A developmentally regulated, nervous system-specific gene in Xenopus encodes a putative RNA-binding protein.

A gene from Xenopus laevis that is expressed specifically in the nervous system beginning at the stage of neural plate formation has been isolated and several cDNAs have been sequenced. The sequence of the predicted protein contains two copies of a presumed RNA-binding domain, each of which includes two short conserved motifs characteristic for ribonucleoproteins (RNPs), called the RNP-1 and RNP-2 consensus sequences. We name this gene Xenopus nrp-1, for nervous system-specific RNP protein-1. Sequence comparisons suggest that the nrp-1 protein is a heterogeneous nuclear RNP protein, but it is clearly distinct from previously reported hnRNP proteins such as the A1, A2/B1, and C1 proteins. nrp-1 RNA undergoes an alternative splicing event giving rise to two predicted protein isoforms that differ from each other by seven amino acids. In situ hybridization to tadpole brain shows that the nrp-1 gene is expressed in the ventricular zone where cell proliferation takes place. The occurrence of an RNP protein with nervous system-limited expression suggests that it may be involved in the tissue-specific control of RNA processing.     

Distinct functions of the closely related tandem RNA-recognition motifs of hnRNP A1.

hnRNP A1 regulates alternative splicing by antagonizing SR proteins. It consists of two closely related, tandem RNA-recognition motifs (RRMs), followed by a glycine-rich domain. Analysis of variant proteins with duplications, deletions, or swaps of the RRMs showed that although both RRMs are required for alternative splicing function, each RRM plays distinct roles, and their relative position is important. Surprisingly, RRM2 but not RRM1 could support this function when duplicated, despite their very similar structure. Specific RNA binding and annealing are not sufficient for hnRNP A1 alternative splicing function. These observations, together with phylogenetic and structural data, suggest that the two RRMs are quasi-symmetric but functionally nonequivalent modules that evolved as components of a single bipartite domain.     

Isolation and characterization of a novel,low abundance hnRNP protein: A0.

Pre-messenger RNA is bound by a variety of proteins to form large heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. As defined by immunoprecipitation and two-dimensional gel electrophoresis, there appear to be more than 20 abundant hnRNP proteins ranging in size from 34 kDa to 120 kDa. One major class, the A/B family, is typified by its characteristic primary structure containing two RNA binding domains followed by a glycine-rich C-terminus. We report the cloning and characterization of a novel, low-abundance member of the A/B family named hnRNP A0. This protein was affinity isolated using a biotinylated RNA probe [G4(AU3)4A] designed to select a 32-kDa protein implicated in mRNA stability in mammalian cells. hnRNP A0 is a basic protein with a predicted mass of 31.7 kDa and an isoelectric point of 10.1. Comparative protease mapping shows that it is not the AUUUA binding protein we intended to clone. A0 is present in hnRNP complexes and is encoded by a gene distinct from that of any previously cloned A/B family member.     

A nuclear localization domain in the hnRNP A1 protein

The heterogeneous nuclear RNP (hnRNP) A1 protein is one of the major pre-mRNA/mRNA binding proteins in eukaryotic cells and one of the most abundant proteins in the nucleus. It is localized to the nucleoplasm and it also shuttles between the nucleus and the cytoplasm. The amino acid sequence of A1 contains two RNP motif RNA-binding domains (RBDs) at the amino terminus and a glycine-rich domain at the carboxyl terminus. This configuration, designated 2x RBD-Gly, is representative of perhaps the largest family of hnRNP proteins. Unlike most nuclear proteins characterized so far, A1 (and most 2x RBD-Gly proteins) does not contain a recognizable nuclear localization signal (NLS). We have found that a segment of ca. 40 amino acids near the carboxyl end of the protein (designated M9) is necessary and sufficient for nuclear localization; attaching this segment to the bacterial protein beta- galactosidase or to pyruvate kinase completely localized these otherwise cytoplasmic proteins to the nucleus. The RBDs and another RNA binding motif found in the glycine-rich domain, the RGG box, are not required for A1 nuclear localization. M9 is a novel type of nuclear localization domain as it does not contain sequences similar to classical basic-type NLS. Interestingly, sequences similar to M9 are found in other nuclear RNA-binding proteins including hnRNP A2.     

Binding of an RNA trafficking response element to heterogeneous nuclear ribonucleoproteins A1 and A2

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binds a 21-nucleotide myelin basic protein mRNA response element, the A2RE, and A2RE-like sequences in other localized mRNAs, and is a trans-acting factor in oligodendrocyte cytoplasmic RNA trafficking. Recombinant human hnRNPs A1 and A2 were used in a biosensor to explore interactions with A2RE and the cognate oligodeoxyribonucleotide. Both proteins have a single site that bound oligonucleotides with markedly different sequences but did not bind in the presence of heparin. Both also possess a second, specific site that bound only A2RE and was unaffected by heparin. hnRNP A2 bound A2RE in the latter site with a K(d) near 50 nm, whereas the K(d) for hnRNP A1 was above 10 microm. UV cross-linking assays led to a similar conclusion. Mutant A2RE sequences, that in earlier qualitative studies appeared not to bind hnRNP A2 or support RNA trafficking in oligodendrocytes, had dissociation constants above 5 microm for this protein. The two concatenated RNA recognition motifs (RRMs), but not the individual RRMs, mimicked the binding behavior of hnRNP A2. These data highlight the specificity of the interaction of A2RE with these hnRNPs and suggest that the sequence-specific A2RE-binding site on hnRNP A2 is formed by both RRMs acting in cis.     

Characterization of the major hnRNP proteins from Drosophila melanogaster

To better understand the role(s) of hnRNP proteins in the process of mRNA formation, we have identified and characterized the major nuclear proteins that interact with hnRNAs in Drosophila melanogaster. cDNA clones of several D. melanogaster hnRNP proteins have been isolated and sequenced, and the genes encoding these proteins have been mapped cytologically on polytene chromosomes. These include the hnRNP proteins hrp36, hrp40, and hrp48, which together account for the major proteins of hnRNP complexes in D. melanogaster (Matunis et al., 1992, accompanying paper). All of the proteins described here contain two amino-terminal RNP consensus sequence RNA-binding domains and a carboxyl-terminal glycine-rich domain. We refer to this configuration, which is also found in the hnRNP A/B proteins of vertebrates, as 2 x RBD-Gly. The sequences of the D. melanogaster hnRNP proteins help define both highly conserved and variable amino acids within each RBD and support the suggestion that each RBD in multiple RBD-containing proteins has been conserved independently and has a different function. Although 2 x RBD-Gly proteins from evolutionarily distant organisms are conserved in their general structure, we find a surprising diversity among the members of this family of proteins. A mAb to the hrp40 proteins crossreacts with the human A/B and G hnRNP proteins and detects immunologically related proteins in divergent organisms from yeast to man. These data establish 2 x RBD-Gly as a prevalent hnRNP protein structure across eukaryotes. This information about the composition of hnRNP complexes and about the structure of hnRNA-binding proteins will facilitate studies of the functions of these proteins.     

hnRNP A2, a potential ssDNA/RNA molecular adapter at the telomere

The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multi-tasking protein that acts in the cytoplasm and nucleus. We have explored the possibility that this protein is associated with telomeres and participates in their maintenance. Rat brain hnRNP A2 was shown to have two nucleic acid binding sites. In the presence of heparin one site binds single-stranded oligodeoxyribonucleotides irrespective of sequence but not the corresponding oligoribonucleotides. Both the hnRNP A2-binding cis-acting element for the cytoplasmic RNA trafficking element, A2RE, and the ssDNA telomere repeat match a consensus sequence for binding to a second sequence-specific site identified by mutational analysis. hnRNP A2 protected the telomeric repeat sequence, but not the complementary sequence, against DNase digestion: the glycine-rich domain was found to be necessary, but not sufficient, for protection. The N-terminal RRM (RNA recognition motif) and tandem RRMs of hnRNP A2 also bind the single-stranded, template-containing segment of telomerase RNA. hnRNP A2 colocalizes with telomeric chromatin in the subset of PML bodies that are a hallmark of ALT cells, reinforcing the evidence for hnRNPs having a role in telomere maintenance. Our results support a model in which hnRNP A2 acts as a molecular adapter between single-stranded telomeric repeats, or telomerase RNA, and another segment of ssDNA.     

Purification and domain structure of core hnRNP proteins A1 and A2 and their relationship to single-stranded DNA-binding proteins

Protein A1 (Mr approximately 32,000), a major glycine-rich protein of heterogeneous nuclear ribonucleoproteins (hnRNP), was purified to near homogeneity under nondenaturing conditions from HeLa cells. Limited proteolysis of the native protein yields a trypsin-resistant N-terminal nucleic acid-binding domain about 195 amino acids long which has a primary structure nearly identical to that of the 195-amino acid-long single-stranded DNA (ssDNA)-binding protein UP1 (Mr 22,162) from calf thymus (Williams, K.R., Stone, K. L., LoPresti, M.B., Merrill, B. M., and Planck, S.R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5666-5670). 45 of the 61 glycine residues of A1 are present in the trypsin-sensitive C-terminal domain of the protein which contains no sequences homologous to UP1. Protein A2, another major glycine-rich core hnRNP protein from HeLa, has a domain structure analogous to A1 and appears to be related to ssDNA-binding proteins UP1-B from calf liver and HDP-1 from mouse myeloma in a way similar to the A1/UP1 relationship. In contrast to ssDNA-binding proteins, A1 binds preferentially to RNA over ssDNA and exhibits no helix-destabilizing activity.     

Solution structure of the two RNA recognition motifs of hnRNP A1 using segmental isotope labeling: how the relative orientation between RRMs influences the nucleic acid binding topology

Human hnRNP A1 is a multi-functional protein involved in many aspects of nucleic-acid processing such as alternative splicing, micro-RNA biogenesis, nucleo-cytoplasmic mRNA transport and telomere biogenesis and maintenance. The N-terminal region of hnRNP A1, also named unwinding protein 1 (UP1), is composed of two closely related RNA recognition motifs (RRM), and is followed by a C-terminal glycine rich region. Although crystal structures of UP1 revealed inter-domain interactions between RRM1 and RRM2 in both the free and bound form of UP1, these interactions have never been established in solution. Moreover, the relative orientation of hnRNP A1 RRMs is different in the free and bound crystal structures of UP1, raising the question of the biological significance of this domain movement. In the present study, we have used NMR spectroscopy in combination with segmental isotope labeling techniques to carefully analyze the inter-RRM contacts present in solution and subsequently determine the structure of UP1 in solution. Our data unambiguously demonstrate that hnRNP A1 RRMs interact in solution, and surprisingly, the relative orientation of the two RRMs observed in solution is different from the one found in the crystal structure of free UP1 and rather resembles the one observed in the nucleic-acid bound form of the protein. This strongly supports the idea that the two RRMs of hnRNP A1 have a single defined relative orientation which is the conformation previously observed in the bound form and now observed in solution using NMR. It is likely that the conformation in the crystal structure of the free form is a less stable form induced by crystal contacts. Importantly, the relative orientation of the RRMs in proteins containing multiple-RRMs strongly influences the RNA binding topologies that are practically accessible to these proteins. Indeed, RRM domains are asymmetric binding platforms contacting single-stranded nucleic acids in a single defined orientation. Therefore, the path of the nucleic acid molecule on the multiple RRM domains is strongly dependent on whether the RRMs are interacting with each other. The different nucleic acid recognition modes by multiple-RRM domains are briefly reviewed and analyzed on the basis of the current structural information.     

Heterogeneous nuclear ribonucleoprotein complexes from Xenopus laevis oocytes and somatic cells.

HnRNP proteins have been implicated in most stages of cellular mRNA metabolism, including processing, nucleocytoplasmic transport, stability, and localization. Several hnRNP proteins are also known to participate in key early developmental decisions. In order to facilitate functional studies of these pre-mRNA- and mRNA-binding proteins in a vertebrate organism amenable to developmental studies and experimental manipulation, we identified and purified the major hnRNP proteins and isolated the hnRNP complex from Xenopus laevis oocytes and somatic cells. Using affinity chromatography and immunological methods, we isolated a family of >15 abundant single-stranded nucleic acid-binding proteins, which range in apparent molecular weight from approximately 20 kDa to >150 kDa, and with isoelectric points from <5 to >8. Monoclonal antibodies revealed that a subset of these proteins are major hnRNP proteins in both oocytes and somatic cells in culture, and include proteins related to human hnRNP A2/B1/B2 and hnRNP K. UV crosslinking in living cells demonstrated that these proteins bind poly(A)+ RNA in vivo. Immunopurification using a monoclonal antibodyto X. aevishnRNPA2 resulted in the isolation of RNP complexes that contain a specific subset of single-stranded nucleic acid-binding proteins. The protein composition of complexes isolated from somatic cells and from oocyte germinal vesicles was similar, suggesting that the overall properties and functions of hnRNP proteins in these two cell types are comparable. These findings, together with the novel probes generated here, will also facilitate studies of the function of vertebrate RNA-binding proteins using the well characterized X. laevis oocyte and early embryo as experimental systems.     

Cloning and sequence analysis of a human type A/B hnRNP protein

A cDNA encoding a 284 residue long type A/B hnRNP protein has been cloned. This protein, previously referred to as type C [(1987) J. Biol. Chem. 262, 17126-17137], is an RNA unwinding protein from HeLa 40S hnRNP with a high affinity for G- followed by U-rich sequences. The N-terminal part of the protein contains two consensus RNA binding domains present in a number of other RNA binding proteins. The C-terminal part is glycine-rich and contains a potential ATP/GTP binding loop. The distribution of charged amino acids is highly uneven and there are multiple potential phosphorylation sites.     

Multiple type A/B heterogeneous nuclear ribonucleoproteins (hnRNPs) can replace hnRNP A1 in mouse hepatitis virus RNA synthesis

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has previously been shown to bind mouse hepatitis virus (MHV) RNA at the 3' end of both plus and minus strands and modulate MHV RNA synthesis. However, a mouse erythroleukemia cell line, CB3, does not express hnRNP A1 but still supports MHV replication, suggesting that alternative proteins can replace hnRNP A1 in cellular functions and viral infection. In this study, we set out to identify these proteins. UV cross-linking experiments revealed that several CB3 cellular proteins similar in size to hnRNP A1 interacted with the MHV RNA. These proteins were purified by RNA affinity column with biotinylated negative-strand MHV leader RNA and identified by mass spectrometry to be hnRNP A2/B1, hnRNP A/B, and hnRNP A3, all of which belong to the type A/B hnRNPs. All of these proteins contain amino acid sequences with strong similarity to the RNA-binding domains of hnRNP A1. Some of these hnRNPs have previously been shown to replace hnRNP A1 in regulating RNA splicing. These proteins displayed MHV RNA-binding affinity and specificity similar to those of hnRNP A1. hnRNP A2/B1, which is predominantly localized to the nucleus and shuttles between the nucleus and the cytoplasm, was shown to relocalize to the cytoplasm in MHV-infected CB3 cells. Furthermore, overexpression of hnRNP A/B in cells enhanced MHV RNA synthesis. Our findings demonstrate that the functions of hnRNP A1 in MHV RNA synthesis can be replaced by other closely related hnRNPs, further supporting the roles of cellular proteins in MHV RNA synthesis.     

Molecular characterization of a murine, major A/B type hnRNP protein: mBx

We have previously identified a discrete hnRNP polypeptide of the A/B type, named mBx, as an abundant protein species in murine cells. The molecular characterization of this protein is now accomplished. From all evidence provided, mBx polypeptide represents a new gene product, distinct from the known members of the A/B family A1 and A2/B1. It is, instead, mostly related to a still hypothetical human protein of A/B type, as well as to the Xenopus hnRNPA3 protein species.     

Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to influence the structure of hnRNA and participate in the processing of hnRNA to mRNA. The hnRNP U protein is an abundant nucleoplasmic phosphoprotein that is the largest of the major hnRNP proteins (120 kDa by SDS-PAGE). HnRNP U binds pre-mRNA in vivo and binds both RNA and ssDNA in vitro. Here we describe the cloning and sequencing of a cDNA encoding the hnRNP U protein, the determination of its amino acid sequence and the delineation of a region in this protein that confers RNA binding. The predicted amino acid sequence of hnRNP U contains 806 amino acids (88,939 Daltons), and shows no extensive homology to any known proteins. The N-terminus is rich in acidic residues and the C-terminus is glycine-rich. In addition, a glutamine-rich stretch, a putative NTP binding site and a putative nuclear localization signal are present. It could not be defined from the sequence what segment of the protein confers its RNA binding activity. We identified an RNA binding activity within the C-terminal glycine-rich 112 amino acids. This region, designated U protein glycine-rich RNA binding region (U-gly), can by itself bind RNA. Furthermore, fusion of U-gly to a heterologous bacterial protein (maltose binding protein) converts this fusion protein into an RNA binding protein. A 26 amino acid peptide within U-gly is necessary for the RNA binding activity of the U protein. Interestingly, this peptide contains a cluster of RGG repeats with characteristic spacing and this motif is found also in several other RNA binding proteins. We have termed this region the RGG box and propose that it is an RNA binding motif and a predictor of RNA binding activity.     

HnRNP A/B蛋白D亚群的生物学功能

核不均一核糖核蛋白（heterogeneous nuclear ribonucleoproteins, hnRNPs）是一类结合DNA和RNA的核蛋白,并能在核质间穿梭. A/B型hnRNPs（heterogeneous nuclear ribonucleoproteins,hnRNP A/B）是hnRNPs中研究最为清楚的一大类别,生物信息学分析hnRNP A/B可以区分成A和D两个亚群,已鉴定的D亚群主要成员包括hnRNP AB, D和DL. hnRNP A/B的D亚群均含有2个保守的RNA结合结构域（RNA binding domain, RBD）和1个Gly富集区（glycine-rich domain, GRD）,成员间的主要区别在于N端和C端的长度和序列不同. D亚群与pre-mRNA和其它蛋白质结合成微粒系统,参与pre-mRNA的加工、稳定、核输出以及翻译过程. 此外,D亚群也能结合单链和双链DNA,参与转录起始和端粒的稳定. 因此,hnRNP A/B的 D亚群在各个阶段影响基因的表达,在神经系统发育、肿瘤的发生发展及衰老过程中发挥着多样性的功能.     

Distinct RNP complexes of shuttling hnRNP proteins with pre-mRNA and mRNA: candidate intermediates in formation and export of mRNA

Nascent pre-mRNAs associate with hnRNP proteins in hnRNP complexes, the natural substrates for mRNA processing. Several lines of evidence indicate that hnRNP complexes undergo substantial remodeling during mRNA formation and export. Here we report the isolation of three distinct types of pre-mRNP and mRNP complexes from HeLa cells associated with hnRNP A1, a shuttling hnRNP protein. Based on their RNA and protein compositions, these complexes are likely to represent distinct stages in the nucleocytoplasmic shuttling pathway of hnRNP A1 with its bound RNAs. In the cytoplasm, A1 is associated with its nuclear import receptor (transportin), the cytoplasmic poly(A)-binding protein, and mRNA. In the nucleus, A1 is found in two distinct types of complexes that are differently associated with nuclear structures. One class contains pre-mRNA and mRNA and is identical to previously described hnRNP complexes. The other class behaves as freely diffusible nuclear mRNPs (nmRNPs) at late nuclear stages of maturation and possibly associated with nuclear mRNA export. These nmRNPs differ from hnRNPs in that while they contain shuttling hnRNP proteins, the mRNA export factor REF, and mRNA, they do not contain nonshuttling hnRNP proteins or pre-mRNA. Importantly, nmRNPs also contain proteins not found in hnRNP complexes. These include the alternatively spliced isoforms D01 and D02 of the hnRNP D proteins, the E0 isoform of the hnRNP E proteins, and LRP130, a previously reported protein with unknown function that appears to have a novel type of RNA-binding domain. The characteristics of these complexes indicate that they result from RNP remodeling associated with mRNA maturation and delineate specific changes in RNP protein composition during formation and transport of mRNA in vivo.     

Fractionation of constituents of ribonucleoproteins containing heterogeneous nuclear ribonucleic acid.

A method of fractionation of hnRNP constituents adaptable to large-scale preparation is presented. It is based on differential resistance to salt dissociation of the two classes of units of hnRNP, the 30--50S monoparticles and the heterogeneous complexes. The monoparticle proteins were released from hnRNP by 0.4 M NaCl. They were separated from the salt-resistant RNP corresponding to the heterogeneous complexes in three steps: chromatography on DEAE-cellulose, high-speed centrifugation, and Bio-Gel chromatography. The latter chromatography permitted a first fractionation of monoparticle proteins according to molecular weight. Such fractions may serve for purification of individual proteins of molecular weight below 80 000. After the two first steps, two fractions of salt-resistant RNP were obtained. In addition to heterogeneous RNA up to 30 S, small nuclear RNAs were detected which represented 6% of total RNA. The protein pattern was complex, and no clear-cut segregation of groups of proteins could be observed between the two fractions. They were both highly enriched in phosphoproteins as compared to nomoparticle proteins. In another fraction corresponding to the void volume of Bio-Gel chromatography, one-third of the RNA was small nuclear RNA. It is suggested that this fraction contains snRNP in addition to free proteins of molecular weight above 80 000 and to salt-resistant RNP similar to those described above but of small size.     

Heterogeneous nuclear ribonucleoprotein A3, a novel RNA trafficking response element-binding protein

The cis-acting response element, A2RE, which is sufficient for cytoplasmic mRNA trafficking in oligodendrocytes, binds a small group of rat brain proteins. Predominant among these is heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor for cytoplasmic trafficking of RNAs bearing A2RE-like sequences. We have now identified the other A2RE-binding proteins as hnRNP A1/A1(B), hnRNP B1, and four isoforms of hnRNP A3. The rat and human hnRNP A3 cDNAs have been sequenced, revealing the existence of alternatively spliced mRNAs. In Western blotting, 38-, 39-, 41-, and 41.5-kDa components were all recognized by antibodies against a peptide in the glycine-rich region of hnRNP A3, but only the 41- and 41.5-kDa bands bound antibodies to a 15-residue N-terminal peptide encoded by an alternatively spliced part of exon 1. The identities of these four proteins were verified by Edman sequencing and mass spectral analysis of tryptic fragments generated from electrophoretically separated bands. Sequence-specific binding of bacterially expressed hnRNP A3 to A2RE has been demonstrated by biosensor and UV cross-linking electrophoretic mobility shift assays. Mutational analysis and confocal microscopy data support the hypothesis that the hnRNP A3 isoforms have a role in cytoplasmic trafficking of RNA.