A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability.

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.     

Paxillin associates with poly(A)-binding protein 1 at the dense endoplasmic reticulum and the leading edge of migrating cells.

Using mass spectrometry we have identified proteins which co-immunoprecipitate with paxillin, an adaptor protein implicated in the integrin-mediated signaling pathways of cell motility. A major component of paxillin immunoprecipitates was poly(A)-binding protein 1, a 70-kDa mRNA-binding protein. Poly(A)-binding protein 1 associated with both the alpha and beta isoforms of paxillin, and this was unaffected by RNase treatment consistent with a protein-protein interaction. The NH(2)-terminal region of paxillin (residues 54-313) associated directly with poly(A)-binding protein 1 in cell lysates, and with His-poly(A)-binding protein 1 immobilized in microtiter wells. Binding was specific, saturable and of high affinity (K(d) of approximately 10 nm). Cell fractionation studies showed that at steady state, the bulk of paxillin and poly(A)-binding protein 1 was present in the "dense" polyribosome-associated endoplasmic reticulum. However, inhibition of nuclear export with leptomycin B caused paxillin and poly(A)-binding protein 1 to accumulate in the nucleus, indicating that they shuttle between the nuclear and cytoplasmic compartments. When cells migrate, poly(A)-binding protein 1 colocalized with paxillin-beta at the tips of lamellipodia. Our results suggest a new mechanism whereby a paxillin x poly(A)-binding protein 1 complex facilitates transport of mRNA from the nucleus to sites of protein synthesis at the endoplasmic reticulum and the leading lamella during cell migration.     

Primary structure of human nuclear ribonucleoprotein particle C proteins: conservation of sequence and domain structures in heterogeneous nuclear RNA, mRNA, and pre-rRNA-binding proteins.

In the eucaryotic nucleus, heterogeneous nuclear RNAs exist in a complex with a specific set of proteins to form heterogeneous nuclear ribonucleoprotein particles (hnRNPs). The C proteins, C1 and C2, are major constituents of hnRNPs and appear to play a role in RNA splicing as suggested by antibody inhibition and immunodepletion experiments. With the use of a previously described partial cDNA clone as a hybridization probe, full-length cDNAs for the human C proteins were isolated. All of the cDNAs isolated hybridized to two poly(A)+ RNAs of 1.9 and 1.4 kilobases (kb). DNA sequencing of a cDNA clone for the 1.9-kb mRNA (pHC12) revealed a single open reading frame of 290 amino acids coding for a protein of 31,931 daltons and two polyadenylation signals, AAUAAA, approximately 400 base pairs apart in the 3' untranslated region of the mRNA. DNA sequencing of a clone corresponding to the 1.4-kb mRNA (pHC5) indicated that the sequence of this mRNA is identical to that of the 1.9-kb mRNA up to the first polyadenylation signal which it uses. Both mRNAs therefore have the same coding capacity and are probably transcribed from a single gene. Translation in vitro of the 1.9-kb mRNA selected by hybridization with a 3'-end subfragment of pHC12 demonstrated that it by itself can direct the synthesis of both C1 and C2. The difference between the C1 and C2 proteins which results in their electrophoretic separation is not known, but most likely one of them is generated from the other posttranslationally. Since several hnRNP proteins appeared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as multiple antigenically related polypeptides, this raises the possibility that some of these other groups of hnRNP proteins are also each produced from a single mRNA. The predicted amino acid sequence of the protein indicates that it is composed of two distinct domains: an amino terminus that contains what we have recently described as a RNP consensus sequence, which is the putative RNA-binding site, and a carboxy terminus that is very negatively charged, contains no aromatic amino acids or prolines, and contains a putative nucleoside triphosphate-binding fold, as well as a phosphorylation site for casein kinase type II. The RNP consensus sequence was also found in the yeast poly(A)-binding protein (PABP), the heterogeneous nuclear RNA-binding proteins A1 and A2, and the pre-rRNA binding protein C23. All of these proteins are also composed of at least two distinct domains: an amino terminus, which possesses one or more RNP consensus sequences, and a carboxy terminus, which is unique to each protein, being very acidic in the C proteins and rich in glycine in A1, and C23 and rich in proline in the poly(A)-binding protein. These findings suggest that the amino terminus of these proteins possesses a highly conserved RNA-binding domain, whereas the carboxy terminus contains a region essential to the unique function and interactions of each of the RNA-binding proteins.     

The 36-kilodalton embryonic-type cytoplasmic polyadenylation element-binding protein in Xenopus laevis is ElrA, a member of the ELAV family of RNA-binding proteins.

The translational activation of several maternal mRNAs in Xenopus laevis is dependent on cytoplasmic poly(A) elongation. Messages harboring the UUUUUAU-type cytoplasmic polyadenylation element (CPE) in their 3' untranslated regions (UTRs) undergo polyadenylation and translation during oocyte maturation. This CPE is bound by the protein CPEB, which is essential for polyadenylation. mRNAs that have the poly(U)12-27 embryonic-type CPE (eCPE) in their 3' UTRs undergo polyadenylation and translation during the early cleavage and blastula stages. A 36-kDa eCPE-binding protein in oocytes and embryos has been identified by UV cross-linking. We now report that this 36-kDa protein is ElrA, a member of the ELAV family of RNA-binding proteins. The proteins are identical in size, antibody directed against ElrA immunoprecipitates the 36-kDa protein, and the two proteins have the same RNA binding specificity in vitro. C12 and activin receptor mRNAs, both of which contain eCPEs, are detected in immunoprecipitated ElrA-mRNP complexes from eggs and embryos. In addition, this in vivo interaction requires the eCPE. Although a number of experiments failed to define a role for ElrA in cytoplasmic polyadenylation, the expression of a dominant negative ElrA protein in embryos results in an exogastrulation phenotype. The possible functions of ElrA in gastrulation are discussed.     

RNA-binding properties of a translational activator, the adenovirus L4 100-kilodalton protein.

The adenovirus L4 100-kDa nonstructural protein (100K protein) is required for efficient initiation of translation of viral late mRNA species during the late mRNA species during the late phase of infection (B. W. Hayes, G. C. Telling, M. M. Myat, J. F. Williams, and S. J. Flint, J. Virol. 64:2732-2742, 1990). The RNA-binding properties of this protein were analyzed in an immunoprecipitation assay with the 100K-specific monoclonal antibody 2100K-1 (C. L. Cepko and P. A. Sharp, Virology 129:137-154, 1983). Coprecipitation of the 100K protein and 3H-infected cell RNA was demonstrated. The RNA-binding activity of the 100K protein was inhibited by single-stranded DNA but not by double-stranded DNA, double-stranded RNA, or tRNA. Competition assays were used to investigate the specificity with which the 100K protein binds to RNA in vitro. Although the protein exhibited a strong preference for the ribohomopolymer poly(U) or poly(G), no specific binding to viral mRNA species could be detected; uninfected or adenovirus type 5-infected HeLa cell poly(A)-containing and poly(A)-lacking RNAs were all effective inhibitors of binding of the protein to viral late mRNA. Similar results were obtained when the binding of the 100K protein to a single, in vitro-synthesized L2 mRNA was assessed. The poly(U)-binding activity of the 100K protein was used to compare the RNA-binding properties of the 100K protein prepared from cells infected by adenovirus type 5 and the H5ts1 mutant (B. W. Hayes, G. C. Telling, M. M. Myat, J. F. Williams, and S. J. Flint, J. Virol. 64:2732-2742, 1990). A temperature-dependent decrease in H5ts1 100K protein binding was observed, correlating with the impaired translational function of this protein in vivo. By contrast, wild-type 100K protein RNA binding was unaffected by temperature. These data suggest that the 100K protein acts to increase the translational efficiency of viral late mRNA species by a mechanism that involves binding to RNA.     

Different isoforms of PRIP-interacting protein with methyltransferase domain/trimethylguanosine synthase localizes to the cytoplasm and nucleus

A protein family including the recently identified PIMT/Tgs1 (PRIP-interacting protein with methyltransferase domain/trimethylguanosine synthase) was identified by searching databases for homologues of a newly identified Drosophila protein with RNA-binding activity and methyltransferase domain. Antibodies raised against a short peptide of the mammalian homologue show a 90-kDa isoform expressed specifically in rat brain and testis and a 55-kDa form expressed ubiquitously. In HeLa cells, the larger isoform of the protein is nuclear and associated with a 600-kDa complex, while the smaller isoform is mainly cytoplasmic and co-localizes to the tubulin network. Inhibition of PIMT/Tgs1 expression by siRNA in HeLa cells resulted in an increase in the percentage of cells in G2/M phases. In yeast two-hybrid and in vitro GST pull down experiments, the conserved C-terminal region of PIMT/Tgs1 interacted with the WD domain containing EED/WAIT-1 that acts as a polycomb-type repressor in the nucleus and also binds to integrins in the cytoplasm. Our experiments, together with earlier data, indicate that isoforms of the PIMT/Tgs1 protein with an RNA methyltransferase domain function both in the nucleus and in the cytoplasm and associate with both elements of the cytoskeletal network and nuclear factors known to be involved in gene regulation.     

Poly(L-proline)-binding proteins from chick embryos are a profilin and a profilactin

Two poly(L-proline)-binding proteins (PBP-1 and PBP-2) were purified from chick embryos by using a poly(L-proline)-agarose column. PBP-1 was composed of two different polypeptides (molecular masses of 42 kDa and 15 kDa). The molar ratio of the two proteins in the complex was 1:1. The other poly(L-proline)-binding protein, PBP-2, was the 15-kDa protein itself. The 42-kDa protein was confirmed to be an actin from the amino acid composition, by immunochemical evidence and by its ability to self-polymerize. In addition, the 42 + 15-kDa protein complex (PBP-1) inhibited DNase I, just as a monomeric actin did. The amino acid composition of the 15-kDa protein was similar to that of mammalian profilin and it inhibited the salt-induced polymerization of rabbit skeletal muscle actin. Therefore, we conclude that the two poly(L-proline)-binding proteins from the chick embryo are a profilactin and a profilin in chick embryo. The ability of profilactin to bind poly(L-proline) must be due to profilin itself, because the profilin has a greater affinity for poly(L-proline) than does profilactin. Additionally, both the monomeric and filamentous actin from rabbit skeletal muscle have no affinity for poly(L-proline).     

The cellular polypeptide p57 (pyrimidine tract-binding protein) binds to multiple sites in the poliovirus 5' nontranslated region.

Initiation of translation of poliovirus RNA by ribosomal entry into an internal segment of the 742-nucleotide (nt)-long 5' nontranslated region involves trans-acting factors, including p57, a 57-kDa polypeptide which has been identified as the pyrimidine tract-binding protein (PTB). A UV cross-linking assay was used to compare the RNA-binding properties of the p57 present in various mammalian cytoplasmic extracts with those of purified murine p57 and recombinant human PTB. Three noncontiguous p57-binding sites were located within the poliovirus 5' nontranslated region, between nt 70 and 288, and 443 and 539 (domain V), and 630 and 730. With the same assay, a novel 34-kDa polypeptide was identified that bound nt 1 to 629 specifically. A single A-->G substitution of nt 480 which attenuates poliovirus did not alter UV cross-linking of p57 to domain V. Although UV cross-linking of p57 to the internal ribosome entry site was specifically reduced by competition with poly(U) but not by competition with poly(C), poly(G), and poly(A) homoribopolymers, the presence of a polyuridine tract was not a sufficient determinant for binding of RNA to the p57 present in cytoplasmic extracts, nor was the polypyrimidine tract downstream of domain V necessary for binding to this site.     

In vitro reconstitution of messenger ribonucleoprotein particles from globin messenger RNA and cytosol proteins

Deproteinized globin poly(A) + mRNAs reassociate readily in vitro with soluble RNA-binding proteins of the cytosol; reconstituted messenger ribonucleoprotein complexes are obtained which are very similar to native globin polyribosomal-mRNP as far as bouyant density in Cs2SO4 and the composition of proteins which can be crosslinked to the mRNA are concerned. Proteins thus identified bind specifically to mRNA and not to ribosomal RNA or any synthetic oligonucleotides, with one exception: a 78-kDa protein could be cross-linked to poly(A).