Major RNA-binding Protein Research Articles

Heterogeneous nuclear ribonucleoprotein a1 binds to the 3'-untranslated region and mediates potential 5'-3'-end cross talks of mouse hepatitis virus RNA.

The 3'-untranslated region (3'-UTR) of mouse hepatitis virus (MHV) RNA regulates the replication of and transcription from the viral RNA. Several host cell proteins have previously been shown to interact with this regulatory region. By immunoprecipitation of UV-cross-linked cellular proteins and in vitro binding of the recombinant protein, we have identified the major RNA-binding protein species as heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). A strong hnRNP A1-binding site was located 90 to 170 nucleotides from the 3' end of MHV RNA, and a weak binding site was mapped at nucleotides 260 to 350 from the 3' end. These binding sites are complementary to the sites on the negative-strand RNA that bind another cellular protein, polypyrimidine tract-binding protein (PTB). Mutations that affect PTB binding to the negative strand of the 3'-UTR also inhibited hnRNP A1 binding on the positive strand, indicating a possible relationship between these two proteins. Defective-interfering RNAs containing a mutated hnRNP A1-binding site have reduced RNA transcription and replication activities. Furthermore, hnRNP A1 and PTB, both of which also bind to the complementary strands at the 5' end of MHV RNA, together mediate the formation of an RNP complex involving the 5'- and 3'-end fragments of MHV RNA in vitro. These studies suggest that hnRNP A1-PTB interactions provide a molecular mechanism for potential 5'-3' cross talks in MHV RNA, which may be important for RNA replication and transcription.

Nucleolin and YB-1 are required for JNK-mediated interleukin-2 mRNA stabilization during T-cell activation

Regulated mRNA turnover is a highly important process, but its mechanism is poorly understood. Using interleukin-2 (IL-2) mRNA as a model, we described a role for the JNK-signaling pathway in stabilization of IL-2 mRNA during T-cell activation, acting via a JNK response element (JRE) in the 5' untranslated region (UTR). We have now identified two major RNA-binding proteins, nucleolin and YB-1, that specifically bind to the JRE. Binding of both proteins is required for IL-2 mRNA stabilization induced by T-cell activation signals and for JNK-induced stabilization in a cell-free system that duplicates essential features of regulated mRNA decay. Nucleolin and YB-1 are required for formation of an IL-2 mRNP complex that responds to specific mRNA stabilizing signals.

Nucleolin functions in the first step of ribosomal RNA processing.

The first processing step of precursor ribosomal RNA (pre-rRNA) involves a cleavage within the 5' external transcribed spacer. This processing requires sequences downstream of the cleavage site which are perfectly conserved among human, mouse and Xenopus and also several small nucleolar RNAs (snoRNAs): U3, U14, U17 and E3. In this study, we show that nucleolin, one of the major RNA-binding proteins of the nucleolus, is involved in the early cleavage of pre-rRNA. Nucleolin interacts with the pre-rRNA substrate, and we demonstrate that this interaction is required for the processing reaction in vitro. Furthermore, we show that nucleolin interacts with the U3 snoRNP. Increased levels of nucleolin, in the presence of the U3 snoRNA, activate the processing activity of a S100 cell extract. Our results suggest that the interaction of nucleolin with the pre-rRNA substrate might be a limiting step in the primary processing reaction. Nucleolin is the first identified metazoan proteinaceous factor that interacts directly with the rRNA substrate and that is required for the processing reaction. Potential roles for nucleolin in the primary processing reaction and in ribosome biogenesis are discussed.

Ordered aggregation of ribonucleic acids by the human immunodeficiency virus type 1 nucleocapsid protein.

The nucleocapsid protein NCp7, which is the major genomic RNA binding protein of human immunodeficiency virus type 1, plays an important role in several key steps of the viral life cycle. Many of the NCp7 activities, notably the nucleic acid annealing and the genomic RNA wrapping ones, are thought to be linked to a nonspecific binding of NCp7 to its nucleic acid targets. The mechanism of these activities is still debated but several clues are in favor of an intermediate aggregation of nucleic acids by NCp7. To check and characterize the nucleic acid aggregating properties of NCp7, we investigated the interaction of NCp7 with the model RNA homopolymer, polyA, by quasielastic light scattering and optical density measurements. The ordered growth of monodisperse large particles independently of the nucleic acid size and the almost complete covering of polyA by NCp7 strongly suggested an ordered aggregation mechanism. The aggregate kinetics of growth in the optimum protein concentration range (> or = 2 microM) were governed by a so-called Ostwald ripening mechanism limited by transfer of NCp7-covered polyA complexes from small to large aggregates. The aggregation process was strongly dependent on both Na+ and Mg2+ concentrations, the optimum concentrations being in the physiological range. Similar conclusions held true when polyA was replaced by 16S + 23S ribosomal RNA, suggesting that the NCp7 aggregating properties were only poorly dependent on the nucleic acid sequence and structure. Finally, as in the NCp7 annealing activities, the basic regions of NCp7, but not the zinc fingers, were found critical in nucleic acid aggregation. Taken together, our data indicate that NCp7 is a highly efficient nucleic acid aggregating agent and strengthen the hypothesis that aggregation may constitute a transient step in various NCp7 functions.

Localization of nucleolin binding sites on human and mouse pre-ribosomal RNA.

Nucleolin, a major RNA binding protein of the nucleolus is found associated mainly to the pre-ribosomal particles and is absent from the cytoplasmic mature ribosomes. The role of this protein in ribosome biogenesis remains largely unknown, and is likely to be reflected by its RNA binding properties. Nucleolin contains in its central domain four RNA recognition motifs (RRM, also called RBD for RNA binding domain) which are conserved among different species. RNA binding studies have revealed that nucleolin interacts specifically with a short stem loop structure called NRE (nucleolin recognition element). We show that nucleolin extracted from human, hamster and mouse cells interacts with the same specificity and affinity to a mouse 5'ETS (external transcribed spacer) RNA fragment which contains a NRE motif. A similar structure within the human 5'ETS is also efficiently recognized by mouse nucleolin. We identified putative NRE not only in the 5'ETS but also in the 3'ETS, ITS (internal transcribed spacer) and in the 18S and 28S RNA sequences. This is in agreement with in vivo cross-linking data and a previous immunocytological analysis of ribosomal transcription units. Interestingly, we found that all the NRE localized in the 28S region are within the variable domains. Despite considerable sequence divergence of these domains, several of the NRE have sequences perfectly conserved between these two species. This suggests that these nucleolin binding sites might be functionally important, in particular for ribosome biogenesis.

Assignment of the human gene encoding heterogeneous nuclear RNA ribonucleoprotein I (PTB) to chromosome 14q23-q24.1.

Heterogeneous nuclear ribonucleoprotein type I (hnRNP I), also known as the polypyrimidine tract binding protein (PTB), is a 59.6-kDa nuclear protein that binds pre-mRNAs in specific regions of the hnRNA-protein complexes, sensitive to micrococcal nuclease. A 15,000-bp genomic clone encompassing part of the PTB cDNA, used as a biotinylated probe in a fluorescence in situ hybridization (FISH) experiment, has shown that the human PTB gene is localized on bands 14q23-q24.1. In the nucleus of eukaryotic cells, nascent heterogeneous nuclear RNAs (hnRNAs) associate with a distinct set of abundant nuclear proteins to form heterogeneous nuclear ribonuclear proteins (hnRNP) complexes. HnRNP complexes immunopurified from HeLa cells are composed of at least 20 major RNA binding proteins, designated A1 (34 kDa) through U (120 kDa). HnRNP complexes are thought to be substrate for transcriptional processing events. 19 refs., 2 figs.

Alteration of DNA and RNA Binding Activity of Human Telomere Binding Proteins Occurs during Cellular Senescence

The loss of telomere sequences during in vitro and in vivo aging suggests that mechanisms affecting telomere length may have important consequences in cellular senescence. In this study, we have found that the activity of single-stranded telomere binding proteins is increased in nuclear extracts prepared from senescent human diploid fibroblasts compared to actively growing cells. Since single-stranded telomere binding proteins are closely related to RNA binding proteins, we examined the binding activity of several major RNA binding proteins to RNA by uv cross-linking. The level of activity was greatly diminished and the overall pattern of uv cross-linked products were altered in extracts prepared from senescent cells. Furthermore, Western analysis revealed a concurrent decrease in senescent extracts of the protein level for many RNA binding proteins, including those which bind to telomere sequence. Although the reduction in the level of these proteins parallels the reduced activity in RNA binding, the paradoxical increased telomere binding activity exhibited by extracts from older cells suggests a complex relationship between these proteins with RNA and DNA. Moreover, the reduced RNA binding activity of these proteins indicates that the biochemical function of several RNA binding proteins is compromised during cellular senescence, raising an intriguing possibility that a change in pre-mRNA metabolism may contribute to the multitude of changes in gene expression observed in cellular senescence.

Evidence that XR family interspersed RNA may regulate translation in Xenopus oocytes.

It has been shown that about two thirds of Xenopus oocyte or sea urchin egg cytoplasmic poly(A)+ RNA contains interspersed repetitive sequences. The functional significance of this interspersed RNA has remained unknown. Here the function of a subfamily of interspersed RNA (XR family; McGrew and Richter, 1989: Dev Biol 134:267-270) in Xenopus oocytes was studied. We found that the elimination of T7 XR (one of the two complementary strands of the XR repeat) interspersed RNA by complementary oligodeoxynucleotides significantly inhibited protein synthesis. On the other hand, the injection of in vitro synthesized T7 XR RNA stimulated translation. Moreover, the insertion of the T7 XR RNA sequence into globin mRNA repressed the translation of the globin mRNA. In order to explain these results, we analyzed interactions between the XR interspersed RNA and oocyte proteins. We found that the major XR RNA binding proteins were p56 and p60, which could be the known mRNA "masking" proteins that bind mRNA and inhibit translation. Further, a 42 kD protein has been identified that appears to bind T7 XR RNA relatively specifically, although it interacts with mRNA with a lower affinity. Based on all of these data, we have proposed that interspersed RNA may be involved in regulating translation by competing with mRNA to interact with certain proteins that can regulate translation.

RNA-Binding Characteristics of a Ribonucleoprotein from Spinach Chloroplast.

A chloroplast (nuclear-encoded) RNA-binding protein (28RNP) was previously purified from spinach (Spinacia oleracea). This 28RNP was found to be the major RNA-binding protein co-purified during the isolation scheme of 3[prime] end RNA-processing activity of several chloroplastic genes. To learn more about the possible involvement of 28RNP in the 3[prime] end RNA-processing event, we investigated the RNA-binding properties and the location of the protein in the chloroplast. We found that recombinant Escherichia coliexpressed 28RNP binds with apparently the same affinity to every chloroplastic 3[prime] end RNA that was analyzed, as well as to RNAs derived from the 5[prime] end or the coding region of some chloroplastic genes. Differences in the RNA-binding affinities for some chloroplastic 3[prime] end RNAs were observed when the recombinant 28RNP was compared with the "native" 28RNP in the chloroplast-soluble protein extract. In addition, we found that the 28RNP is not associated with either thylakoid-bound or soluble polysomes in which a great portion of the chloroplast rRNA and mRNA are localized. These results suggest that the native 28RNP binds specifically to certain RNA molecules in the chloroplast in which other components (possibly proteins) and/or posttranslational modifications are involved in determining RNA-binding specificity of the 28RNP.

Nucleolin promotes secondary structure in ribosomal RNA

The effect of nucleolin on the secondary structure of RNA was studied using circular dichroism (CD). Nucleolin caused decreases in the main positive bands and shifts to higher wavelengths in the CD spectra of synthetic polynucleotides such as poly(G) and poly(A) indicating helix destabilizing activity. In contrast, nucleolin effected increases in signal and shifts to lower wavelengths of the peaks of CD spectra of ribosomal RNA, suggesting enhancement of secondary structure. Another major nucleolar RNA binding protein, B23, had helix destabilizing activity but did not enhance RNA secondary structure. It is proposed that nucleolin promotes formation of secondary structure in preribosomal RNA during the early stages of ribosome biogenesis.

Association of glyceraldehyde-3-phosphate dehydrogenase with mono- and polyribosomes of rabbit reticulocytes.

It has been shown recently that glyceraldehyde-3-phosphate dehydrogenase (GAPD) is one of the three major RNA-binding proteins of rabbit reticulocytes [Ryazanov, A. G. (1985) FEBS Lett. 192, 131-134]. It was suggested that, due to its RNA-binding capacity, GAPD can form loose dynamic complexes with polyribosomes. This communication reports that a considerable amount of GAPD activity can be found in the mono- and polyribosome fraction after sucrose gradient centrifugation of rabbit reticulocyte lysate. An increase of ionic strength, as well as the addition of exogenous RNA to the extract, result in the removal of GAPD from the complex with mono- and polyribosomes. It appears that GAPD forms the complex with polyribosomes due to the interaction with some exposed RNA regions of these structures. Although the interaction of GAPD with ribosomes is weak, it can be detected under physiological ionic conditions by the difference boundary sedimentation velocity technique. Association of GAPD with mono- and polyribosomes can be prevented by a low concentration (10 microM) of NADH, but not NAD+. A nitrocellulose filter binding assay also shows that NADH has a stronger inhibitory effect on the enzyme-RNA complex formation, as compared with NAD+. We propose that the RNA-mediated association of GAPD with mono- and polyribosomes can provide compartmentation of the energy-supplying system on these structures within the cell. This can maintain a high local concentration of ATP and GTP near the sites of protein synthesis.

Glyceraldehyde-3-phosphate dehydrogenase is one of the three major RNA-binding proteins of rabbit reticulocytes

One of the 3 major RNA-binding proteins of rabbit reticulocytes, a polypeptide of 36 kDa, is identified as glyceraldehyde-3-phosphate dehydrogenase (GAPD). This fact was deduced from the identity of molecular masses, one-dimensional peptide maps and isoelectric points of the 36 kDa protein and GAPD from rabbit muscle. It is concluded that GAPD can bind rather unspecifically different RNAs and polynucleotides. This means that GAPD, like other RNA-binding proteins, can form loose dynamic complexes with polyribosomes. Association of such a kind may be used for compartmentation of glycolysis near polyribosomes.