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At least 19 recordsLinked to original sources
PubMed1991

Characterization of cDNAs encoding the polypyrimidine tract-binding protein.

The polypyrimidine tract of mammalian introns is recognized by a 62-kD protein (pPTB). Mutations in the polypyrimidine tract that reduce the binding of pPTB also reduce the efficiency of formation of the pre-spliceosome complex containing U2 snRNP. The PTB protein was purified to homogeneity by affinity chromatography on a matrix containing poly(U), and peptide sequence was used to isolate several cDNAs. Because a variety of cell types express mRNA complementary to these cDNAs, PTB may be a ubiquitous splicing factor. Three classes of cDNAs were identified, on the basis of the presence of additional sequences at an internal position. This variation in sequence probably reflects alternative splicing of the PTB pre-mRNA and produces mRNAs encoding the prototype PTB protein, a form of PTB protein containing 19 additional residues, and a truncated form of PTB protein with a novel carboxyl terminus. A murine homolog of pPTB has been characterized previously as a DNA-binding protein. Sequence comparisons indicate that pPTB is distantly related to the hnRNP L protein and that these two proteins should be considered as members of a novel family of RNA-binding proteins.

Amino Acid Sequence↗
PubMed1995

Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins.

In higher eukaryotes, the polypyrimidine-tract (Py-tract) adjacent to the 3' splice site is recognized by several proteins, including the essential splicing factor U2AF65, the splicing regulator Sex-lethal (Sxl), and polypyrimidine tract-binding protein (PTB), whose function is unknown. Iterative in vitro genetic selection was used to show that these proteins have distinct sequence preferences. The uridine-rich degenerate sequences selected by U2AF65 are similar to those present in the diverse array of natural metazoan Py-tracts. In contrast, the Sxl-consensus is a highly specific sequence, which can help explain the ability of Sxl to regulate splicing of transformer pre-mRNA and autoregulate splicing of its own pre-mRNA. The PTB-consensus is not a typical Py-tract; it can be found in certain alternatively spliced pre-mRNAs that undergo negative regulation. Here it is shown that PTB can regulate alternative splicing by selectively repressing 3' splice sites that contain a PTB-binding site.

Animals↗
PubMed1991

Characterization and molecular cloning of polypyrimidine tract-binding protein: a component of a complex necessary for pre-mRNA splicing.

alpha-Tropomyosin exons 2 and 3 are spliced in a mutually exclusive manner. Exon 3 is included as the default exon in the mRNA of most cell types, whereas exon 2 is only included in the mRNA of smooth muscle cells. The primary determinant for the default selection of exon 3 is the branchpoint/polypyrimidine tract. This element upstream of exon 3 clearly and effectively outcompetes the corresponding element upstream of exon 2. To identify trans-acting factors that bind to this important cis element, we used UV cross-linking to identify a 57-kD protein whose binding characteristics directly correlate with 3'-splice-site selection in cis-competition splicing assays. This protein appears to be identical to polypyrimidine tract-binding protein. In this report we have used oligonucleotides derived from peptide sequences to isolate and sequence cDNA clones encoding this 57.2-kD protein. The primary sequence reveals a novel protein with significant homology to other RNA-binding proteins. Expression of the mRNA is detected in all tissues and cells examined, although its levels exhibit tissue-specific and developmental regulation. Using a biochemical complementation assay, we have found that this protein, along with a 100-kD protein, exists as part of a large complex that is required to rescue splicing from depleted nuclear extracts.

Amino Acid Sequence↗
PubMed1995

Interaction of polypyrimidine tract-binding protein with the 5' noncoding region of the hepatitis C virus RNA genome and its functional requirement in internal initiation of translation.

Initiation of translation of the human hepatitis C virus (HCV) RNA genome occurs by internal ribosome entry into the 5' noncoding region (5'NCR) in a cap-independent manner. The internal ribosome entry site of the HCV 5'NCR has been previously defined to encompass almost the entire 5'NCR. Here we report the interaction of polypyrimidine tract-binding protein (PTB) at three distinct regions within the 5'NCR by UV cross-linking assays. All three regions contain a consensus polypyrimidine tract motif. The evidence for the interaction of recombinant PTB at multiple sites within the 5'NCR is based on the use of 5'NCR mutants as competitors and by direct UV cross-linking of the mutant RNAs. Furthermore, the PTB isomers from HeLa nuclear extracts interact with the HCV 5'NCR, as shown by immunoprecipitation of a UV cross-linked complex with anti-PTB serum. Immunodepletion of PTB from translation lysates suggested the functional requirement for PTB during translation initiation of the HCV RNA. Addition of purified PTB to immunodepleted lysates did not restore translation mediated by the HCV 5'NCR, indicating the requirement of PTB-associated factors that were removed during immunodepletion.

Base Sequence↗
PubMed1995

Polypyrimidine tract-binding protein and heterogeneous nuclear ribonucleoprotein A1 bind to human T-cell leukemia virus type 2 RNA regulatory elements.

Efficient expression of human T-cell leukemia virus (HTLV) and human immunodeficiency virus structural proteins requires Rx and Rev proteins, respectively. Decreased expression of Gag and Env appears to be due, in part, to intragenic RNA sequences, termed cis-acting repressive sequences (CRS), and may be mediated by binding of specific cellular factors. We demonstrated previously that two cellular proteins, p60CRS and p40CRS, interact with HTLV type 2.5' long terminal repeat CRS RNA and that the interaction of both proteins with CRS RNA correlates with function (A. C. Black, C. T. Ruland, J. Luo, A. Bakker, J. K. Fraser, and J. D. Rosenblatt, Virology 200:29-41, 1994). By radioimmunoprecipitation of HeLa nuclear proteins UV cross-linked to CRS RNAs with murine monoclonal antibodies, we now show that p40CRS is heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and p60CRS is polypyrimidine tract-binding protein or hnRNP I. These immunoprecipitation results were confirmed by an immunobinding assay with hnRNP I and hnRNP AI antibodies and by cross-competition electrophoretic mobility shift experiments. In addition, we mapped a putative hnRNP A1 binding site in U5 RNA and demonstrated that p40CRS (hnRNP A1) binding to that site correlates with CRS function. Since both hnRNP I and hnRNP A1 have been shown to influence splicing and potentially other steps in RNA processing, the binding of both hnRNP I and hnRNP A1 to HTLV RNA regulatory elements may alter retrovirus RNA processing and may be involved in regulation by Rex.

Base Sequence↗
PubMed1992

hnRNP I, the polypyrimidine tract-binding protein: distinct nuclear localization and association with hnRNAs.

Many hnRNP proteins and snRNPs interact with hnRNA in the nucleus of eukaryotic cells and affect the fate of hnRNA and its processing into mRNA. There are at least 20 abundant proteins in vertebrate cell hnRNP complexes and their structure and arrangement on specific hnRNAs is likely to be important for the processing of pre-mRNAs. hnRNP I, a basic protein of ca. 58,000 daltons by SDS-PAGE, is one of the abundant hnRNA-binding proteins. Monoclonal antibodies to hnRNP I were produced and full length cDNA clones for hnRNP I were isolated and sequenced. The sequence of hnRNP I (59,632 daltons and pI 9.86) demonstrates that it is identical to the previously described polypyrimidine tract-binding protein (PTB) and shows that it is highly related to hnRNP L. The sequences of these two proteins, I and L, define a new family of hnRNP proteins within the large superfamily of the RNP consensus RNA-binding proteins. Here we describe experiments which reveal new and unique properties on the association of hnRNP I/PTB with hnRNP complexes and on its cellular localization. Micrococcal nuclease digestions show that hnRNP I, along with hnRNP S and P, is released from hnRNP complexes by nuclease digestion more readily than most other hnRNP proteins. This nuclease hypersensitivity suggests that hnRNP I is bound to hnRNA regions that are particularly exposed in the complexes. Immunofluorescence microscopy shows that hnRNP I is found in the nucleoplasm but in addition high concentrations are detected in a discrete perinucleolar structure. Thus, the PTB is one of the major proteins that bind pre-mRNAs; it is bound to nuclease-hypersensitive regions of the hnRNA-protein complexes and shows a novel pattern of nuclear localization.

Amino Acid Sequence↗
PubMed1995

A perinucleolar compartment contains several RNA polymerase III transcripts as well as the polypyrimidine tract-binding protein, hnRNP I.

We have investigated the subcellular organization of the four human Y RNAs. These RNAs, which are transcribed by RNA polymerase III, are usually found complexed with the Ro autoantigen, a 60-kD protein. We designed 2'-OMe oligoribonucleotides that were complementary to accessible single-stranded regions of Y RNAs within Ro RNPs and used them in fluorescence in situ hybridization. Although all four Y RNAs were primarily cytoplasmic, oligonucleotides directed against three of the RNAs hybridized to discrete structures near the nucleolar rim. We have termed these structures "perinucleolar compartments" (PNCs). Double labeling experiments with appropriate antisera revealed that PNCs are distinct from coiled bodies and fibrillar centers. Co-hybridization with a genomic DNA clone spanning the human Y1 and Y3 genes showed that PNCs are not stably associated with the transcription site for these Y RNAs. Although 5S rDNA was often located near the nucleolar periphery, PNCs are not associated with 5S gene loci. Two additional pol III transcripts, the RNA components of RNase P and RNase MRP, did colocalize within PNCs. Most interestingly, the polypyrimidine tract-binding protein hnRNP I/PTB was also concentrated in this compartment. Possible roles for this novel nuclear subdomain in macromolecular assembly and/or nucleocytoplasmic shuttling of these five pol III transcripts, along with hnRNP I/PTB, are discussed.

Base Sequence↗
PubMed2025

Proteins that recognize unique features of U7 snRNA and may substitute for Gemin5 in the assembly of U7-specific Sm ring.

U7 snRNA is a 60 nucleotide component of U7 snRNP, a multisubunit endonuclease that cleaves precursors of metazoan replication-dependent histone mRNAs at the 3' end, hence generating mature histone mRNAs. The Sm site in U7 snRNA differs from the Sm site in spliceosomal snRNAs and promotes the assembly of a unique Sm ring containing Lsm10 and Lsm11 instead of the spliceosomal SmD1 and SmD2 proteins. While the spliceosomal-type Sm site is recognized by Gemin5, a subunit of the SMN complex, the identity of the protein that recognizes the unusual Sm site of U7 snRNA resulting in the incorporation of Lsm10 and Lsm11 has not been determined. Here, we looked for proteins in mammalian extracts that interact with U7 snRNA and identified polypyrimidine tract-binding protein 1 (PTBP1) and insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) as two major proteins with this characteristic. The binding of PTBP1 and IGF2BP3 to U7 snRNA depends on its unique Sm site and on the upstream CUCUUU motif that base-pairs with histone pre-mRNAs and defines substrate specificity of U7 snRNP. Among proteins that bind U7 snRNA, we also identified hnRNP A1. We show that hnRNP A1 interacts with the SMN protein of the SMN complex, a likely prerequisite for the protein that substitutes for Gemin5 in the assembly of U7-specific Sm ring. Our results also suggest a mechanism that explains why Gemin5 does not bind the Sm site of U7 snRNA.

RNA, Small Nuclear↗
PubMed1994

Polypyrimidine tract sequences direct selection of alternative branch sites and influence protein binding.

IVS1, an intron derived from the rat fibronectin gene, is spliced inefficiently in vitro, involving the use of three alternative branch sites. Mutation of one branch point site, BP3, so as to increase complementarity to U2 snRNA resulted in exclusive use of that site and improved splicing efficiency, indicating that the wild type BP3 site is one determinant of poor IVS1 splicing. Deletions within the polypyrimidine tract had a variable effect on splicing efficiency and altered the pattern of branch site usage. Selection of each branch site was influenced negatively by purine substitutions ca. 20 nucleotides downstream. It is proposed that all three IVS1 branch sites are pyrimidine tract-dependent. Pyrimidine tract deletions also influenced the crosslinking of PTB (the polypyrimidine tract-binding protein), hnRNP C, and splicing factor U2AF65. All three proteins bound preferentially to distinct regions within the polypyrimidine tract and thus are candidates for mediating pyrimidine tract-dependent branch site selection. The findings indicate the complexity of the IVS1 polypyrimidine tract and suggest a crucial role for this region in modulating branch site selection and IVS1 splicing.

Alternative Splicing↗
PubMed1990

A 62,000 molecular weight spliceosome protein crosslinks to the intron polypyrimidine tract.

Incubation in HeLa nuclear extract of a 32P-labeled 61 nucleotide-long RNA corresponding to the lariat branch site/polypyrimidine tract/3' splice site of the first intron of human beta-globin pre-mRNA led to the crosslinking of a single protein of approximately 62,000 mol. wt. (p62). p62 corresponds to a polypyrimidine tract-binding protein recently described by Garcia-Blanco et al. (Genes & Dev. 3: 1874-1886, 1989). Crosslinking of p62 to the 61 nt RNA was highly sequence specific. No p62 crosslinking was observed with a 60 nt pGEM vector RNA, a 63 nt RNA antisense to the 61-mer or a 72 nt U2 RNA sequence. p62 crosslinking to the 61 nt RNA was competed by unlabeled 61 nt RNA, by beta-globin pre-mRNA containing intron 1, and by poly(U) and poly(C), but was competed to a lesser extent or not at all by pGEM RNA, a beta-globin RNA lacking intron 1, or poly(A). Experiments with mutated RNAs revealed that neither the lariat branch site adenosine nor the 3' splice site were required for p62 crosslinking to polypyrimidine tract-containing RNA. Elimination of the polypyrimidine tract reduced p62 crosslinking, as did mutation of a polypyrimidine tract UU dinucleotide to GA. However, replacement of a pyrimidine-rich tract immediately adjacent (3') to the lariat branch site with a 57% A + G pGEM vector RNA sequence also significantly reduced p62 crosslinking, indicating the involvement of both this pyrimidine-rich region and the classical polypyrimidine tract adjacent to the 3' splice site. The sites of protein interaction were further defined by RNase H protection experiments, the results of which confirmed the patterns of p62 crosslinking to mutant RNAs. p62 crosslinking was efficiently competed by a DNA oligonucleotide having the same sequence as the 61 nt RNA, showing that p62 requires neither ribose 2' OH groups nor uracil bases for its interaction with the polypyrimidine tract. p62 was not crosslinked to double-stranded 61 nt RNA. Q-Sepharose chromatography of HeLa nuclear extract yielded an unbound fraction (QU) in which p62 was the only polypyrimidine tract-crosslinkable protein and a bound fraction (QB) in which, surprisingly, several non-p62 proteins were crosslinkable to the polypyrimidine tract RNA. Yet, when the two Q-Sepharose fractions were combined, p62 strongly out-competed the otherwise-crosslinkable QB proteins for polypyrimidine tract RNA crosslinking. This indicates that p62 may have the highest affinity and/or crosslinking efficiency for the intron polypyrimidine tract of any HeLa nuclear protein.

Base Sequence↗
PubMed1995

Regulation of alternative 3' splice site selection by constitutive splicing factors.

We have devised an in vitro splicing assay in which the mutually exclusive exons 2 and 3 of alpha-tropomyosin act as competing 3' splice sites for joining to exon 1. Splicing in normal HeLa cell nuclear extracts results in almost exclusive joining of exons 1 and 3. Splicing in decreased nuclear extract concentrations and decreased ionic strength results in increased 1-2 splicing. We have used this assay to determine the role of three constitutive pre-mRNA splicing factors on alternative 3' splice site selection. Polypyrimidine tract binding protein (PTB) was found to inhibit the splicing of introns containing a strong binding site for this factor. However, the inhibitory effect of PTB could be partially reversed if pre-mRNAs were preincubated with U2 auxiliary factor (U2AF) prior to splicing in PTB-supplemented extracts. For alpha-tropomyosin, regulation of splicing by PTB and U2AF primarily affected the joining of exons 1-3 with no dramatic increases in 1-2 splicing being detected. Preincubation of pre-mRNAs with SR proteins led to small increases in 1-2 splicing. However, if pre-mRNAs were preincubated with SR proteins followed by splicing in PTB-supplemented extracts, there was a nearly complete reversal of the normal 1-2 to 1-3 splicing ratios. Thus, multiple pairwise, and sometimes antagonizing, interactions between constitutive pre-mRNA splicing factors and the pre-mRNA can regulate 3' splice site selection.

Base Sequence↗
PubMed2026

PTBP1 at the host-virus interface: mechanistic roles in viral RNA translation, replication, and immune modulation.

Viruses require the involvement of host RNA binding proteins for completion of important steps of their life cycle. Polypyrimidine tract binding protein 1 (PTBP1) is an RNA-binding protein found ubiquitously which performs important regulatory functions like alternative splicing, RNA stability, RNA localization, and translation by virtue of its four RRMs and shuttling between nucleus and cytoplasm. There is increasing evidence showing that many viruses make use of such regulatory roles of PTBP1 to facilitate their gene expression and replication. This review describes the existing mechanistic knowledge about the PTBP1 functions during viral infection, paying attention to the role of PTBP1 in viral RNA translation, viral RNA genome replication, and regulation of host antiviral response. Special attention is paid to the regulation by PTBP1 of IRES-dependent translation of enteroviruses and hepatitis C virus, as well as to the PTBP1 contribution to RNA stabilization, long-distance RNA interactions, and genome cyclization of flaviviruses such as dengue virus and Japanese encephalitis virus. Recent data on the PTBP1 function in coronavirus RNA metabolism are discussed as well. Furthermore, the role of PTBP1 in being both proviral and antiviral is reviewed in terms of innate immunity signalling pathways, stress granule biology, and virus-host interaction. Finally, we will explore the possibility of PTBP1 being used as a host-directed antiviral drug target despite the hurdles in doing so considering its multifunctionality as an essential cellular RNA-binding protein.

Polypyrimidine Tract-Binding Protein↗
PubMed1992

The rat poly pyrimidine tract binding protein (PTB) interacts with a single-stranded DNA motif in a liver-specific enhancer.

We characterized and purified a protein from rat liver which specifically binds to a DNA motif present in a liver-specific enhancer of the rat tyrosine aminotransferase (TAT) gene, when offered as single-stranded DNA. Binding is highly sequence-specific and coincides with a region known to be essential for function of the enhancer. Microsequencing revealed that this protein is the rat homologue of the mouse and human poly Pyrimidine Tract binding protein (PTB), which has been shown to bind to premRNA and may participate in RNA splicing. This finding was corroborated by subsequent Western blot experiments using a PTB-specific antibody. These findings indicate a possible dual role for this protein in RNA processing and transcription.

Amino Acid Sequence↗
PubMed1995

Alternative translation initiation of the Moloney murine leukemia virus mRNA controlled by internal ribosome entry involving the p57/PTB splicing factor.

Moloney murine leukemia virus (Mo-MuLV) genomic mRNA codes for two gag precursors by alternative initiations of translation. An AUG codon governs the synthesis of the retroviral capsid proteins precursor, whereas a CUG codon directs the synthesis of a glycosylated cell surface antigen, the gross cell surface antigen. Control of the relative synthesis of the two precursors is crucial for MuLV infectivity and pathology. Furthermore, the MuLV mRNA leader sequence is very long and should inhibit translation according to the classical scanning model. This suggests a different translation initiation mechanism allowing gag efficient expression. We demonstrate, by using bicistronic vectors expressed in COS-7 cells, that the Mo-MuLV mRNA leader drives translation initiation by internal ribosome entry. We have localized the internal ribosome entry site (IRES) between the two initiation codons. This 126 nucleotide long IRES implies an oligopyrimidine tract located 45 nucleotides upstream of AUG codon. UV cross-linking and affinity chromatography experiments show that the PTB/p57 splicing factor specifically interacts with this oligopyrimidine tract. The MuLV IRES controls alternative translation initiation by activating the capsid protein precursor expression. This gag translational enhancer could exist in other retroviruses.

Animals↗
PubMed1995

Cell proteins bind to a linear polypyrimidine-rich sequence within the 5'-untranslated region of rhinovirus 14 RNA.

Members of the picornavirus family initiate translation of their RNA genomes by a cap-independent mechanism in which ribosomes bind to an internal site in the 5' untranslated region (5'-UTR). This unique process requires an internal ribosome entry site (IRES), a highly structured RNA whose function is mediated in part by interactions with cell proteins. The IRES element of human rhinovirus 2 (HRV-2) extends from nucleotide (nt) 10 to between nt 544 and 568 and has been shown to interact with two cell proteins, pyrimidine tract-binding protein (pPTB) and p97. To map the specific regions of HRV-14 RNA that bind cell proteins, mobility shift, UV cross-linking and Western immunoblot analyses were performed. The results indicate that an RNA sequence from nt 538 to 591 interacts with pPTB and La, two proteins previously shown to functionally interact with the IRES elements of several picornaviruses. Two additional proteins, p97 and p68, were also cross-linked to nt 538 to 591 RNA. These four proteins interact with a putatively unstructured portion of the 5'-UTR that contains a polypyrimidine tract and has been shown to be present at the 3' border of sequences that are essential for IRES function of HRV-2. These protein-RNA interactions are likely to play a role in internal initiation of translation.

Base Sequence↗
PubMed1994

Binding of eukaryotic initiation factor-2 and trans-acting factors to the 5' untranslated region of encephalomyocarditis virus RNA.

The encephalomyocarditis virus 5' untranslated region (EMC 5' UTR) has a binding site for eukaryotic initiation factor eIF-2. Mutations in the 3' end or deletion of the 5' end of the internal ribosomal entry site had a negative effect on the binding of eIF-2 to the EMC 5' UTR. The binding of eIF-2 to the mutant 5' UTRs was completely inhibited by the addition of competitor tRNA. Cross-linking of the EMC 5' UTR with proteins from rabbit reticulocyte lysates showed binding of trans-acting factors p52 and p57. Deletions in the 5' end of the internal ribosomal entry site resulted in a loss of the ability to bind trans-acting factor p57, in accordance with literature data, while p52 binding to these deletion mutants was weak compared to the wildtype EMC 5' UTR. Mutations in the 3' part of the 5' UTR of EMC still resulted in binding of both trans-acting factors, as with wild type RNA, but binding was more sensitive to competitor tRNA when compared to the binding of p52/p57 to the wild type 5' UTR.

Base Sequence↗
PubMed1992

Polypyrimidine tract binding protein interacts with sequences involved in alternative splicing of beta-tropomyosin pre-mRNA.

Previous studies of alternative splicing of the rat beta-tropomyosin gene have shown that nonmuscle cells contain factors that block the use of the skeletal muscle exon 7 (Guo, W., Mulligan, G. J., Wormsley, S., and Helfman, D. M. (1991) Genes & Dev. 5, 2095-2106). Using an RNA mobility-shift assay we have identified factors in HeLa cell nuclear extracts that specifically interact with sequences responsible for exon blockage. Here we present the purification to apparent homogeneity of a protein that exhibits these sequence specific RNA binding properties. This protein is identical to the polypyrimidine tract binding protein (PTB) which other studies have suggested is involved in the recognition and efficient use of 3'-splice sites. PTB binds to two distinct functional elements within intron 6 of the beta-tropomyosin pre-mRNA: 1) the polypyrimidine tract sequences required for the use of branch points associated with the splicing of exon 7, and 2) the intron regulatory element that is involved in the repression of exon 7. Our results demonstrate that the sequence requirements for PTB binding are different than previously reported and shows that PTB binding cannot be predicted solely on the basis of pyrimidine content. In addition, PTB fails to bind stably to sequences within intron 5 and intron 7 of beta-TM pre-mRNA, yet forms a stable complex with sequences in intron 6, which is not normally spliced in HeLa cells in vitro and in vivo. The nature of the interactions of PTB within this regulated intron reveals several new details about the binding specificity of PTB and suggests that PTB does not function exclusively in a positive manner in the recognition and use of 3'-splice sites.

Alternative Splicing↗