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EVOLUTIONARY HOMOLOGS part 2/2

hnRNP involvement in mRNA nuclear-cytoplasmic transport

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. Several of the abundant hnRNP proteins, including A1, are shown to 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. It is proposed that the cytoplasmic ribonucleoprotein complex of mRNA with hnRNP proteins is the substrate of nuclear-cytoplasmic transport of mRNA (Piñol-Roma, 1992).

hnRNP A1 (34 kDa) is an RNA binding protein consisting of two tandemly arranged RNA binding domains C-terminally linked to a glycine-rich auxiliary domain (2 x RBD-Gly). A1 belongs to the set of polypeptides that bind nascent hnRNA in the nucleus to form the so called hnRNP complexes. These complexes seem to be involved both in pre-mRNA processing and in the nuclear export of mRNA. In fact A1, along with other hnRNP proteins, is exported from the nucleus probably bound to mRNA and is immediately re-imported. A1 nuclear re-import, which requires active transcription, is not mediated by a canonical nuclear localisation signal (NLS). To identify the determinants of A1 subcellular localisation an expression vector was developed for studying the localisation, in transiently transfected cells, of the different structural motifs of A1 fused to a small reporter protein (chloramphenicol acetyltransferase, CAT; 26 kDa). A 30 amino acid sequence in the glycine-rich domain (YNDFGNYNNQSSNFGPMKGGNFGGRSSGPY), which bears no resemblance to canonical NLS, is necessary and sufficient to target the protein to the nucleus. These data suggest that this targeting sequence might act by mediating the interaction of A1 with a NLS-containing nuclear import complex. On the other hand, the nuclear export of A1 requires at least one RNA binding domain in accord with the hypothesis that A1 exits from the nucleus bound to mRNA. A mechanism is proposed for the nucleo-cytoplasmic shuttling of A1 that envisages a specific role for the different structural domains and can explain the dependence of nuclear import from active transcription (Weighardt, 1995).

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). 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 (Siomi, 1995).

Pre-mRNAs are associated with hnRNPs, and these proteins play important roles in the biogenesis of mRNAs. The hnRNP A1 is one of the most abundant hnRNPs, and although localized primarily in the nucleoplasm, shuttles continuously between the nucleus and the cytoplasm. A 38 amino acid domain within A1, termed M9, which bears no resemblance to classical nuclear localization signal (NLS) sequences, localizes A1 to the nucleus. M9 is also a nuclear export signal; placing M9 on a protein that is otherwise restricted to the nucleus, the nucleoplasmin core domain (NPc), efficiently exports it to the cytoplasm in a temperature-dependent manner. In contrast, classical NLSs cannot promote the export of NPc. These findings demonstrate that there is a signal-dependent, temperature-sensitive nuclear export pathway and strengthen the suggestion that A1 and other shuttling hnRNPs function as carriers for RNA during export to the cytoplasm (Michael, 1995).

Targeting of most nuclear proteins to the cell nucleus is initiated by interaction between the classical nuclear localization signals (NLSs) contained within them and the importin NLS receptor complex. A novel 38 amino acid transport signal in the hnRNP A1 protein, termed M9, confers bidirectional transport across the nuclear envelope. M9-mediated nuclear import occurs by a novel pathway that is independent of the well-characterized, importin-mediated classical NLS pathway. Additionally, a specific M9-interacting protein, termed transportin, has been identified which binds to wild-type M9 but not to transport-defective M9 mutants. Transportin is a 90 kDa protein, distantly related to importin beta, and it mediates the nuclear import of M9-containing proteins. These findings demonstrate that there are at least two receptor-mediated nuclear protein import pathways. Furthermore, as hnRNP A1 likely participates in mRNA export, it raises the possibility that transportin is a mediator of this process as well (Pollard, 1996).

The connection between RNA and protein export from the nucleus was examined in the budding yeast Saccharomyces cerevisiae. NPL3 encodes an RNA-binding protein that shuttles in and out of the nucleus. Export of poly(A)+ RNA has been shown previously to be blocked in np13-1 mutants. To understand the role of Np13p in RNA export, a novel assay has been developed that effectively uncouples nuclear protein export from reimport. Np13p is shown to satisfy several of the predicted requirements for a protein carrier for mRNA export. Temperature-sensitive mutations in the RNA recognition motifs of Np13p result in nuclear accumulation of poly(A)+ RNA. One such mutation prevents nuclear export of Np13p. Moreover, Np13p export depends on ongoing RNA polymerase II transcription. Export ceases in either the presence of the RNA synthesis inhibitor thiolutin or in a temperature-sensitive RNA polymerase (rpb1) mutant. Together, these findings support a model in which Np13p exits the nucleus in association with poly(A)+ RNA, deposits the RNA in the cytoplasm, and is rapidly reimported for another cycle of export (Lee, 1996).

In yeast, four factors (CF I, CF II, PF I, and PAP) are required for accurate pre-mRNA cleavage and polyadenylation in vitro. CF I can be separated further into CF IA and CF IB. CF IB is the 73-kD Hrp1 protein. Recombinant Hrp1p made in Escherichia coli provides full CF IB function in both cleavage and poly(A) addition assays. Consistent with the presence of two RRM-type motifs, Hrp1p can be UV cross-linked to RNA, and this specific interaction requires the (UA)6 polyadenylation efficiency element. Furthermore, the CF II factor enhances the binding of Hrp1p to the RNA precursor. A temperature-sensitive mutant in HRP1 yields mRNAs with shorter poly(A) tails when grown at the nonpermissive temperature. Genetic analyses indicate that Hrp1p interacts with Rna15p and Rna14p, two components of CF 1A. The HRP1 gene was originally isolated as a suppressor of a temperature-sensitive npl3 allele, a gene encoding a protein involved in mRNA export. Like Npl3p, Hrp1p shuttles between the nucleus and cytoplasm, providing a potential link between 3'-end processing and mRNA export from the nucleus (Kessler, 1997).

Segregation of mRNAs in the cytoplasm of polar cells has been demonstrated for proteins involved in Xenopus and Drosophila oogenesis, and for some proteins in somatic cells. It is assumed that vectorial transport of the messages is generally responsible for this localization. The mRNA encoding the basic protein of central nervous system myelin is selectively transported to the distal ends of the processes of oligodendrocytes, where it is anchored to the myelin membrane and translated. This transport is dependent on a 21-nucleotide cis-acting segment of the 3'-untranslated region (RTS). Proteins that bind to this cis-acting segment have now been isolated from extracts of rat brain. A group of six 35-42-kDa proteins bind to a 35-base oligoribonucleotide incorporating the RTS, but not to several oligoribonucleotides with the same composition but randomized sequences, thus establishing specificity for the base sequence in the RTS. The most abundant of these proteins has been identified as heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a 36-kDa member of a family of proteins that are primarily, but not solely, intranuclear. This protein is most abundant in samples from rat brain and testis, with lower amounts in other tissues. It was separated from the other polypeptides by using reverse-phase HPLC and shown to retain preferential association with the RTS. In cultured oligodendrocytes, hnRNP A2 was demonstrated to be distributed throughout the nucleus, cell soma, and processes (Hoek, 1998).

The structural and accessory proteins of human immunodeficiency virus type 1 are expressed by unspliced or partially spliced mRNAs. Efficient transport of these mRNAs from the nucleus requires the binding of the viral nuclear transport protein Rev to an RNA stem-loop structure called the RRE (Rev response element). However, the RRE does not permit Rev to stimulate the export of unspliced mRNAs from the efficiently spliced beta-globin gene in the absence of additional cis-acting RNA regulatory signals. The p17gag gene instability (INS) element contains RNA elements that can complement Rev activity. In the presence of the INS element and the RRE, Rev permits up to 30 % of the total beta-globin mRNA to be exported to the cytoplasm as unspliced mRNA. A minimal sequence of 30 nt derived from the 5' end of the p17 gag gene INS element (5' INS) is functional and permits the export to the cytoplasm of 14% of the total beta-globin mRNA as unspliced pre-mRNA. Gel mobility shift assays and UV cross-linking experiments have shown that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and a cellular RNA-binding protein of 50 kDa form a complex on the 5' INS. Mutants in the 5' INS that prevent hnRNP A1 and 50 kDa protein binding are inactive in the transport assay. To confirm that the hnRNP A1 complex is responsible for INS activity, a synthetic high-affinity binding site for hnRNP A1 was also analysed. When the high affinity hnRNP A1 binding site is inserted into the beta-globin reporter, Rev is able to increase the cytoplasmic levels of unspliced mRNAs to 14%. In contrast, the mutant hnRNP A1 binding site, or binding sites for hnRNP C and L are unable to stimulate Rev-mediated RNA transport. It is concluded that hnRNP A1 is able to direct unspliced globin pre-mRNA into a nuclear compartment where it is recognised by Rev and then transported to the cytoplasm (Najera, 1999).

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an abundant nuclear protein that plays an important role in pre-mRNA processing and mRNA export from the nucleus. A1 shuttles rapidly between the nucleus and the cytoplasm, and a 38-amino acid domain, M9, serves as the bidirectional transport signal of A1. Recently, a 90-kD protein, transportin, was identified as the mediator of A1 nuclear import. In this study, transportin is shown to mediate the nuclear import of additional hnRNP proteins, including hnRNP F. A novel transportin homolog, transportin2, has been isolated and sequenced which may differ from transportin1 in its substrate specificity. Immunostaining shows that transportin1 is localized both in the cytoplasm and the nucleoplasm, and nuclear rim staining is also observed. The nuclear localization of A1 is dependent on ongoing RNA polymerase II transcription. Interestingly, a pyruvate kinase-M9 fusion, which normally localizes in the nucleus, also accumulates in the cytoplasm when RNA polymerase II is inhibited. Thus, M9 itself is a specific sensor for transcription-dependent nuclear transport. Transportin1-A1 complexes can be isolated from the cytoplasm and the nucleoplasm, but transportin1 is not detectable in hnRNP complexes. RanGTP causes dissociation of A1-transportin1 complexes in vitro. Thus, it is likely that after nuclear import, A1 dissociates from transportin1 by RanGTP and becomes incorporated into hnRNP complexes, where A1 functions in pre-mRNA processing (Siomi, 1997).

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, a yeast two-hybrid system was used to search for proteins that interact with yTRN. In an exhaustive screen of the S. cerevisiae genome, the most frequently selected open reading frame was the nuclear mRNA-binding protein, Nab2p. A ca.-50-amino-acid region in Nab2p, termed NAB35, was delineated 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. Additional substrates for TRN are reported 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 (Siomi, 1998).

In the larval salivary glands of C. tentans, it is possible to visualize by electron microscopy how Balbiani ring (BR) pre-mRNA associates with proteins to form pre-mRNP particles, how these particles move to and through the nuclear pore, and how the BR RNA is engaged in the formation of giant polysomes in the cytoplasm. C. tentans hrp36 is an abundant protein in the BR particles that it is similar to the mammalian hnRNP A1. By immuno-electron microscopy it is demonstrated that hrp36 is added to BR RNA concomitant with transcription, remains in nucleoplasmic BR particles, and is translocated through the nuclear pore still associated with BR RNA. It appears in the giant BR RNA-containing polysomes, where it remains as an abundant protein in spite of ongoing translation (Visa, 1996).

hnRNP involvement in mRNA turnover

mRNA turnover is an important regulatory component of gene expression and is significantly influenced by ribonucleoprotein (RNP) complexes which form on the mRNA. Studies of human alpha-globin mRNA stability have identified a specific RNP complex (alpha-complex) which forms on the 3' untranslated region (3'UTR) of the mRNA and appears to regulate the erythrocyte-specific accumulation of alpha-globin mRNA. One of the protein activities in this multiprotein complex is a poly(C)-binding activity which consists of two proteins, alphaCP1 and alphaCP2. Neither of these proteins, individually or as a pair, can bind the alpha-globin 3'UTR unless they are complexed with the remaining non-poly(C) binding proteins of the alpha-complex. With the yeast two-hybrid screen, a second alpha-complex protein was identified. This protein is a member of the previously identified A+U-rich (ARE) binding/degradation factor (AUF1) family of proteins, which are also known as the heterogeneous nuclear RNP (hnRNP) D proteins. These proteins are referred to as AUF1/hnRNP-D. Thus, a protein implicated in ARE-mediated mRNA decay is also an integral component of the mRNA stabilizing alpha-complex. The interaction of AUF1/hnRNP-D is more efficient with alphaCP1 relative to alphaCP2 both in vitro and in vivo, suggesting that the alpha-complex might be dynamic rather than a fixed complex. AUF1/hnRNP-D could, therefore, be a general mRNA turnover factor involved in both stabilization and decay of mRNA (Kiledjian, 1997).

Monocyte adherence results in the rapid transcriptional activation and mRNA stabilization of numerous mediators of inflammation and tissue repair. While the enhancer and promoter elements associated with transcriptional activation have been studied, mechanisms linking adhesion, mRNA stabilization, and translation are unknown. GROalpha and interleukin-1beta (IL-1beta) mRNAs are highly labile in nonadhered monocytes but stabilize rapidly after adherence. GROalpha and IL-1beta transcripts both contain A+U-rich elements (AREs) in the 3' untranslated region (UTR) which have been directly associated with rapid mRNA turnover. To determine if the GROalpha ARE region is recognized by factors associated with mRNA degradation, mobility gel shift analyses was carried out using a series of RNA probes encompassing the entire GROalpha transcript. Stable complexes are formed only with the proximal 3' UTR which contain the ARE region. The two slower-moving complexes are rapidly depleted following monocyte adherence but not direct integrin engagement. Deadherence reactivates the two largest ARE-binding complexes and destabilize IL-1beta transcripts. Antibody supershift studies demonstrate that both of these ARE RNA-binding complexes contained AUF1. The formation of these complexes and the accelerated mRNA turnover are phosphorylation-dependent events, as both are induced in adherent monocytes by the tyrosine kinase inhibitor genistein and the p38 MAP kinase inhibitor of IL-1beta translation, SK&F 86002. These results demonstrate that cell adhesion and deadhesion rapidly and reversibly modify both cytokine mRNA stability and the RNA-binding complexes associated with AUF1 (Sirenko, 1997).

AU-rich RNA-destabilizing elements (AREs) have become a paradigm for studying cytoplasmic mRNA turnover in mammalian cells. Though many RNA-binding proteins have been shown to bind to AREs in vitro, trans-acting factors that participate in the in vivo destabilization of cytoplasmic RNA by AREs remains unknown. Experiments were performed to investigate the cellular mechanisms and to identify potential trans-acting factors for ARE-directed mRNA decay. These experiments identified hnRNP D, a heterogeneous nuclear ribonucleoprotein (hnRNP) capable of shuttling between the nucleus and cytoplasm, as an RNA destabilizing protein in vivo in ARE-mediated rapid mRNA decay. These results show that the ARE destabilizing function is dramatically impeded during hemin-induced erythroid differentiation and not in TPA-induced megakaryocytic differentiation of human erythroleukemic K562 cells. A sequestration of hnRNP D into a hemin-induced protein complex, termed hemin-regulated factor or HRF, correlates well with the loss of ARE-destabilizing function in the cytoplasm. Further experiments show that in hemin-treated cells, ectopic expression of hnRNP D restores the rapid decay directed by the ARE. The extent of destabilizing effect varies among the four isoforms of hnRNP D, with p37 and p42 displaying the most profound effect. These results demonstrate a specific cytoplasmic function for hnRNP D as an RNA-destabilizing protein in ARE-mediated decay pathway. These in vivo findings support an emerging idea that shuttling hnRNP proteins have not only a nuclear but also a cytoplasmic function in mRNA metabolism. The data further imply that shuttling hnRNP proteins define, at least in part, the nuclear history of individual mRNAs and thereby influence their cytoplasmic fate (Loflin, 1999).

In both cell culture based model systems and in the failing human heart, beta-adrenergic receptors (beta-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3'-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. The mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT1-MF2 smooth muscle cells and its abundance is regulated by beta-AR agonist stimulation. In human heart, AUF1 mRNA and protein is significantly increased in individuals with myocardial failure, a condition associated with increases in the beta-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (approximately 50%) in the abundance of beta1-AR mRNA and protein. In DDT1-MF2 cells, where agonist-mediated destabilization of beta2-AR mRNA was first described, exposure to beta-AR agonist results in a significant increase in AUF1 mRNA and protein (approximately 100%). Conversely, agonist exposure significantly decreases (approximately 40%) beta2-adrenergic receptor mRNA abundance. AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3'-untranslated region of the human beta1-AR mRNA and purified, recombinant p37AUF1 protein also binds to beta1-AR 3'-untranslated region mRNA (Pende, 1996).

hnRNP involvement in maintenance of telomeres

Telomeric DNA of mammalian chromosomes consists of several kilobase-pairs of tandemly repeated sequences with a terminal 3' overhang in single-stranded form. Maintaining the integrity of these repeats is essential for cell survival; telomere attrition is associated with chromosome instability and cell senescence, whereas stabilization of telomere length correlates with the immortalization of somatic cells. Telomere elongation is carried out by telomerase, an RNA-dependent DNA polymerase which adds single-stranded TAGGGT repeats to the 3' ends of chromosomes. While proteins that associate with single-stranded telomeric repeats can influence tract lengths in yeast, equivalent factors have not yet been identified in vertebrates. The heterogeneous nuclear ribonucleoprotein A1is shown to participate in telomere biogenesis. A mouse cell line deficient in A1 expression harbours telomeres that are shorter than those of a related cell line expressing normal levels of A1. Restoring A1 expression in A1-deficient cells increases telomere length. Telomere elongation is also observed upon introduction of exogenous UP1, the amino-terminal fragment of A1. While both A1 and UP1 bind to vertebrate single-stranded telomeric repeats directly and with specificity in vitro, only UP1 can recover telomerase activity from a cell lysate. These findings establish A1/UP1 as the first single-stranded DNA binding protein involved in mammalian telomere biogenesis and suggest possible mechanisms by which UP1 may modulate telomere length (LaBranche, 1998).

hnRNPs bind to DNA: hnRNPs involvement in the immunoglobulin heavy chain switch reaction

The B cell-specific, sequence-specific duplex DNA-binding protein LR1 is a transcriptional activator and may also function in heavy chain class switch recombination. LR1 is composed of two polypeptides, a 106-kDa subunit that is nucleolin, and a 45-kDa subunit that is a specific isoform of hnRNP D. hnRNP D and nucleolin both contain canonical RNA binding domains (RBDs also called RRMs) and Arg-Gly-Gly (RGG) motifs. Although these motifs are not commonly associated with sequence-specific recognition of duplex DNA, nonetheless LR1 binds duplex DNA with high affinity (KD = 1.8 nM) and clear sequence specificity. Two RBD-RGG proteins can therefore combine to produce a sequence-specific duplex DNA-binding protein (Dempsey, 1998b).

The immunoglobulin heavy chain switch regions contain multiple runs of guanines on the top (nontemplate) DNA strand. LR1, a B cell-specific, duplex DNA binding factor, binds tightly and specifically to synthetic oligonucleotides containing G-G base pairs (KD = 0.25 nM). LR1 also binds to single-stranded G-rich sequences (KD approximately 10 nM). The two subunits of LR1, nucleolin and hnRNP D, bind with high affinity to G4 DNA (KD = 0.4 and 0.5 nM, respectively). LR1 therefore contains two independent G4 DNA binding domains. It is proposed that LR1 binds with G-G-paired structures that form during the transcription of the S regions and that this binding is a prerequisite to recombination in vivo. Interactions of donor and acceptor S regions with subunits of the LR1 could juxtapose the switch regions for recombination (Dempsey, 1999).

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