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 proteins and the nuclear export of mRNA

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.     

Interactions of 40LoVe within the ribonucleoprotein complex that forms on the localization element of Xenopus Vg1 mRNA

Proline rich RNA-binding protein (Prrp), which associates with mRNAs that employ the late pathway for localization in Xenopus oocytes, was used as bait in a yeast two-hybrid screen of an expression library. Several independent clones were recovered that correspond to a paralog of 40LoVe, a factor required for proper localization of Vg1 mRNA to the vegetal cortex. 40LoVe is present in at least three alternatively spliced isoforms; however, only one, corresponding to the variant identified in the two-hybrid screen, can be crosslinked to Vg1 mRNA. In vitro binding assays revealed that 40LoVe has high affinity for RNA, but exhibits little binding specificity on its own. Nonetheless, it was only found associated with localized mRNAs in oocytes. 40LoVe also interacts directly with VgRBP71 and VgRBP60/hnRNP I; it is the latter factor that likely determines the binding specificity of 40LoVe. Initially, 40LoVe binds to Vg1 mRNA in the nucleus and remains with the RNA in the cytoplasm. Immunohistochemical staining of oocytes shows that the protein is distributed between the nucleus and cytoplasm, consistent with nucleocytoplasmic shuttling activity. 40LoVe is excluded from the mitochondrial cloud, which is used by RNAs that localize through the early (METRO) pathway in stage I oocytes; nonetheless, it is associated with at least some early pathway RNAs during later stages of oogenesis. A phylogenetic analysis of 2×RBD hnRNP proteins combined with other experimental evidence suggests that 40LoVe is a distant homolog of Drosophila Squid.     

PTB Binds to the 3’ Untranslated Region of the Human Astrovirus Type 8: A Possible Role in Viral Replication

The 3′ untranslated region (3′UTR) of human astroviruses (HAstV) consists of two hairpin structures (helix I and II) joined by a linker harboring a conserved PTB/hnRNP1 binding site. The identification and characterization of cellular proteins that interact with the 3′UTR of HAstV-8 virus will help to uncover cellular requirements for viral functions. To this end, mobility shift assays and UV cross-linking were performed with uninfected and HAstV-8-infected cell extracts and HAstV-8 3′UTR probes. Two RNA-protein complexes (CI and CII) were recruited into the 3′UTR. Complex CII formation was compromised with cold homologous RNA, and seven proteins of 35, 40, 45, 50, 52, 57/60 and 75 kDa were cross-linked to the 3′UTR. Supermobility shift assays indicated that PTB/hnRNP1 is part of this complex, and 3′UTR-crosslinked PTB/hnRNP1 was immunoprecipitated from HAstV-8 infected cell-membrane extracts. Also, immunofluorescence analyses revealed that PTB/hnRNP1 is distributed in the nucleus and cytoplasm of uninfected cells, but it is mainly localized perinuclearly in the cytoplasm of HAstV-8 infected cells. Furthermore, the minimal 3′UTR sequences recognized by recombinant PTB are those conforming helix I, and an intact PTB/hnRNP1-binding site. Finally, small interfering RNA-mediated PTB/hnRNP1 silencing reduced synthesis viral genome and virus yield in CaCo2 cells, suggesting that PTB/hnRNP1 is required for HAstV replication. In conclusion, PTB/hnRNP1 binds to the 3′UTR HAstV-8 and is required or participates in viral replication.     

PROTEINS IN NUCLEOCYTOPLASMIC INTERACTIONS : II. Turnover and Changes in Nuclear Protein Distribution with Time and Growth

In previous studies, we showed that essentially all the proteins of the Amoeba proteus nucleus could be classified either as Rapidly Migrating Proteins (RMP), which shuttle between nucleus and cytoplasm continuously at a relatively rapid rate during interphase, or as Slow Turnover Proteins (STP), which seem to move hardly at all during interphase. In this paper, we report on the kinetics and direction of the movement of both classes of protein, as well as on aspects of their localization, with and without growth. The effects of growth were observed with and without cell division. These nuclear proteins have been studied in several ways: by transplantation of labeled nuclei into unlabeled cells and noting the rate of distribution to cytoplasm and host cell nuclei; by repeated amputation of cytoplasm from labeled cells—with and without initially labeled cytoplasm—each amputation being followed by refeeding on unlabeled food; by noting the redistribution of the various protein classes following growth and cell division. The data show (a) labeled RMP equilibrate between a grafted labeled nucleus and an unlabeled host nucleus in ca. 3 hr, but are detectable in the latter less than 30 min after the operation; (b) STP label does, indeed, leave the nucleus and does so at a rate of ca. 25% of the nuclear total per cell generation (ca. 36–40 hr at 23°C); (c) the cytoplasm appears to have a reserve of material that is converted to RMP; (d) when labeled cells are amputated just before they would have divided and are refed unlabeled food after each amputation, there is a loss of 20–25% of the nuclear protein label with each amputation; (e) under the latter circumstances, an essentially complete turnover of all nuclear protein can be demonstrated.     

Heterogeneous nuclear ribonucleoprotein D/AUF1 interacts with heterogeneous nuclear ribonucleoprotein L

Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is one of the principal pre-mRNA-binding proteins found in human cells. The hnRNP L protein is fairly abundant. However, it is not restricted to the nucleus, and instead shuttles between the nucleus and the cytoplasm. It is composed of 558 amino acid residues and harbours four loosely conserved RNP-consensus RNA-binding domains. In an attempt to characterize the interaction occurring between cellular proteins and hnRNP L, yeast two-hybrid screening was conducted using a HeLa cDNA library. Some of the cDNA clones were found to harbour a partial human hnRNP D/AUF1 cDNA (GeneBank accession number NM_031369). In this study, we determined that hnRNP L interacts specifically with the hnRNP D/AUF1 in the yeast two-hybrid system. This interaction was verified via an in vitro pull-down assay.     

p35 interacts with α‐tubulin and organelle proteins: Nuclear translocation of p35 in dying cells

We identified heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2, hnRNP A1, the translocase of the transporter outer membrane 40 (TOM40), and α‐tubulin as new interaction partners of anti‐apoptotic protein p35 using MS‐based functional proteomics with GST‐p35 fusion protein as a bait, and using a pull‐down assay with p35‐6His followed by Western blot analysis. p35 was localized in the cytoplasm and in distinct organelles such as the nucleus and mitochondria. p35 was more abundant in the cytoplasm than it was in the nucleus. It co‐localized with α‐tubulin in the cytoplasm in the absence of a death stimulus. However, while cells were undergoing death induced by actinomycin D, cytoplasmic p35 was translocated into the nucleus; this process was inhibited by deletions of the N‐ and C‐terminal domains containing leucine‐rich motifs. Gene delivery of p35 using recombinant adenoviruses inhibited cytoplasmic compartmentalization of hnRNP C1/C2 and hnRNP A1 in dying cells. This study demonstrated translocation of p35 into the nuclei, as well as protection of the hnRNPs from redistribution in cells undergoing death. We propose an active role for p35 in maintaining the integrity of nuclear proteins during cell death.     

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.     

A Highlights from MBoC Selection: Quantitative Analysis of the Mechanism of Endocytic Actin Patch Assembly and Disassembly in Fission Yeast

We used quantitative confocal microscopy to measure the numbers of 16 proteins tagged with fluorescent proteins during assembly and disassembly of endocytic actin patches in fission yeast. The peak numbers of each molecule that accumulate in patches varied <30–50% between individual patches. The pathway begins with accumulation of 30–40 clathrin molecules, sufficient to build a hemisphere at the tip of a plasma membrane invagination. Thereafter precisely timed waves of proteins reach characteristic peak numbers: endocytic adaptor proteins (∼120 End4p and ∼230 Pan1p), activators of Arp2/3 complex (∼200 Wsp1p and ∼340 Myo1p) and ∼300 Arp2/3 complexes just ahead of a burst of actin assembly into short, capped and highly cross-linked filaments (∼7000 actins, ∼200 capping proteins, and ∼900 fimbrins). Coronin arrives last as all other components disperse upon patch internalization and movement over ∼10 s. Patch internalization occurs without recruitment of dynamins. Mathematical modeling, described in the accompanying paper (Berro et al., 2010, MBoC 21: 2803–2813), shows that the dendritic nucleation hypothesis can account for the time course of actin assembly into a branched network of several hundred filaments 100–200 nm long and that patch disassembly requires actin filament fragmentation in addition to depolymerization from the ends.     

Functional Conservation of the Transportin Nuclear Import Pathway in Divergent Organisms

Human transportin1 (hTRN1) is the nuclear import receptor for a group of pre-mRNA/mRNA-binding proteins (heterogeneous nuclear ribonucleoproteins [hnRNP]) represented by hnRNP A1, which shuttle continuously between the nucleus and the cytoplasm. hTRN1 interacts with the M9 region of hnRNP A1, a 38-amino-acid domain rich in Gly, Ser, and Asn, and mediates the nuclear import of M9-bearing proteins in vitro. Saccharomyces cerevisiae transportin (yTRN; also known as YBR017c or Kap104p) has been identified and cloned. To understanding the nuclear import mediated by yTRN, we searched with a yeast two-hybrid system for proteins that interact with it. In an exhaustive screen of the S. cerevisiae genome, the most frequently selected open reading frame was the nuclear mRNA-binding protein, Nab2p. We delineated a ca.-50-amino-acid region in Nab2p, termed NAB35, which specifically binds yTRN and is similar to the M9 motif. NAB35 also interacts with hTRN1 and functions as a nuclear localization signal in mammalian cells. Interestingly, yTRN can also mediate the import of NAB35-bearing proteins into mammalian nuclei in vitro. We also report on additional substrates for TRN as well as sequences of Drosophila melanogaster, Xenopus laevis, and Schizosaccharomyces pombe TRNs. Together, these findings demonstrate that both the M9 signal and the nuclear import machinery utilized by the transportin pathway are conserved in evolution.     

A Role for the M9 Transport Signal of hnRNP A1 in mRNA Nuclear Export

Among the nuclear proteins associated with mRNAs before their export to the cytoplasm are the abundant heterogeneous nuclear (hn) RNPs. Several of these contain the M9 signal that, in the case of hnRNP A1, has been shown to be sufficient to signal both nuclear export and nuclear import in cultured somatic cells. Kinetic competition experiments are used here to demonstrate that M9-directed nuclear import in Xenopus oocytes is a saturable process. Saturating levels of M9 have, however, no effect on the import of either U snRNPs or proteins carrying a classical basic NLS. Previous work demonstrated the existence of nuclear export factors specific for particular classes of RNA. Injection of hnRNP A1 but not of a mutant protein lacking the M9 domain inhibited export of mRNA but not of other classes of RNA. This suggests that hnRNP A1 or other proteins containing an M9 domain play a role in mRNA export from the nucleus. However, the requirement for M9 function in mRNA export is not identical to that in hnRNP A1 protein transport.The transport of macromolecules between the nucleus and cytoplasm is a bi-directional process. The best understood aspect is the import of nuclear proteins that carry a basic nuclear localization signal (NLS)¹ like the simple NLS found in SV-40 T antigen or the bipartite NLS found in nucleoplasmin (Dingwall and Laskey, 1991). Proteins of this class are recognized by the heterodimeric importin receptor, composed of importin α and importin β (for review see Powers and Forbes, 1994; Melchior and Gerace, 1995; Görlich and Mattaj, 1996). The NLS binds directly to the importin α subunit. The importin NLS protein complex docks at the cytoplasmic face of the nuclear pore complex in an energy-independent manner (Newmeyer and Forbes, 1988; Richardson et al., 1988). Subsequently, the small GTPase Ran/TC4 (Melchior et al., 1993; Moore and Blobel, 1993) and a protein of unknown function named variously pp15, p10, or NTF2 (Moore and Blobel, 1994; Paschal and Gerace, 1995) are required for translocation of the NLS-containing complex through the nuclear pore complex.A second major class of imported macromolecules are the uracil rich small nuclear (U sn) RNPs. They do not have a basic NLS but instead have a bipartite nuclear targeting signal. This is composed of an essential signal formed when the Sm core proteins bind to the U snRNA and an additional signal, the trimethyl-guanosine (m₃G) cap, which depending on the cell type or the U snRNA is either essential or required for optimal U snRNP import efficiency (Fischer and Lührmann, 1990; Hamm et al., 1990; Fischer et al., 1993). Kinetic competition experiments have supported the conclusion that U snRNPs require different limiting factors than do NLS-containing proteins for their import and that U snRNPs do not bind to importin α (Fischer et al., 1991, 1993; Michaud and Goldfarb, 1991; van Zee et al., 1993). There is also preliminary evidence that additional different receptors may be required for the nuclear uptake of other RNA species (Michaud and Goldfarb, 1992).Similarly, RNA export from the nucleus relies on recognition of the RNA or RNP export substrates by saturable factors (Zasloff, 1983; Bataillé et al., 1990; Jarmolowski et al., 1994). As for import, evidence for the existence of RNA class-specific export receptors has been obtained from kinetic competition experiments (Jarmolowski et al., 1994). Two RNA-binding proteins have been directly shown to function in RNA export, a nuclear cap binding protein complex in the case of U snRNAs (Izaurralde et al., 1995a ) and the HIV-1 Rev protein in the case of RNAs containing a rev response element (Fischer et al., 1994, 1995). In the case of mRNAs, the best candidates for export mediators are the heterogeneous nuclear (hn) RNP proteins (for review see Piñol-Roma and Dreyfuss, 1993; Izaurralde and Mattaj, 1995).About 20 different hnRNP proteins have been characterized in vertebrate cells (for review see Dreyfuss et al., 1993). The association of hnRNP proteins with mRNA in the nucleus and the cytoplasm suggests that they may regulate and/or facilitate different aspects of gene expression. The possibility that hnRNP proteins might be directly involved in the nucleocytoplasmic trafficking of mRNA molecules was suggested by the observation that several hnRNP proteins, including A1, A2, D, E, I, and K shuttle continuously and rapidly between the nucleus and the cytoplasm and are associated with mRNA in both compartments (Piñol-Roma and Dreyfuss 1992, 1993; Michael et al., 1995a , b). Of these, the best studied example is hnRNP A1. An A1-like hnRNP protein has been shown by immunoelectron microscopy to be associated with a specific mRNA in transit to the cytoplasm through the nuclear pore complex in the insect Chironomus tentans (Visa et al., 1996a ). In mammalian cells, the amount of A1 which is in constant flux between nucleus and cytoplasm is striking. It has been estimated that at least 120,000 molecules of A1 are exported to the cytoplasm per minute but then rapidly reimported such that the steady state localization of A1 is nuclear (Michael et al., 1995a ). Taken together, these results suggest that A1 and other shuttling hnRNP proteins such as A2, D, E, I, and K could play a significant role in the transport of mRNA from the nucleus to the cytoplasm.One key in understanding how hnRNPs may facilitate mRNA export is to determine the signals that mediate their shuttling, i.e., their import into and exit from the nucleus. The nucleocytoplasmic transport of A1 has been recently studied in detail, and the signals that mediate shuttling have been identified (Michael et al., 1995b ; Siomi and Dreyfuss, 1995; Weighardt et al., 1995). Nuclear import of A1 is determined by a 38-amino acid sequence, termed M9, located near the COOH terminus of the protein between amino acids 268 and 305. Its fusion to cytoplasmic reporter proteins such as pyruvate kinase resulted in rapid import of the fusion protein into the nucleus (Siomi and Dreyfuss, 1995). However, the A1 NLS has no sequence similarity to classical protein NLSs such as that of SV-40 large T antigen or nucleoplasmin (Siomi and Dreyfuss, 1995).Surprisingly, M9 also acts as a nuclear export signal (NES). In heterokaryon shuttling assays this domain is necessary and sufficient to allow the export of heterologous proteins, such as the nucleoplasmin core domain (NPLc), which are normally retained in the nucleus (Michael et al., 1995b ). Thus, M9 alone can account for the shuttling of A1. Other hnRNPs such as A2 and B1 bear sequences with striking similarities to M9 (Siomi and Dreyfuss, 1995). Mutagenesis experiments indicate that the NES and NLS activities of M9 are either identical or overlapping as mutants which block M9 NLS activity also abolish NES activity (Michael et al., 1995b ). It is therefore possible that M9 is recognized in the nucleus and the cytoplasm by the same receptor.The second category of NES described was first found in the HIV-1 Rev protein and the inhibitor of protein kinase A (Fischer et al., 1995; Wen et al., 1995; Bogerd et al., 1996; for review see Gerace, 1995). These short, leucine-rich NES sequences bear no relationship to the primary sequence of M9. Furthermore, saturation of the export factor recognized by the Rev NES has no effect on mRNA export (Fischer et al., 1995). A model for mRNA export has been postulated on the basis of the hnRNP data described above. In this model, NES/NLS containing hnRNPs bind in the nucleus to mRNA molecules and deliver them, via the export pathway they access, to the cytoplasm. In the cytoplasm these hnRNP proteins dissociate from the mRNA and return to the nucleus. To further test this model we have analyzed the transport of hnRNP A1 and mRNA in Xenopus laevis oocytes. The oocyte offers a unique opportunity to manipulate specific import or export pathways, like that accessed by M9, and examine the effect on mRNA nuclear export. By using this approach we show here that M9 is, as in somatic cells, a functional NLS in oocytes. Moreover, competition studies indicate that M9 defines a novel class of NLS, since saturation of the M9- mediated import pathway does not interfere with the two previously identified import pathways used by classical NLS-bearing proteins or m₃G-capped-spliceosomal U snRNPs. Injection of an excess of hnRNP A1 but not of a mutant form of the protein lacking the M9 domain, resulted in a specific inhibition of mRNA export, demonstrating that the M9 domain is recognized by a saturable component of the mRNA export machinery. The export of other cellular RNAs such as U snRNAs and tRNA was, in contrast, not affected. Further analysis of mutant hnRNP A1 proteins provides evidence that M9 recognition during mRNA export differs from its recognition during protein transport.     

Non-random localization of ribonucleoprotein (RNP) structures within an adenovirus mRNA precursor.

Heterogeneous nuclear protein complexes (hnRNP) containing the precursor RNA from the adenovirus early region 2 were analysed to determine the specificity of protein-RNA interaction. RNA precursor sequences were present in isolated hnRNP complexes and endogenous 30S particles. At least 20-40 bases long fragments were protected when RNase A was used to remove unprotected RNA sequences in hnRNA complexes. Similarly around 40 bases of RNA were protected in 30S particles. These sequences represent discrete regions of the adenovirus genome. Especially sequences complementary to the EcoRI-F fragment encoding the first leader and the major intron for the DNA binding protein (DBP) RNA precursor, were analysed in detail. Tentatively, sequences resistant to RNase A were located in the middle of the intron and at the splice-donor junction of the first leader of the DBP precursor RNA. The same sequences were identified irrespective whether hnRNP complexes or 30S particles were used suggesting that 30S particles originate from hnRNP complexes. A 38.000 dalton protein appears to be in direct contact with RNA sequences complementary to the EcoRI-F fragment.