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.     

The hnRNP F protein: unique primary structure, nucleic acid-binding properties, and subcellular localization.

More than 20 different heterogeneous nuclear ribonucleoproteins (hnRNPs) are associated with pre-mRNAs in the nucleus of mammalian cells and these proteins appear to influence pre-mRNA processing and other aspects of mRNA metabolism and transport. The arrangement of hnRNP proteins on pre-mRNAs is likely to be unique for each RNA and may be determined by the different RNA-binding preferences of each of these proteins. hnRNP F (M(r) = 53 kD, pI = 6.1) and hnRNP H (M(r) = 56 kD, pI = 6.7-7.1) are abundant components of immunopurified hnRNP complexes and they have distinct nucleic acid binding properties. Unlike other hnRNP proteins which display a varying range of affinities for different ribonucleotidehomopolymers and ssDNA, hnRNP F and hnRNP H bind only to poly(rG) in vitro. hnRNP F and hnRNP H were purified from HeLa cells by poly(rG) affinity chromatography and oligonucleotides derived from peptide sequences were used to isolate a cDNA encoding hnRNP F. The predicted amino acid sequence of hnRNP F revealed a novel protein with three repeated domains related to the RNP consensus sequence RNA-binding domain. Monoclonal antibodies produced against bacterially expressed hnRNP F were specific for both hnRNP F and hnRNP H and recognized related proteins in divergent organisms, including in the yeast Saccharomyces cerevisiae. hnRNP F and hnRNP H are thus highly related immunologically and they share identical peptides. Interestingly, immunofluorescence microscopy revealed that hnRNP F and hnRNP H are concentrated in discrete regions of the nucleoplasm, in contrast to the general nucleoplasmic distribution of previously characterized hnRNP proteins. The unique RNA-binding properties, amino acid sequence and distinct intranuclear localization of hnRNP F and hnRNP H make them novel hnRNP proteins that are likely to be important for the processing of RNAs containing guanosine-rich sequences.     

Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein.

At least 20 major proteins make up the ribonucleoprotein (RNP) complexes of heterogeneous nuclear RNA (hnRNA) in mammalian cells. Many of these proteins have distinct RNA-binding specificities. The abundant, acidic heterogeneous nuclear RNP (hnRNP) K and J proteins (66 and 64 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are unique among the hnRNP proteins in their binding preference: they bind tenaciously to poly(C), and they are the major oligo(C)- and poly(C)-binding proteins in human HeLa cells. We purified K and J from HeLa cells by affinity chromatography and produced monoclonal antibodies to them. K and J are immunologically related and conserved among various vertebrates. Immunofluorescence microscopy with antibodies shows that K and J are located in the nucleoplasm. cDNA clones for K were isolated, and their sequences were determined. The predicted amino acid sequence of K does not contain an RNP consensus sequence found in many characterized hnRNP proteins and shows no extensive homology to sequences of any known proteins. The K protein contains two internal repeats not found in other known proteins, as well as GlyArgGlyGly and GlyArgGlyGlyPhe sequences, which occur frequently in many RNA-binding proteins. Overall, K represents a novel type of hnRNA-binding protein. It is likely that K and J play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences.     

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.     

hnRNP G: sequence and characterization of a glycosylated RNA-binding protein.

The autoantigen p43 is a nuclear protein initially identified with autoantibodies from dogs with a lupus-like syndrome. Here we show that p43 is an RNA-binding protein, and identify it as hnRNP G, a previously described component of heterogeneous nuclear ribonucleoprotein complexes. We demonstrate that p43/hnRNP G is glycosylated, and identify the modification as O-linked N-acetylglucosamine. A full-length cDNA clone for hnRNP G has been isolated and sequenced, and the predicted amino acid sequence for hnRNP G shows that it contains one RNP-consensus RNA binding domain (RBD) at the amino terminus and a carboxyl domain rich in serines, arginines and glycines. The RBD of human hnRNP G shows striking similarities with the RBDs of several plant RNA-binding proteins.     

The nucleolar protein, B-36, contains a glycine and dimethylarginine-rich sequence conserved in several other nuclear RNA-binding proteins

The amino terminal sequence of the 34 kD nucleolar protein B-36 isolated from the slime mold Physarum polycephalum has been determined. This portion of B-36 is rich in glycine, phenylalanine and the modified amino acid asymmetrical dimethylarginine (DMA) and is 65% identical to that for fibrillarin, a similar and potentially homologous 34 kD nucleolar protein from rat. The terminus of B-36 contains an interesting nine amino acid sequence, Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA-Gly, which is precisely repeated three times in the 110 kD nucleolar protein nucleolin. Similar sequences have also been reported in a yeast nucleolar protein (SSB-1) and several hnRNP proteins (rat A1 and brine shrimp GRP33). The conserved nature of this unusual sequence is suggestive of an important function which may include RNA-binding since several of these proteins share this feature.     

Mutational definition of RNA-binding and protein-protein interaction domains of heterogeneous nuclear RNP C1

The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of approximately 80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.     

Identification of two RNA-binding proteins in Balbiani ring premessenger ribonucleoprotein granules and presence of these proteins in specific subsets of heterogeneous nuclear ribonucleoprotein particles.

Balbiani ring (BR) granules are premessenger ribonucleoprotein particles (RNPs) generated in giant chromosomal puffs, the BRs, in the larval salivary glands of the dipteran chironomus tentans. Monoclonal antibodies were raised against nuclear proteins collected on a single-stranded-DNA-agarose affinity column, and two of them were used to identify RNA-binding proteins in BR granules. First, in Western blots (immunoblots), one of the antibodies recognized a 36-kDa protein and the other recognized a 45-KDa protein. Second, both antibodies bound to the BRs in immunocytological experiments. It was shown in cross-linking experiments that the two proteins are associated with heterogeneous nuclear RNP (hnRNP) complexes extracted from C. tentans nuclei. By immunoelectron microscopy of isolated and partly unfolded BR RNPs, it was specifically demonstrated that the BR granules contain the two proteins and, in addition, that both proteins are distributed frequently along the RNP fiber of the particles. Thus, the 36- and 45-KDa proteins are likely to be abundant, RNA-binding proteins in the BR particles. To elucidate to what extent the two proteins are also present in other hnRNPs, we studied the binding of the antibodies to chromosomal puffs in general. It was observed that many puffs in addition to the BRs harbor the two proteins, but there are also puffs containing only one of the components, either the 36- or the 45-kDa protein. We conclude that the two proteins are not randomly bound to all hnRNPs but that each of them seems to be linked to a specific subset of the particles.     

The hnRNP C proteins contain a nuclear retention sequence that can override nuclear export signals

Nascent pre-mRNAs associate with the abundant heterogeneous nuclear RNP (hnRNP) proteins and remain associated with them throughout the time they are in the nucleus. The hnRNP proteins can be divided into two groups according to their nucleocytoplasmic transport properties. One group is completely restricted to the nucleus in interphase cells, whereas the other group, although primarily nuclear at steady state, shuttles between the nucleus and the cytoplasm. Nuclear export of the shuttling hnRNP proteins is mediated by nuclear export signals (NESs). Mounting evidence indicates that NES-bearing hnRNP proteins are mediators of mRNA export. The hnRNP C proteins are representative of the nonshuttling group of hnRNP proteins. Here we show that hnRNP C proteins are restricted to the nucleus not because they lack an NES, but because they bear a nuclear retention sequence (NRS) that is capable of overriding NESs. The NRS comprises approximately 78 amino acids and is largely within the auxiliary domain of hnRNP C1. We suggest that the removal of NRS-containing hnRNP proteins from pre- mRNA/mRNA is required for mRNA export from the nucleus and is an essential step in the pathway of gene expression.     

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.     

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.     

A cDNA clone of the hnRNP C proteins and its homology with the single-stranded DNA binding protein UP2.

A cDNA clone which expresses a protein that cross-reacts immunologically with the human C1 and C2 hnRNP core proteins has been isolated. The clone was selected by a sensitive immunochemical assay employing an avidin-biotin complex for detection, and identified as a clone for the hnRNP C proteins by a highly sensitive antibody select assay that is described here. The clone contains 677 nucleotides, and, as shown by northern blotting, is derived from a 1.5 Kb poly(A)+ mRNA. There are regions of strong homology between the human and mouse genes, weak homology is seen with chicken DNA, and very little, if any, homology can be detected with Drosophila, Artemia, sea urchin, or yeast DNAs. Two peptides (a total of 24 amino acids) of the calf thymus single-stranded DNA binding protein UP2 show perfect homology with the deduced amino acid sequence of the clone, suggesting that UP2 is related to the hnRNP C proteins. There is also a region that has a sequence very similar to two regions of the single-stranded DNA binding protein UP1 that contain proposed DNA binding sites.     

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.     

RNP-T,a ribonucleoprotein from Arabidopsis thaliana,contains two RNP-80 motifs and a novel acidic repeat arranged in an α-helix conformation

We have isolated a 1148 bp long cDNA clone encoding an RNA-binding protein in Arabidopsis. Several partial cDNA clones were isolated by screening an Arabidopsis gt11 expression library for the binding of DNA. One of these clones was used as a probe to isolate a full-length clone. The 329 amino acid protein, termed RNP-T, contains in its carboxy terminus two adjacent RNP-80 motifs, a previously described 80 amino acid long conserved putative RNA-binding domain. Each RNP-80 motif includes both consensus short sequences, RNP1 and RNP2, which are separated by 33 amino acids. We have identified an acidic domain of 54 amino acids, which is located amino-terminal to the RNP-80 motifs. Seven tandem repeats of a hexamer are present within this domain. This acidic domain has a potential -helix conformation. We propose that the acidic patch might play a role in protein-protein interaction.