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.

RNA-binding proteins as regulators of gene expression.

A plethora of post-transcriptional mechanisms are involved in essential steps in the pathway of genetic information expression in eukaryotes. These processes are specified by cis-acting signals on RNAs and are mediated by specific trans-acting factors, including RNA-binding proteins and small complementary RNAs. Recent information has begun to define the molecular mechanisms by which RNA-binding proteins recognize specific RNA sequences and influence the processing and function of RNA molecules.

Specific sequences in the fragile X syndrome protein FMR1 and the FXR proteins mediate their binding to 60S ribosomal subunits and the interactions among them.

Fragile X syndrome, the most common form of hereditary mental retardation, usually results from lack of expression of the FMR1 gene. The FMR1 protein is a cytoplasmic RNA-binding protein. The RNA-binding activity of FMR1 is an essential feature of FMR1, as fragile X syndrome can also result from the expression of mutant FMR1 protein that is impaired in RNA binding. Recently, we described two novel cytoplasmic proteins, FXR1 and FXR2, which are both very similar in amino acid sequence to FMR1 and which also interact strongly with FMR1 and with each other. To understand the function of FMR1 and the FXR proteins, we carried out cell fractionation and sedimentation experiments with monoclonal antibodies to these proteins to characterize the complexes they form. Here, we report that the FMR1 and FXR proteins are associated with ribosomes, predominantly with 60S large ribosomal subunits. The FXR proteins are associated with 60S ribosomal subunits even in cells that lack FMR1 and that are derived from a fragile X syndrome patient, indicating that FMR1 is not required for this association. We delineated the regions of FMR1 that mediate its binding to 60S ribosomal subunits and the interactions among the FMR1-FXR family members. Both regions contain sequences predicted to have a high propensity to form coiled coil interactions, and the sequences are highly evolutionarily conserved in this protein family. The association of the FMR1, FXR1, and FXR2 proteins with ribosomes suggests they have functions in translation or mRNA stability.

Long cellular repeats flanking a defective HTLV-I provirus: implication for site-targeted integration.

Retroviruses generally integrate as proviruses which are flanked by long-terminal repeats (LTRs) on both 5' and 3' ends. Since these LTRs are required for the efficient integration mediated by the viral integrase, it is believed that defective proviruses with a single LTR are normally formed by deletion after integration. However, we found no deletion of cellular sequences around the integration site of such a defective HTLV-1. Rather, we identified 99 bp-long direct repeats adjacent to both ends of the defective provirus. The repeated cellular sequences contained a potential poly(A) signal followed by a retroviral primer-binding-site-like sequence. The presence of the direct repeats of cellular sequences can be explained by the integration of the defective virus through homologous recombination between cellular and viral read-through sequences.

A cellular protein which is coprecipitated with HTLV-I rex protein in the presence of the target mRNA.

We examined the cellular protein(s) which can associate with Rex protein of human T cell leukemia virus type I (HTLV-I), using Rex-maltose binding protein (MBP) fusion protein. Immunoprecipitation of RexMBP with anti-MBP antibody revealed that a 24 kD protein (p24) associated with RexMBP only in the presence of Rex-responsive mRNA. The fact that p24 was present in both the nucleus and the cytoplasm is consistent with a role of Rex in the nucleo-cytoplasmic transport of viral mRNAs. P24 did not interact with nonfunctional Rex mutant proteins even if they had RNA binding activity in vitro. These results suggest the possible involvement of p24 in the Rex function through a complex formation with Rex on Rex-responsive mRNA.

Cis/trans-activation of the interleukin-9 receptor gene in an HTLV-I-transformed human lymphocytic cell.

The MT-2 cell-line, which had been established through in vitro cell to cell transmission of human T-cell leukemia virus type I (HTLV-I) among human primary lymphocytes, was shown to possess multiple copies of integrated proviruses, including defective proviral genomes. By analysing a genomic clone, we identified the integration site of a single HTLV-I long terminal repeat (LTR) in the interleukin-9 (IL-9) receptor (IL-9R) gene. The integrated HTLV-I-LTR was shown to be functional as a promoter and the integration site was located in an intron upstream of the first coding exon of the IL-9R gene. Upon analysis of total cellular RNA, specific expression of HTLV-I-LTR Il-9R chimeric mRNAs in MT-2 cells was demonstrated. Cloning and characterization of these cDNAs have identified HTLV-I-IL-9R chimeric splicing, using either intact or alternative splice sites within the IL-9R gene. The potential roles of multiple interactions between IL-9, IL-9R and HTLV-I in the monoclonal expansion and transformation of MT-2 cells are explored.

Analysis of a novel defective HTLV-I provirus and detection of a new HTLV-I-induced cellular transcript.

HTLV-I generally integrates at least one full-length copy in adult T-cell leukemia (ATL) cells. A group of patients without full-length provirus have a unique conserved truncation of the provirus which retains env-pX-3'LTR. Tumor cells of a patient from this group were genetically analyzed. Analysis of the 5' and 3' cellular flanking region adjacent to the provirus suggest that the defective provirus was integrated immediately downstream of a promoter of an unknown cellular gene. The activity of the promoter was weak but was responsive to Tax-like HTLV-I LTR. The provirus may have utilized it as a substitute for the 5'LTR and thus 3'LTR may have become an alternative promoter for the cellular gene, which may give similar viral-cellular interactions to that of general cases with full-length proviruses. Surprisingly, the 3' cellular flanking region which is thought to be controlled originally by the promoter is constitutively expressed specifically in an HTLV-I producing ATL cell line HUT1O2G, in which the corresponding region is not modified by provirus. The detection of this HTLV-I-induced transcript provides a probe to find an HTLV-I inducible unknown cellular gene that may be related to the pathogenesis of ATL.

Molecular mechanisms of fragile X syndrome.

Fragile X syndrome is the most common form of inherited mental retardation Mutations which abolish expression of an X-linked gene, FMR1, result in pathogenesis of the disease. FMR1 encodes a cytoplasmic RNA-binding protein which interacts with two autosomal homologs, FXR1 and FXR2. These proteins are highly expressed in neurons. In addition, the FMR1/FXR proteins are associated with ribosomes. Given their RNA-binding activity and association with ribosomes, these proteins are hypothesized to bind to specific RNAs and regulate their expression at translational levels in a manner critical for correct development of neurons. Much progress has been made in FMR1 research over the past several years, but little light has yet to be shed on the physiological function of these proteins. It will be critical to define the biochemical properties of these proteins, and identify potential downstream targets to clarify the molecular mechanisms underlying the potential roles of these proteins in translation. A basic understanding of the function of this new family of RNA-binding proteins should then allow us to begin to address the question of how the lack of FMR1 expression leads to symptoms in fragile X syndrome.

Effects of a highly basic region of human immunodeficiency virus Tat protein on nucleolar localization.

Human immunodeficiency virus type 1 encodes a positive trans-activator protein, Tat, which is located predominantly in the cell nucleolus. To study the role of the basic region of Tat in nucleolar localization, we constructed fusion genes encoding serially deleted segments of Tat joined to the amino-terminal end of the Escherichia coli beta-galactosidase molecule. We show that the basic region of Tat was sufficient for nuclear localization but not for nucleolar localization. Addition of three amino acids (59, 60, and 61) of the Tat sequence at the C-terminal end of the basic region was necessary for the chimeric beta-galactosidase to localize in the nucleus as well as in the nucleolus. We demonstrate that a short amino acid sequence (G-48 RKKRRQRRRA HQ N-61), when fused to the amino terminus of beta-galactosidase, can act as a nucleolar localization signal.

The protein product of the fragile X gene, FMR1, has characteristics of an RNA-binding protein.

Fragile X syndrome is one of the most common human genetic diseases and the most common cause of hereditary mental retardation. The gene that causes fragile X syndrome, FMR1, was recently identified and sequenced and found to encode a putative protein of unknown function. Here we report that FMR1 contains two types of sequence motifs recently found in RNA-binding proteins: an RGG box and two heterogeneous nuclear RNP K homology domains. We also demonstrate that FMR1 binds RNA in vitro. Using antibodies to FMR1, we detect its expression in divergent organisms and in cells of unaffected humans, but fragile X-affected patients express little or no FMR1. These findings demonstrate that FMR1 expression is directly correlated with the fragile X syndrome and suggest that anti-FMR1 antibodies will be important for diagnosis of fragile X syndrome. Furthermore, the RNA binding activity of FMR1 opens the way to understanding the function of FMR1.

Sequence requirements for nucleolar localization of human T cell leukemia virus type I pX protein, which regulates viral RNA processing.

The posttranscriptional regulator (p27x-III) of human T cell leukemia virus type I (HTLV-I) is located predominantly in the cell nucleolus. A highly basic amino-terminal sequence (NH2-Met-Pro-Lys-Thr-Arg-Arg-Arg-Pro-Arg-Arg-Ser-Gln-Arg-Lys-Arg-Pro-Pro -Thr- Pro) in this protein, when fused to the amino termini of beta-galactosidase and p40x of HTLV-I, acts as an autonomous signal capable of directing the hybrid proteins to the cell nucleolus.

Essential role for KH domains in RNA binding: impaired RNA binding by a mutation in the KH domain of FMR1 that causes fragile X syndrome.

The KH domain is an evolutionarily conserved sequence motif present in many RNA-binding proteins, including the pre-mRNA-binding (hnRNP) K protein and the fragile X mental retardation gene product (FMR1). We assessed the role of KH domains in RNA binding by mutagenesis of KH domains in hnRNP K and FMR1. Conserved residues of all three hnRNP K KH domains are required for its wild-type RNA binding. Interestingly, while fragile X syndrome is usually caused by lack of FMR1 expression, a previously reported mutation in a highly conserved residue of one of its two KH domains (Ile-304-->Asn) also results in mental retardation. We found that the binding of this mutant protein to RNA is severely impaired. These results demonstrate an essential role for KH domains in RNA binding. Furthermore, they strengthen the connection between fragile X syndrome and loss of the RNA binding activity of FMR1.

Functional comparison of transactivation by human retrovirus rev and rex genes.

The effect of rev-responsive element deletion on human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) gene expression was examined. The phenotypes of HIV-1 and HIV-2 provirus DNAs lacking the rev-responsive element, as determined by transfection experiments, were indistinguishable from those of virus DNAs carrying rev gene mutations. By using rev-response elements derived from these two viruses, we developed two monitoring systems to evaluate the functionality of HIV-1 rev, HIV-2 rev, and human T-lymphotropic virus type I rex. In both systems, HIV-1 rev and human T-lymphotropic virus type I rex transactivated HIV-2 very efficiently. On the contrary, HIV-2 rev and human T-lymphotropic virus type I rex were poor activators of HIV-1. No functional replacement of rex by HIV-2 rev was observed.

A region of basic amino-acid cluster in HIV-1 Tat protein is essential for trans-acting activity and nucleolar localization.

The trans-acting factor of human immunodeficiency virus (HIV), Tat, has a basic amino-acid cluster that is highly conserved among different HIV isolates. We have examined the effects of mutations in the basic region of Tat on its trans-acting activity and cellular localization. Introduction of a stop codon immediately preceding the basic region abolished the activity, while the truncated mutant with the basic region retained some activity. The basic region of Tat was replaceable with that of Rev (another trans-acting factor of HIV) but not with that of adenovirus Ela nor cellular enzyme. The result of immunofluorescence analysis revealed a correlation between the nuclear, especially nucleolar, accumulation and the activities of mutant Tat proteins.

Nucleolar targeting signal of human T-cell leukemia virus type I rex-encoded protein is essential for cytoplasmic accumulation of unspliced viral mRNA.

The posttranscriptional regulator (rex) of human T-cell leukemia virus type I is known to be located predominantly in the cell nucleolus and to induce the accumulation of gag and env viral mRNAs. The N-terminal 19 amino acids of rex-encoded protein (Rex) has been shown to be sufficient to direct hybrid proteins to the cell nucleolus. We have studied the function of the nucleolar targeting signal (NOS) of rex by using full-length proviral DNA and mutant rex expression plasmids. Partial deletions of the NOS sequence abolished the accumulation of unspliced cytoplasmic mRNA, although the gene products of rex mutants were found in the nucleoplasm. These results indicate that NOS sequence, or nucleolar localization of Rex, is essential for Rex function.

Functional similarity of HIV-I rev and HTLV-I rex proteins: identification of a new nucleolar-targeting signal in rev protein.

We have tested the functional compatibility between rev protein of human immunodeficiency virus type I (HIV-I) and rex protein of human T-cell lymphotropic virus type I (HTLV-I). Each protein recognized the other's cis-acting sequence, albeit at reduced levels. Both proteins localize predominantly in the nucleolus. We have identified a new nucleolar-targeting signal in rev protein, which was homologous to that of rex protein. The sequence [35-RQARRNRRRRWRERQR-50] in rev protein, when fused to the amino-terminus of beta-galactosidase, directed the hybrid protein to the cell nucleolus. A deletion mutant which lacks several amino acid residues within the signal failed to function in the CAT assay system. These results demonstrate that the nucleolar targeting signals are essential for the functions of Rev and Rex.

The pre-mRNA binding K protein contains a novel evolutionarily conserved motif.

The K protein is among the major pre-mRNA-binding proteins (hnRNPs) in vertebrate cell nuclei. It binds tenaciously to cytidine-rich sequences and is the major oligo(rC/dC)-binding protein in vertebrate cells. We have cloned a cDNA of the Xenopus laevis hnRNP K and determined its sequence. The X.laevis hnRNP K is a 47 kD protein that is remarkably similar to its human 66 kD counterpart except for two large internal deletions. The sequence of hnRNP K contains a 45 amino acid repeated motif which is almost completely conserved between the X.laevis and human proteins. We found that this repeated motif, the KH motif (for K homology), shows significant homology to several proteins some of which are known nucleic acids binding proteins. The homology is particularly strong with the archeabacterial ribosomal protein S3 and with the saccharomyces cerevisiae protein MER1 which is required for meiosis-specific splicing of the MER 2 transcript. As several of the proteins that contain the KH motif are known to bind RNA, this domain may be involved in RNA binding.

Transportin: nuclear transport receptor of a novel nuclear protein import pathway.

Many nuclear proteins are imported into the cell nucleus by the "classical" nuclear localization signal (NLS)-mediated import pathway. In this pathway, a sequence rich in basic residues in the protein interacts with a heterodimeric complex termed importin and this, along with the GTPase Ran, mediates nuclear import of the NLS-bearing protein. The heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein contains a novel nuclear localization sequence, termed M9, that does not contain any clusters of basic residues. Very recently, we showed that M9 directs import into the nucleus by a novel protein import pathway distinct from the classical NLS pathway. A 90-kilodalton protein termed transportin was identified as a protein that specifically interacts with wild-type M9 but not transport-defective M9 mutants. Transportin and an ATP-regenerating system were found to be necessary and sufficient for import of M9-containing proteins in an in vitro import assay. In this report, we provide additional evidence that transportin can interact directly with M9-containing proteins and also show that it can mediate import of full-length hnRNP A1. In addition, Ran, or a Ran-binding protein, is identified as a second protein component of this novel nuclear import pathway. Transportin relatives from Saccharomyces cerevisiae which likely serve as additional nuclear transport receptors are described.

Preferential transcription of HTLV-I LTR in cell-free extracts of human T cells producing HTLV-I viral proteins.

The promoters of the adenovirus 2 major late gene, the mouse beta-globin gene, the mouse immunoglobulin VH gene and the LTR of the human T-lymphotropic retrovirus type I were tested for their transcription activities in cell-free extracts of four cell lines; HeLa, CESS (Epstein-Barr virus-transformed human B cell line), MT-1 (HTLV-I-infected human T cell line without viral protein synthesis), and MT-2 (HTLV-I-infected human T cell line producing viral proteins). LTR was preferentially transcribed in the extracts of MT-2 although the other three genes were transcribed with relatively constant efficiencies in different extracts. The results agree well with the previous in vivo studies on the promoter activity of HTLV-I LTR. Mixing of HeLa and MT-2 extracts revealed the presence of a LTR-specific stimulating activity in MT-2 extracts.