fundamental mechanism for splice site selection in alternative splicing. Here we report human alternative GC-AG introns (for the first time from any species), and show that while constitutive GC-AG introns do possess strong signals at their donor sites, a large subset of alternative GC-AG introns possess weak consensus sequences at their donor sites. Surprisingly, this subset of alternative isoforms shows strong consensus at acceptor exon positions 1 and 2. The improved consensus at the acceptor exon can facilitate a strong interaction with U5 snRNA, which tethers the two exons for ligation during the second step of splicing. Further, these isoforms nearly always possess alternative acceptor sites and exhibit particularly weak polypyrimidine tracts characteristic of AG-dependent introns. The acceptor exon nucleotides are part of the consensus required for the U2AF(35)-mediated recognition of AG in such introns. Such improved consensus at acceptor exons is not found in either normal or alternative GT-AG introns having weak donor sites or weak polypyrimidine tracts. The changes probably reflect mechanisms that allow GC-AG alternative intron isoforms to cope with two conflicting requirements, namely an apparent need for differential splice strength to direct the choice of alternative sites and a need for improved donor signals to compensate for the central mismatch base pair (C-A) in the RNA duplex of U1 snRNA and the pre-mRNA. The other important findings include (i) one in every twenty alternative introns is a GC-AG intron, and (ii) three of every five observed GC-AG introns are alternative isoforms.

Alternative Splicing↗

PubMed1996

Absence of imprinting in U2AFBPL, a human homologue of the imprinted mouse gene U2afbp-rs.

The mouse gene U2 auxiliary factor binding protein related sequence (U2afbp-rs) has previously been shown to be genomically imprinted with monoallelic expression from the paternal allele. To determine if the human homologue is imprinted and contains conserved structural features which regulate imprinting, we isolated genomic clones from a human P1-derived artificial chromosome (PAC) library that map to human chromosome 5q22-31, a region syntenic to the proximal portion of mouse chromosome 11 where U2afbp-rs resides. A genomic subclone was isolated which contained an open reading frame with high homology to the mouse gene. This subclone also maintained the intronless character of the mouse gene. A KpnI polymorphism within the open reading frame of the gene was found to occur in 21% (8/38) of the alleles tested from human placental tissue samples. RT-PCR analysis of human placentas using the KpnI polymorphism to determine the parental origin of the alleles indicates biallelic expression of the human chromosome 5 U2AFBPL gene.

Alleles↗

PubMed2000

The oocyte-specific methylated region of the U2afbp-rs/U2af1-rs1 gene is dispensable for its imprinted methylation.

Imprinted genes harbor discrete regions which are differentially methylated in gametes; usually the final differential methylation patterns in adults are established during embryogenesis through modifications of the initial methylation patterns in gametes. Previous reports have shown that a 200-bp region termed region II within the CpG island of the mouse imprinted U2afbp-rs gene is methylated in oocytes but not in sperm, suggesting that this region is a center for the propagation of methylated CpGs on the maternal allele and is also a candidate for an imprinting control element. To determine whether region II is required for the imprinted methylation of this gene at the endogenous locus, we generated mice carrying a deletion of this region. We herein show that parental methylation differences still exist in the CpG island on the region II-deleted allele. These findings suggest that region II is dispensable for the imprinted methylation of the U2afbp-rs gene.

Animals↗

PubMed1995

Stage-specific induction and regulation by genomic imprinting of the mouse U2afbp-rs gene during preimplantation development.

The mouse U2afbp-rs gene is imprinted and expressed exclusively from the paternal allele in the adult. To determine whether the U2afbp-rs gene is regulated by imprinting in the early embryo and to examine its normal temporal pattern of expression, we used a new quantitative RT-PCR approach to compare U2afbp-rs gene expression between androgenetic and gynogenetic embryos and to monitor U2afbp-rs expression during normal preimplantation embryogenesis. The U2afbp-rs gene is transcriptionally induced transiently at the 2-cell stage, making it one of the first genes ever identified with this pattern of regulation. At the 2-cell stage, androgenones express approximately twice as much U2afbp-rs mRNA as normal embryos, while gynogenones express much less. Thus, genomic imprinting regulates the U2afbp-rs gene from the time of its initial induction, distinguishing it from some other autosomal imprinted genes that have been analyzed (e.g., Igf2r and Igf2). Because the function of the U2afbp-rs protein has not been determined, the significance of transient induction of the U2afbp-rs gene at the 2-cell stage is not clear. Our data indicate that abundant expression of the U2afbp-rs gene is not essential for early development since gynogenones form blastocysts at a high frequency.

Animals↗

PubMed1998

Repeat-induced point mutations in Pad-1, a putative RNA splicing factor from Neurospora crassa, confer dominant lethal effects on ascus development.

We describe the characterization of a gene, Pad-1, from Neurospora crassa which displays sequence characteristics of the RS class of hnRNA-binding proteins (hnRNP) and mRNA splicing factors. This is the first report of the isolation of a putative hnRNP gene from N. crassa. PAD-1 showed 30% identity and 57% similarity to a protein, HCC1, which was isolated using autoantibodies from patients suffering from hepatocellular carcinoma. Both HCC1 and PAD-1 show amino acid sequence similarities to the human splicing factor, U2AF65. Mutations induced in Pad-1 by repeat-induced point (RIP) mutation show dominant effects on ascus and ascospore formation, a novel phenotypic class of RIP mutants. A mutant isolated from the Pad-1 RIP cross displayed a severe vegetative growth defect and dominant effects on ascus development, indicating that Pad-1 is essential for both asexual and sexual development.

Amino Acid Sequence↗