Complementary change in cis determinants and trans factors in the evolution of an mRNP stability complex

RNA-protein (RNP) complexes play significant roles in the fate and expression of mRNAs. The prolonged half-life of human alpha-globin mRNA, a major determinant of normal erythroid differentiation, is dependent on the assembly of a sequence-specific 3'-untranslated region (3'UTR) RNP (alpha-complex). We demonstrate that the stability of murine alpha-globin mRNA is controlled by a parallel mechanism. Unexpectedly, however, the respective 3'UTR RNP complexes that stabilize the h(alpha)- and m(alpha)-globin mRNAs differ in structure. While the cis determinants in both species are encoded in polypyrimidine tracks, the human determinant is C-rich (CCUCC motif) while the mouse alpha-3'UTR consists of an equal distribution of Cs and Us (CCUUCU motif). The protein components of the corresponding human and murine alpha-complexes differ in a complementary manner: the previously described 39 kDa poly(C) binding protein (PCBP) present in the human alpha-complex is replaced in the mouse alpha-complex by a 48 kDa cytoplasmic poly(CU) binding protein (CUBP). These results reveal that drift in the primary sequences of the alpha-globin mRNA 3'UTR polypyrimidine tracks in a comparison between mouse and human is paralleled by an alteration in the composition of the corresponding trans-acting components. Surprisingly, these structurally distinct complexes appear to perform the identical function of stabilizing the corresponding alpha-globin mRNAs.     

Purification of a RNA-binding protein from rat liver. Identification as ferritin L chain and determination of the RNA/protein binding characteristics

In cultured rat hepatocytes the degradation of phosphoenolpyruvate carboxykinase mRNA might be regulated by protein(s), which by binding to the mRNA alter its stability. The 3'-untranslated region of phosphoenolpyruvate carboxykinase mRNA as a potential target was used to select RNA-binding protein(s) from rat liver by the use of gel retardation assays. A cytosolic protein was isolated, which bound to the phosphoenolpyruvate carboxykinase mRNA 3'-untranslated region and other in vitro synthesized RNAs. The protein was purified to homogeneity; it had an apparent molecular mass of 400 kDa and consisted of identical subunits with an apparent size of 24.5 kDa. Sequence analysis of a tryptic peptide from the 24.5-kDa protein revealed its identity with rat ferritin light chain. Binding of ferritin to RNA was abolished after phosphorylation with cAMP-dependent protein kinase and was augmented after dephosphorylation with alkaline phosphatase. Weak binding was observed in extracts from okadaic acid-treated cultured hepatocytes compared with untreated cells. Preincubation of ferritin with an anti-phosphoserine or an anti-phosphothreonine antibody attenuated binding to RNA, while an anti-phosphotyrosine antibody generated a supershift indicating that phosphoserine and phosphothreonine but not phosphotyrosine residues were in close proximity to the RNA-binding region. Ferritin is the iron storage protein in the liver. Binding of ferritin to RNA was diminished in the presence of increasing iron concentrations, whereas the iron chelator desferal was without effect. It is concluded that ferritin might function as RNA-binding protein and that it may have important functions in the general regulation of cellular RNA metabolism.     

Involvement of the Xenopus laevis Ro60 autoantigen in the alternative interaction of La and CNBP proteins with the 5'UTR of L4 ribosomal protein mRNA

In vertebrates the synthesis of ribosomal proteins is co-ordinately regulated at the translational level. The 5'-untranslated region (5'UTR) of this class of mRNAs contains conserved regions that are necessary and sufficient for translational regulation. Recently, we found that two proteins, the Xenopus laevis La autoantigen and the cellular nucleic acid binding protein (CNBP), are able to bind in vitro a pyrimidine tract at the 5' end and a downstream region, respectively. These regions are considered the common cis-acting elements of translational regulation. It was previously observed that the binding of both these putative trans-acting factors to their RNA sequences is assisted by a protease-sensitive factor(s) that dissociates from the complex after its formation. Here we provide evidence that the requirement for an ancillary factor assisting La binding to the pyrimidine tract of ribosomal protein mRNAs is typical of this RNA, and secondly that it may involve an RNA recognition motif of the La protein not clearly characterized previously. We also show that the Ro60 autoantigen is involved in the common factor activity necessary for the binding of La and CNBP proteins to their respective sequences. In addition, our findings suggest that an RNA also participates in this process. We show that CNBP can multimerise and that it binds to the 5'UTR as a dimer. Both La and CNBP compete for the interaction with the factor, and their binding to the 5'UTR is mutually exclusive. Our results from the binding analysis of mutations in the 5'UTR, which are known to disrupt the translational control in vivo, suggest a model in which the protein interactions and the 5'UTR RNA structure may co-operate in regulating the translational fate of ribosomal protein mRNAs.     

Interaction of proteins with the mRNA for ribosomal protein L1 in Xenopus: structural characterization of in vivo complexes and identification of proteins that bind in vitro to its 5'UTR

Xenopus r-protein mRNAs are known to be coordinately regulated at the translational level. To find out if RNA/protein interactions are involved in this control mechanism, we have characterized the particles containing the translationally repressed rp-mRNA and we have investigated the proteins that specifically bind to this type of mRNA. By sedimentation analysis and isopycnic centrifugation we have found that the repressed rp-mRNAs are assembled in slow sedimenting complexes where the RNA is prevalent over the protein mass (2.3 to 1). This composition is maintained also after in vitro reconstitution of the particle. We carried out also a detailed analysis of in vitro RNA/protein complex formation by focusing our attention on the 5'UTR, very similar in different rp-mRNAs and important in the translational regulation. We describe specific interactions of L1 mRNA with four proteins. The binding site of two of them, 57 kD and 47 kD, is in the typical pyrimidine sequence at the 5' end and is position dependent. Proteins of the same size interact also with the analogous region of r-protein S1 and L14 mRNA, not with unrelated RNAs. Binding of two other proteins, 31 kD and 24 kD, in the downstream region of the 5'UTR was also observed. The most evident 57 kD protein has been partially purified. Although the binding of these proteins to the r-protein mRNA 5'UTR is specific, their involvement in the translation regulation remains to be proved.     

Interaction of Theiler's virus with intermediate filaments of infected cells

Theiler's murine encephalomyelitis virus is a neurotropic murine picornavirus which replicates permissively and causes a cytopathic effect in the BHK-21 cell line. We examined the interactions between the GDVII and DA strains of Theiler's virus and BHK-21 host cell proteins in a virus overlay assay. We observed binding of the virions to two proteins of approximately 60 kDa. These proteins were microsequenced and identified as desmin and vimentin, two main components of the intermediate filament network. The association between desmin or vimentin and virions was demonstrated by immunoprecipitation. Anti-desmin and anti-vimentin monoclonal antibodies precipitated GDVII or DA virions from extracts of infected BHK-21 cells. The intracellular distributions of virions and of the desmin and vimentin intermediate filaments of BHK-21 cells were investigated by two-color immunofluorescence confocal microscopy. Following infection, the intermediate filament network was rearranged into a shell-like structure which surrounded a viral inclusion. Finally, close contact between GDVII virus particles and 10-nm intermediate filaments was observed by electron microscopy.     

A conserved 90 nucleotide element mediates translational repression of nanos RNA

Correct formation of the Drosophila body plan requires restriction of nanos activity to the posterior of the embryo. Spatial regulation of nanos is achieved by a combination of RNA localization and localization-dependent translation such that only posteriorly localized nanos RNA is translated. Cis-acting sequences that mediate both RNA localization and translational regulation lie within the nanos 3' untranslated region. We have identified a discrete translational control element within the nanos 3' untranslated region that acts independently of the localization signal to mediate translational repression of unlocalized nanos RNA. Both the translational regulatory function of the nanos 3'UTR and the sequence of the translational control element are conserved between D. melanogaster and D. virilis. Furthermore, we show that the RNA helicase Vasa, which is required for nanos RNA localization, also plays a critical role in promoting nanos translation. Our results specifically exclude models for translational regulation of nanos that rely on changes in polyadenylation.     

A 68-nucleotide sequence within the 3' noncoding region of simian hemorrhagic fever virus negative-strand RNA binds to four MA104 cell proteins

The 3' noncoding region (NCR) of the negative-strand RNA [3'(-)NCR RNA] of the arterivirus simian hemorrhagic fever virus (SHFV) is 209 nucleotides (nt) in length. Since this 3' region, designated 3'(-)209, is the site of initiation of full-length positive-strand RNA and is the template for the synthesis of the 5' leader sequence, which is found on both full-length and subgenomic mRNAs, it is likely to contain cis-acting signals for RNA synthesis and to interact with cellular and viral proteins to form replication complexes. Gel mobility shift assays showed that cellular proteins in MA104 S100 cytoplasmic extracts formed two complexes with the SHFV 3'(-)209 RNA, and results from competition gel mobility shift assays demonstrated that these interactions were specific. Four proteins with molecular masses of 103, 86, 55, and 36 kDa were detected in UV-induced cross-linking assays, and three of these proteins (103, 55, and 36 kDa) were also detected by Northwestern blotting assays. Identical gel mobility shift and UV-induced cross-linking patterns were obtained with uninfected and SHFV-infected extracts, indicating that the four proteins detected are cellular, not viral, proteins. The binding sites for the four cellular proteins were mapped to the region between nt 117 and 184 (68-nt sequence) from the 3' end of the SHFV negative-strand RNA. This 68-nt sequence was predicted to form two stem-loops, SL4 and SL5. The 3'(-)NCR RNA of another arterivirus, lactate dehydrogenase-elevating virus C (LDV-C), competed with the SHFV 3'(-)209 RNA in competition gel mobility shift assays. UV-induced cross-linking assays showed that four MA104 cellular proteins with the same molecular masses as those that bind to the SHFV 3'(-)209 RNA also bind to the LDV-C 3'(-)NCR RNA and equine arteritis virus 3'(-)NCR RNA. However, each of these viral RNAs also bound to an additional MA104 protein. The binding sites for the MA104 cellular proteins were shown to be located in similar positions in the LDV-C 3'(-)NCR and SHFV 3'(-)209 RNAs. These data suggest that the binding sites for a set of the cellular proteins are conserved in all arterivirus RNAs and that these cell proteins may be utilized as components of viral replication complexes.     

Regulation of the stability of heat-stable antigen mRNA by interplay between two novel cis elements in the 3' untranslated region

The heat-stable antigen (HSA) is a costimulatory molecule for T-cell activation. Its expression is strictly regulated during lymphocyte development and differentiation. Recent studies using HSA-transgenic mice have demonstrated that this regulated expression is critical for normal development of T and B lymphocytes. However, the mechanisms that control the expression of HSA are largely unknown. HSA mRNA is comprised of a 0.23-kb open reading frame and a 1.5-kb 3' untranslated region (3'UTR). The function of the long 3'UTR has not been addressed. Here we investigate the role of the 3'UTR of HSA mRNA. We show that a 160-bp element, located in the region of nucleotides 1465 to 1625 in the 3'UTR of HSA mRNA, promotes RNA degradation and that this effect is neutralized by a 43-bp fragment approximately 1 kb upstream of the negative cis element. Both positive and negative cis elements in the HSA mRNA are distinct from other sequences that are known to modulate mRNA stability. These results provide direct evidence that the interplay between two novel cis elements in the 3'UTR of HSA mRNA determines cell surface HSA expression by modulating its RNA stability.     

Identification of an mRNA-binding protein and the specific elements that may mediate the pH-responsive induction of renal glutaminase mRNA

Various segments of the 3'-nontranslated region of the renal glutaminase (GA) mRNA were tested for their ability to enhance turnover and pH responsiveness. The combined effects were retained in the 340-base R-2 segment. However, the combined R-1 and R-3 fragments also imparted a partial destabilization and pH responsiveness to a chimeric beta-globin mRNA. RNA electrophoretic mobility shift assays indicated that cytosolic extracts of rat renal cortex contain a protein that binds to the R-2 and R-3 RNAs. The binding observed with the R-2 RNA was mapped to a direct repeat of an 8-base AU sequence. This binding was effectively competed with an excess of the same RNA, but not by adjacent or unrelated RNAs. UV cross-linking experiments identified a 48-kDa protein that binds to the AU repeats of the R-2 RNA. The apparent binding of this protein was greatly reduced in renal cytosolic extracts prepared from acutely acidotic rats. Two related RNA sequences in the R-3 segment also exhibited specific binding. However, the latter binding was more effectively competed by R-2 RNA than by itself, indicating that the homologous sites may be weaker binding sites for the same 48-kDa protein. Thus, a single protein may bind specifically to multiple instability elements within the 3'-nontranslated region of the GA mRNA and mediate its pH-responsive stabilization.     

Expression of inhibin alpha-subunit in horse testis

Inhibin is believed to play roles in the pituitary secretion of FSH and in the paracrine regulation of testicular function. Although it has been generally accepted that inhibin is produced in Sertoli cells, there was a recent evidence for the localization of inhibin in Leydig cells of primates, rat and sheep. However, there is no report on the expression of inhibin in the adult horse testis. Therefore, using immunohistochemistry, western blotting and in situ hybridization techniques, the present study examined inhibin alpha-subunit (Ih-alpha) expression in the adult horse testis. For the detection of Ih-alpha protein, we used anti-porcine Ih-alpha antibody in immunohistochemistry and western blotting. Furthermore, digoxigenin-labeled complementary RNA probes were prepared to detect intracellular messenger RNA (mRNA) of Ih-alpha. Immunostainings for Ih-alpha were found not only in Leydig cells but also in Sertoli cells. The intensity in Leydig cells was stronger than in Sertoli cells. Immunoreactivities for Ih-alpha were found at approximately 46 kDa, 56 kDa and 90 kDa in the homogenates from testicular interstitial tissues. The bands at 56 kDa and 90 kDa agree with previous report, but not at 46 kDa. Signals for mRNA of Ih-alpha by in situ hybridization were detected in Leydig cells and in the basal region of seminiferous epithelium including Sertoli cells. These results suggest that Ih-alpha is expressed in Leydig cells and Sertoli cells of horse testis, and the expression level should be higher in Leydig cells than Sertoli cells.     

Molecular mechanism of regulation of pentylenetetrazol-induced calcium entry by 3'-untranslated region of a seizure-related cDNA, PTZ-17, in Xenopus oocytes

To determine the molecular mechanism of regulation of pentylenetetrazol (PTZ)-induced calcium entry by the seizure-related gene, PTZ-17, the role of the 3'-untranslated region (3'UTR) and also interaction between 3'UTR and intracellular factors were investigated. PTZ-induced calcium inward current in Xenopus oocytes injected with PTZ-17 RNA varied in magnitude among strains of mice: RNA derived from the DBA/2 mouse, which has a high susceptibility to convulsions, showed the largest current and that from the BALB/c mouse with a low susceptibility to convulsions showed no PTZ response. The sequence of 3'UTR showed alterations among mouse strains: 3'UTR of BALB/c showed a sequence alteration from T to G and that of DBA/2 showed a GTG insertion compared with that of B6. The 3'UTR also regulated the translation of chloramphenicol acetyltransferase (CAT) RNA depending on its sequence. A particular region within the 3'UTR demonstrated interaction with 60- and 47-kDa proteins. Sequence alterations in this region corresponded to disappearance or increase in PTZ-induced calcium entry. These findings suggest that a particular region within 3'UTR of the seizure-related gene, PTZ-17, is involved in PTZ-induced calcium entry via interaction between mRNA and specific RNA-binding proteins.     

Beta-actin mRNA-binding proteins associated with the cytoskeletal framework

Association of mRNA with the cytoskeletal framework (CSK) is thought to play a strategic role in the placement of mRNA in the cytoplasm. However, the molecular determinants underlying mRNA/CSK association are completely unknown. To begin addressing this issue, we have employed a binding assay to identify proteins of the CSK compartment of NIH 3T3 cells that bind in-vitro-transcribed 32P-labelled beta-actin mRNA with high affinity. Three proteins, of approximate molecular masses 27, 50 and 97 kDa, were observed to exhibit strong binding. Binding to these proteins took place at physiological salt concentration and withstood washing in 0.5 M salt. Furthermore, binding was unaffected by heparin but was inhibited by unlabelled beta-actin mRNA. Treatment of isolated CSKs with the microfilament-severing agent DNase I abolished all beta-actin mRNA-binding activities, thus suggesting a possible association of beta-actin mRNA with the microfilament network in situ. Removal of the 3' untranslated region (UTR) significantly reduced beta-actin mRNA binding to all three CSK proteins but removal of the 5' UTR mainly affected binding to the 97-kDa species and that to a lesser extent. beta-Tubulin mRNA bound to the same three CSK proteins as did beta-actin mRNA, but with considerably less avidity. In contrast, vimentin mRNA strongly recognized these CSK proteins, and further bound to a group of smaller proteins (< 29 kDa). As beta-actin mRNA, beta-tubulin mRNA and vimentin mRNA have been observed to occupy separate cytoplasmic locales, the proteins detected here may be operative both in binding mRNAs to the CSK in situ, as well as in localizing mRNA in the cytoplasm.     

Molecular cloning and expression of the functional rat C5a receptor

The C5a receptor belongs to the superfamily of G-protein coupled receptors with seven transmembrane segments. In this study we report on the cloning of the rat C5a receptor (ratC5aR). We used a hybridization probe produced by PCR utilizing degenerate primers which corresponded to conserved parts of the human, canine and murine C5a receptor nucleotide sequences and to the published partial amino acid sequence of the rat C5a receptor to screen a rat macrophage cDNA library. We found two overlapping clones containing an open reading frame of 1056 bp, a 3'untranslated region of 683 bp and a 5'untranslated region of 27 bp. The overall nucleotide acid sequence identity, compared to the murine, human and canine C5a receptor sequences, was 85.8, 70.5 and 68.9%, respectively. The greatest diversity exists in the putative extracellular domains, especially in the aminoterminal domain which is assumed to be involved in ligand binding. An N-glycosylation site is present within the N-terminal sequence at residue 6. One of the cDNA containing the 5'untranslated region, the coding sequence and part of the 3'untranslated region was cloned into an eucaryotic expression vector and stably transfected into the rat basophilic leukemia cell line RBL-2H3. Expression of the rat C5a receptor on the surface of these cells could be demonstrated by flow cytometric analysis using FITC-labeled recombinant rat C5a (rrC5a). By measuring the release of calcium from intracellular stores after stimulation with rrC5a it could further be shown that the receptor is functionally coupled. Receptor binding assays showed that rrC5a specifically binds to the ratC5aR with a KD of 0.91 +/- 0.36 and to the human C5a receptor (huC5aR) with a KD of 7.19 +/- 1.56. The determined KD for binding of human C5a (huC5a) to the huCSaR was 2.16 +/- 0.65. No binding of huC5a to the ratC5aR could be observed although high concentrations of this ligand (> 60 nM) promoted chemotaxis of RBL cells transfected with the huC5aR.