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The eukaryotic translation initiation factor 4E (eIF4E) controls gene expression through its effects on mRNA export and cap-dependent translation, both of which contribute to its oncogenic potential. In contrast to its translation function, the mRNA export function of eIF4E is poorly understood. Using an RNP isolation/mass spectrometry approach, we identified candidate cofactors of eIF4E mRNA export including LRPPRC. This protein associates with mRNAs containing the eIF4E-sensitivity element (4E-SE), and its overexpression alters the nuclear export of several eIF4E-sensitive mRNAs. LRPPRC-mediated alteration of eIF4E's mRNA export function requires the integrity of its eIF4E-binding site and it coincides with the subcellular re-distribution of eIF4E. The eIF4E export RNP is distinct in composition from the bulk mRNA export pathway, in that eIF4E- and eIF4E-sensitive mRNAs do not associate with general mRNA export factors such as TAP/NXF1 or REF/Aly. Our data indicate that mRNA export pathways have evolved for specific mRNAs enabling the differential regulation of biochemical pathways by modulating the expression of groups of genes at the level of their export.

The ADAR enzymes mediate the hydrolytic deamination of adenosines in specific RNA substrates and thereby diversify both the transcriptome and the proteome in metazoan species. Three promoters drive the transcription from the ADAR1 gene yielding the ADAR1-A, -B, and -C transcripts, which, in turn, lead to the production of two protein isoforms, namely, iADAR1 and cADAR1. In this study, we establish the presence of a previously unidentified alternative intron within the 5'-end of the common second exon of mRNAs encoding ADAR1 in primate species-a region that can function either as a 5'-UTR or an ORF. In addition, it is shown that the relative expression of the three promoter-specific ADAR1 transcripts is tissue specific and that the novel intron is excised from all transcripts, but at different relative levels indicating a specific regulation of the alternative splicing. Finally, possible functional consequences of the splicing are investigated. From these studies, we conclude that the alternatively spliced ADAR1-A transcript is immune to nonsense-mediated decay although it is a potential substrate. Moreover, this transcript is associated with translating ribosomes, which suggests that a truncated version of iADAR1 is expressed.

LRP130 (also known as LRPPRC) is an RNA-binding protein that is a constituent of postsplicing nuclear RNP complexes associated with mature mRNA. It belongs to a growing family of pentatricopeptide repeat (PPR) motif-containing proteins, several of which have been implicated in organellar RNA metabolism. We show here that only a fraction of LRP130 proteins are in nuclei and are directly bound in vivo to at least some of the same RNA molecules as the nucleocytoplasmic shuttle protein hnRNP A1. The majority of LRP130 proteins are located within mitochondria, where they are directly bound to polyadenylated RNAs in vivo. In vitro, LRP130 binds preferentially to polypyrimidines. This RNA-binding activity maps to a domain in its C-terminal region that does not contain any previously described RNA-binding motifs and that contains only 2 of the 11 predicted PPR motifs. Therefore, LRP130 is a novel type of RNA-binding protein that associates with both nuclear and mitochondrial mRNAs and as such is a potential candidate for coordinating nuclear and mitochondrial gene expression. These findings provide the first identification of a mammalian protein directly bound to mitochondrial RNA in vivo and provide a possible molecular explanation for the recently described association of mutations in LRP130 with cytochrome c oxidase deficiency in humans.

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 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.

Southwestern screening of human fibroblast cDNAs with an upstream element of the alpha2(I) collagen promoter (Box 5A) has led to the identification of a novel gene product (RBMS3). RBMS3 contains two pairs of RNA binding motifs and is very closely related to the structure of the c-myc gene single-strand binding proteins (MSSPs). MSSPs are believed to regulate DNA replication, transcription, apopotosis and cell cycle progression by interacting with the C-MYC protein. Consonant with this postulate, RBMS3 binds in vitro to the minus strand of Box 5A and transactivates transcription in the chimeric GAL4 hybrid system. However, the RBMS3 protein mostly localizes to the cytoplasm of transfected cells, in addition to binding strongly in vitro to synthetic poly-U and poly-A oligoribonucleotides. Finally, overexpression in transfected fibroblasts of RBMS3 with and without a nuclear localization signal has no effect on Box 5A-driven transcription. The results thus exclude RBMS3 involvement in the transcriptional regulation of COL1A2 and strongly suggest a cytoplasmic function of this new member of the MSSP family. As part of the initial characterization of RBMS3 we have also established that the gene resides on human chromosome 3p23-p24 and is widely expressed in the embryo and in the adult organism.

rRNA precursors are bound throughout their length by specific proteins, as the pre-rRNAs emerge from the transcription machinery. The association of pre-rRNA with proteins as ribonucleoprotein (RNP) complexes persists during maturation of 18S, 5.8S, and 28S rRNA, and through assembly of ribosomal subunits in the nucleolus. Preribosomal RNP complexes contain, in addition to ribosomal proteins, an unknown number of nonribosomal nucleolar proteins, as well as small nucleolar RNA-ribonucleoproteins (sno-RNPs). This report describes the use of a specific, rapid, and mild immunopurification approach to isolate and analyze human RNP complexes that contain nonribosomal nucleolar proteins, as well as ribosomal proteins and rRNA. Complexes immunopurified with antibodies to nucleolin-a major nucleolar RNA-binding protein-contain several distinct specific polypeptides that include, in addition to nucleolin, the previously identified nucleolar proteins B23 and fibrillarin, proteins with electrophoretic mobilities characteristic of ribosomal proteins including ribosomal protein S6, and a number of additional unidentified proteins. The physical association of these proteins with one another is mediated largely by RNA, in that the complexes dissociate upon digestion with RNase. Complexes isolated from M-phase cells are similar in protein composition to those isolated from interphase cell nuclear extracts. Therefore, the predominant proteins that associate with nucleolin in interphase remain in RNP complexes during mitosis, despite the cessation of rRNA synthesis and processing in M-phase. In addition, precursor rRNA, as well as processed 18S and 28S rRNA and candidate rRNA processing intermediates, is found associated with the immunopurified complexes. The characteristics of the rRNP complexes described here, therefore, indicate that they represent bona fide precursors of mature cytoplasmic ribosomal subunits.

Pre-mRNAs associate in the nucleus with specific RNA-binding proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. The hnRNP proteins participate directly or indirectly in the processing of pre-mRNAs into mature mRNAs. Recent studies have shown that some hnRNP proteins shuttle continuously between the nucleus and the cytoplasm. The export of shuttling hnRNP proteins from the nucleus is mediated by specific nuclear export sequences (NESs) within the proteins. In addition, shuttling hnRNP proteins appear to remain bound to exported mRNAs in transit through nuclear pores. As discussed in this review, the picture that is emerging is that nuclear export of mRNAs is mediated by the export of NES-containing hnRNP proteins to which they are bound.

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 proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are among the most abundant proteins in the nucleus. They bind nascent pre-mRNAs and remain associated with them through their nuclear processing into mRNA. Recent findings indicate roles for hnRNP proteins in the biogenesis of mRNA and reveal a surprising intracellular localization pathway for these proteins. Several of the hnRNP proteins shuttle continuously between the nucleus and the cytoplasm, and the reaccumulation of the exported hnRNP proteins in the nucleus occurs by a novel process that is dependent on transcription by RNA polymerase II. These findings suggest possible novel functions for hnRNP proteins in the cytoplasm and in the nucleocytoplasmic transport of mRNA.

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.

RNA polymerase II transcripts, heterogeneous nuclear RNAs (hnRNAs), associate in the nucleus with specific proteins that bind premessenger RNA (hnRNP proteins) and with small nuclear ribonucleoprotein particles (snRNPs). These hnRNA-hnRNP-snRNP complexes assemble on nascent transcripts and hnRNA is processed to mRNA in them. HnRNP proteins have been localized to the nucleoplasm and their functions were presumed to be limited to nuclear events in mRNA biogenesis. It was proposed that an exchange of hnRNP for mRNA-binding proteins accompanies transport of mRNA from the nucleus to the cytoplasm. We show here that several of the abundant hnRNP proteins, including A1, shuttle between the nucleus and the cytoplasm. HnRNP proteins may thus also have cytoplasmic functions. Furthermore, when in the cytoplasm, A1 is bound to mRNA and RNA polymerase II transcription is necessary before it can return to the nucleus. We propose that the cytoplasmic ribonucleoprotein complex of mRNA with hnRNP proteins is the substrate of nuclear-cytoplasmic transport of mRNA.

Heterogeneous nuclear RNAs and specific nuclear proteins form heterogeneous nuclear ribonucleoprotein complexes (hnRNPs), one of the most abundant components of the nucleus. In mitosis, as the nuclear envelope breaks down, hnRNPs disperse throughout the cell. At the end of mitosis, hnRNPs dissociate and their proteins are transported into the daughter cell nuclei separately. Some are transported immediately (early group), while others are transported later (late group). Transport of the late group appears to require transcription by RNA polymerase II, in that inhibitors of this polymerase cause the late proteins to remain in the cytoplasm. Thus, there are two modes, transcription-dependent and transcription-independent, for the transport of nuclear proteins.