Polymorphism at position -308 of the tumour necrosis factor alpha gene and rheumatoid arthritis pharmacogenetics.
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Adult↗
Biomedical subjects
M Carmo-Fonseca
Publications and source records attributed to M Carmo-Fonseca.
The rules and roles of nucleocytoplasmic shuttling proteins.
The spatial separation of mRNA synthesis from translation, while providing eukaryotes with the possibility to achieve higher complexity through a more elaborate regulation of gene expression, has set the need for transport mechanisms through the nuclear envelope. In a simplistic view of nucleocytoplasmic transport, nuclear proteins are imported into the nucleus while RNAs are exported to the cytoplasm. The reality is, however, that transport of either proteins or RNAs across the nuclear envelope can be bi-directional. During the past years, an increasing number of proteins have been identified that shuttle continuously back and forth between the nucleus and the cytoplasm. The emerging picture is that shuttling proteins are key factors in conveying information on nuclear and cytoplasmic activities within the cell.
Active Transport, Cell Nucleus↗
REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus.
The REF family of evolutionarily conserved heterogeneous ribonucleoprotein (hnRNP)-like proteins consists of one central RNP-type RNA binding domain flanked by Arg-Gly-rich regions of variable length. Members of this protein family bind directly to RNA and the mRNA export factor TAP/Mex67p, and it has been suggested that they facilitate the recruitment of TAP/Mex67p to cellular mRNPs. We show that the variable regions are necessary for binding of REFs to RNA and to TAP. Antibodies specific to REFs prevent their interaction with RNA in vitro. After microinjection into Xenopus oocytes, these antibodies inhibit mRNA nuclear export. This inhibition of export is observed whether or not the mRNAs are generated by splicing. The antibodies do not interfere with pre-mRNA splicing or with the nuclear export of constitutive transport element (CTE)-containing RNAs (directly mediated by TAP), so REF proteins must play a critical role in mRNA nuclear export, acting downstream of splicing and upstream of TAP/Mex67p. We also show that recombinant REFs stimulate directly the export of mRNAs that are otherwise exported inefficiently. Together, our data indicate that REFs are directly implicated in the export of mRNAs from the nucleus. More generally, we show that spliced and unspliced mRNAs use common export factors to reach the cytoplasm.
Amino Acid Sequence↗
Precursor RNAs harboring nonsense codons accumulate near the site of transcription.
Messenger RNAs containing premature termination codons (PTCs) are selectively eliminated by nonsense-mediated mRNA decay (NMD). Paradoxically, although cytoplasmic ribosomes are the only known species capable of PTC recognition, in mammals many PTC-containing mRNAs are apparently eliminated prior to release from the nucleus. To determine whether PTCs can influence events within the nucleus proper, we studied the immunoglobulin (Ig)-mu and T cell receptor (TCR)-beta genes using fluorescent in situ hybridization (FISH). Alleles containing PTCs, but not those containing a missense mutation or a frameshift followed by frame-correcting mutations, exhibited elevated levels of pre-mRNA, which accumulated at or near the site of transcription. Our data indicate that mRNA reading frame can influence events at or near the site of gene transcription.
Animals↗
Chromosomal G-dark bands determine the spatial organization of centromeric heterochromatin in the nucleus.
Gene expression can be silenced by proximity to heterochromatin blocks containing centromeric alpha-satellite DNA. This has been shown experimentally through cis-acting chromosome rearrangements resulting in linear genomic proximity, or through trans-acting changes resulting in intranuclear spatial proximity. Although it has long been been established that centromeres are nonrandomly distributed during interphase, little is known of what determines the three-dimensional organization of these silencing domains in the nucleus. Here, we propose a model that predicts the intranuclear positioning of centromeric heterochromatin for each individual chromosome. With the use of fluorescence in situ hybridization and confocal microscopy, we show that the distribution of centromeric alpha-satellite DNA in human lymphoid cells synchronized at G(0)/G(1) is unique for most individual chromosomes. Regression analysis reveals a tight correlation between nuclear distribution of centromeric alpha-satellite DNA and the presence of G-dark bands in the corresponding chromosome. Centromeres surrounded by G-dark bands are preferentially located at the nuclear periphery, whereas centromeres of chromosomes with a lower content of G-dark bands tend to be localized at the nucleolus. Consistent with the model, a t(11; 14) translocation that removes G-dark bands from chromosome 11 causes a repositioning of the centromere, which becomes less frequently localized at the nuclear periphery and more frequently associated with the nucleolus. The data suggest that "chromosomal environment" plays a key role in the intranuclear organization of centromeric heterochromatin. Our model further predicts that facultative heterochromatinization of distinct genomic regions may contribute to cell-type specific patterns of centromere localization.
Cell Nucleus↗
Transcription-dependent nucleocytoplasmic distribution of hnRNP A1 protein in early mouse embryos.
A unique feature of certain members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins is that they shuttle continuously between nucleus and cytoplasm and their accumulation in the nucleus is transcription-dependent. An extensively characterised protein of this group is hnRNP A1. To date, most studies addressing the transcription-dependent transport of hnRNP A1 have been performed on cultured cell lines treated with transcription inhibitors. Here we have analysed the nucleocytoplasmic distribution of hnRNP A1 in early mouse embryos, where the haploid pronuclei remain transcriptionally inactive for a period of several hours. Consistent with its small molecular size (36 kDa), the hnRNP A1 protein diffuses passively through the nuclear pores and equilibrates between the nucleus and the cytoplasm of transcriptionally inactive embryos. In contrast, following transcriptional activation the A1 protein becomes accumulated in the nucleus. This accumulation of the A1 protein in the nucleus is blocked by the lectin wheat germ agglutinin (WGA), which binds to nuclear pore proteins and prevents translocation of receptor-cargo complexes through the pores. This indicates that a carrier-mediated transport pathway is required for the concentration of A1 in transcriptionally active nuclei. To further analyse how transcription is coupled to nucleocytoplasmic transport, we transplanted transcriptionally inactive pronuclei into the cytoplasm of transcriptionally active embryos. The results show that the presence of newly synthesised RNAs in the cytoplasm is not sufficient to induce the accumulation of hnRNP A1 in the nucleus. Rather, the appearance of nascent transcripts in the nucleus appears to be the crucial event. Since hnRNP A1 is a shuttling protein, an increase in its steady state nuclear concentration could be the result of either faster nuclear import or slower export to the cytoplasm. We propose that binding of A1 to nascent transcripts retards its export to the cytoplasm and therefore contributes to its concentration in the nucleus.
Animals↗
Identification of two novel RanGTP-binding proteins belonging to the importin beta superfamily.
Nucleo-cytoplasmic transport comprises a large number of distinct pathways, many of which are defined by members of the importin beta superfamily of nuclear transport receptors. These transport receptors all directly interact with RanGTP to modulate the compartment-specific binding of their transport substrates. To identify new members of the importin beta family, we used affinity chromatography on immobilized RanGTP and isolated Ran-binding protein (RanBP) 16 from HeLa cell extracts. RanBP16 and its close human homologue, RanBP17, are distant members of the importin beta family. Like the other members of the transport receptor superfamily, RanBP16 interacts with the nuclear pore complex and is able to enter the nucleus independent of energy and additional nuclear transport receptors.
Amino Acid Sequence↗
Nucleocytoplasmic shuttling of heterodimeric splicing factor U2AF.
The U2 small nuclear ribonucleoprotein auxiliary factor (U2AF) is a heterodimeric splicing factor composed of 65-kDa (U2AF(65)) and 35-kDa (U2AF(35)) subunits. The large subunit of U2AF recognizes the intronic polypyrimidine tract, a sequence located adjacent to the 3' splice site that serves as an important signal for both constitutive and regulated pre-mRNA splicing. The small subunit U2AF(35) interacts with the 3' splice site dinucleotide AG and is essential for regulated splicing. Like several other proteins involved in constitutive and regulated splicing, both U2AF(65) and U2AF(35) contain an arginine/serine-rich (RS) domain. In the present study we determined the role of RS domains in the subcellular localization of U2AF. Both U2AF(65) and U2AF(35) are shown to shuttle continuously between the nucleus and the cytoplasm by a mechanism that involves carrier receptors and is independent from binding to mRNA. The RS domain on either U2AF(65) or U2AF(35) acts as a nuclear localization signal and is sufficient to target a heterologous protein to the nuclear speckles. Furthermore, the results suggest that the presence of an RS domain in either U2AF subunit is sufficient to trigger the nucleocytoplasmic import of the heterodimeric complex. Shuttling of U2AF between nucleus and cytoplasm possibly represents a means to control the availability of this factor to initiate spliceosome assembly and therefore contribute to regulate splicing.
3T3 Cells↗
Nuclear inclusions in oculopharyngeal muscular dystrophy consist of poly(A) binding protein 2 aggregates which sequester poly(A) RNA.
Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of the disease is caused by short (GCG)(8-13) expansions in the PABP2 gene. This gene encodes the poly(A) binding protein 2 (PABP2), an abundant nuclear protein that binds with high affinity to nascent poly(A) tails, stimulating their extension and controlling their length. In this work we report that PABP2 is detected in filamentous nuclear inclusions, which are the pathological hallmark of OPMD. Using both immunoelectron microscopy and fluorescence confocal microscopy, the OPMD-specific nuclear inclusions appeared decorated by anti-PABP2 antibodies. In addition, the inclusions were labeled with antibodies directed against ubiquitin and the subunits of the proteasome and contained a form of PABP2 that was more resistant to salt extraction than the protein dispersed in the nucleoplasm. This suggests that the polyalanine expansions in PABP2 induce a misfolding and aggregation of the protein into insoluble inclusions, similarly to events in neurodegenerative diseases caused by CAG/polyglutamine expansions. No significant differences were observed in the steady-state poly(A) tail length in OPMD and normal myoblasts. However, the nuclear inclusions were shown to sequester poly(A) RNA. This raises the possibility that in OPMD the polyalanine expansions in the PABP2 protein may interfere with the cellular traffic of poly(A) RNA.
Cell Nucleus↗
Molecular mechanisms involved in cisplatin cytotoxicity.
cis-diamminedichloroplatinum(II) or cisplatin is a DNA-damaging agent that is widely used in cancer chemotherapy. Cisplatin cross-links to DNA, forming intra- and interstrand adducts, which bend and unwind the duplex and attract high-mobility-group domain and other proteins. Presumably due to a shielding effect caused by these proteins, the cisplatin-modified DNA is poorly repaired. The resulting DNA damage triggers cell-cycle arrest and apoptosis. Although it is still debatable whether the clinical success of cisplatin relies primarily on its ability to trigger apoptosis, at least two distinct pathways have been proposed to contribute to cisplatin-induced apoptosis in vitro. One involves the tumour-suppressor protein p53, the other is mediated by the p53-related protein p73. Coupling cisplatin damage to apoptosis requires mismatch repair activity, and recent observations further suggest involvement of the homologous recombinatorial repair system. At present it is generally accepted that abortive attempts to repair the DNA lesions play a key role in the cytotoxicity of the drug, and loss of the mismatch repair activity is known to cause cisplatin resistance, a major problem in antineoplastic therapy. Clearly, a better understanding of the signalling networks involved in cisplatin toxicity should provide a rational basis for the development of new therapeutic strategies.
Animals↗
Vesicular stomatitis virus matrix protein inhibits host cell gene expression by targeting the nucleoporin Nup98.
Vesicular stomatitis virus matrix protein (VSV M) has been shown to inhibit both transcription and nucleocytoplasmic transport. We have isolated a mutant form of M, termed M(D), lacking both inhibitory activities. HeLa cells expressing M, but not M(D), accumulate polyadenylated RNAs within the nucleus. Concomitantly, a fraction of M, but not of the M(D) mutant, localizes at the nuclear rim. Additionally, the nucleoporin Nup98 specifically interacts with M but not with M(D). In Nup98(-/-) cells, both the levels of M at the nuclear envelope and its inhibitory effects on host cell-directed expression of reporter genes were significantly reduced. Together, our data demonstrate that VSV M inhibits host cell gene expression by targeting a nucleoporin and primarily blocking nuclear export.
Active Transport, Cell Nucleus↗
Deciphering the cellular pathway for transport of poly(A)-binding protein II.
Poly(A)-binding protein II (PABP2) is an abundant nuclear protein that binds with high affinity to nascent poly(A) tails, stimulating their extension and controlling their length. In the cytoplasm, a distinct protein (PABP1) binds to poly(A) tails and participates in mRNA translation and stability. How cytoplasmic PABP1 substitutes for nuclear PABP2 is still unknown. Here we report that PABP2 shuttles back and forth between nucleus and cytoplasm by a carrier-mediated mechanism. A potential novel type of nuclear localization signal exists at the C-terminus of the protein, a domain that is highly enriched in methylated arginines. PABP2 binds directly to transportin in a RanGTP-sensitive manner, suggesting an involvement of this transport receptor in mediating import of the protein into the nucleus. Although PABP2 is small enough to diffuse passively through the nuclear pores, protein fusion experiments reveal the existence of a facilitated export pathway. Accordingly, no transport of PABP2 to the cytoplasm occurs at 4 degrees C. In contrast, export of PABP2 continues in the absence of transcription, indicating that transport to the cytoplasm is independent of mRNA traffic. Thus, rather than leaving the nucleus as a passive passenger of mRNAs, the data suggest that PABP2 interacts with the nuclear export machinery and may therefore contribute to mRNA transport.
3T3 Cells↗
The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates.
Messenger RNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins implicated in this process include TAP/Mex67p and RAE1/Gle2p and are distinct from the nuclear transport receptors of the beta-related, Ran-binding protein family. Mex67p is essential for mRNA export in yeast. Its vertebrate homolog TAP has been implicated in the export of cellular mRNAs and of simian type D viral RNAs bearing the constitutive transport element (CTE). Here we show that TAP is predominantly localized in the nucleoplasm and at both the nucleoplasmic and cytoplasmic faces of the nuclear pore complex (NPC). TAP interacts with multiple components of the NPC including the nucleoporins CAN, Nup98, Nup153, p62, and with three major NPC subcomplexes. The nucleoporin-binding domain of TAP comprises residues 508-619. In HeLa cells, this domain is necessary and sufficient to target GFP-TAP fusions to the nuclear rim. Moreover, the isolated domain strongly competes multiple export pathways in vivo, probably by blocking binding sites on the NPC that are shared with other transport receptors. Microinjection experiments implicate this domain in the export of specific CTE-containing RNAs. Finally, we show that TAP interacts with transportin and with two proteins implicated in the export of cellular mRNAs: RAE1/hGle2 and E1B-AP5. The interaction of TAP with nucleoporins, its direct binding to the CTE RNA, and its association with two mRNP binding proteins suggest that TAP is an RNA export mediator that may bridge the interaction between specific RNP export substrates and the NPC.
Amino Acid Sequence↗
To be or not to be in the nucleolus.
Compartmentalization has long been known to have a key role in regulation of cellular processes. By keeping enzymes and regulatory complexes in compartments where the delivery of substrate or exit of product is controlled, competing reactions can occur simultaneously in different parts of the cell. Moreover, spatial confinement facilitates the working of molecules participating in reaction chains and is crucial for coupling unfavourable with energetically favourable chemical reactions. Although in many cases intracellular compartmentalization relies on boundaries imposed by membranes, several non-membrane-bounded compartments exist in eukaryotic cells. One of these, the nucleolus, has recently attracted much attention. The emerging view is that molecular confinement in the nucleolus actively contributes to the control of cellular survival and proliferation.
Animals↗
TAP (NXF1) belongs to a multigene family of putative RNA export factors with a conserved modular architecture.
Vertebrate TAP (also called NXF1) and its yeast orthologue, Mex67p, have been implicated in the export of mRNAs from the nucleus. The TAP protein includes a noncanonical RNP-type RNA binding domain, four leucine-rich repeats, an NTF2-like domain that allows heterodimerization with p15 (also called NXT1), and a ubiquitin-associated domain that mediates the interaction with nucleoporins. Here we show that TAP belongs to an evolutionarily conserved family of proteins that has more than one member in higher eukaryotes. Not only the overall domain organization but also residues important for p15 and nucleoporin interaction are conserved in most family members. We characterize two of four human TAP homologues and show that one of them, NXF2, binds RNA, localizes to the nuclear envelope, and exhibits RNA export activity. NXF3, which does not bind RNA or localize to the nuclear rim, has no RNA export activity. Database searches revealed that although only one p15 (nxt) gene is present in the Drosophila melanogaster and Caenorhabditis elegans genomes, there is at least one additional p15 homologue (p15-2 [also called NXT2]) encoded by the human genome. Both human p15 homologues bind TAP, NXF2, and NXF3. Together, our results indicate that the TAP-p15 mRNA export pathway has diversified in higher eukaryotes compared to yeast, perhaps reflecting a greater substrate complexity.
Active Transport, Cell Nucleus↗
Localization of poly(A)-binding protein 2 (PABP2) in nuclear speckles is independent of import into the nucleus and requires binding to poly(A) RNA.
The nuclei of mammalian cells contain domains, termed nuclear speckles, which are enriched in splicing factors and poly(A) RNA. Although nuclear speckles are thought to represent reservoirs from which splicing factors are recruited to sites of transcription and splicing, the presence of poly(A) RNA in these structures remains enigmatic. An additional component of the speckles is poly(A) binding protein 2 (PABP2), a protein that binds with high affinity to nascent poly(A) tails, stimulating their extension and controlling their length. In this work we investigated whether PABP2 contributes to the targeting of poly(A) RNA to the speckles. The results show that localization of PABP2 in speckles is independent of import of the protein into the nucleus. Inhibition of transcription or poly(A) synthesis at the end of mitosis does not affect nuclear import of PABP2 but prevents its localization to speckles. Furthermore, PABP2 mutants with decreased ability to bind to poly(A) fail to localize to speckles. Taken together the results show that PABP2 localizes to the nuclear speckles as a consequence of its binding to poly(A) RNA, contrasting to splicing factors which assemble into speckles in the absence of newly synthesized transcripts.
Biological Transport↗
The spinal muscular atrophy disease gene product, SMN: A link between snRNP biogenesis and the Cajal (coiled) body.
The spliceosomal snRNAs U1, U2, U4, and U5 are synthesized in the nucleus, exported to the cytoplasm to assemble with Sm proteins, and reimported to the nucleus as ribonucleoprotein particles. Recently, two novel proteins involved in biogenesis of small nuclear ribonucleoproteins (snRNPs) were identified, the Spinal muscular atrophy disease gene product (SMN) and its associated protein SIP1. It was previously reported that in HeLa cells, SMN and SIP1 form discrete foci located next to Cajal (coiled) bodies, the so-called "gemini of coiled bodies" or "gems." An intriguing feature of gems is that they do not appear to contain snRNPs. Here we show that gems are present in a variable but small proportion of rapidly proliferating cells in culture. In the vast majority of cultured cells and in all primary neurons analyzed, SMN and SIP1 colocalize precisely with snRNPs in the Cajal body. The presence of SMN and SIP1 in Cajal bodies is confirmed by immunoelectron microscopy and by microinjection of antibodies that interfere with the integrity of the structure. The association of SMN with snRNPs and coilin persists during cell division, but at the end of mitosis there is a lag period between assembly of new Cajal bodies in the nucleus and detection of SMN in these structures, suggesting that SMN is targeted to preformed Cajal bodies. Finally, treatment of cells with leptomycin B (a drug that blocks export of U snRNAs to the cytoplasm and consequently import of new snRNPs into the nucleus) is shown to deplete snRNPs (but not SMN or SIP1) from the Cajal body. This suggests that snRNPs flow through the Cajal body during their biogenesis pathway.
Animals↗
Dbp5, a DEAD-box protein required for mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with CAN/Nup159p.
Dbp5 is a DEAD-box protein essential for mRNA export from the nucleus in yeast. Here we report the isolation of a cDNA encoding human Dbp5 (hDbp5) which is 46% identical to yDbp5p. Like its yeast homologue, hDbp5 is localized within the cytoplasm and at the nuclear rim. By immunoelectron microscopy, the nuclear envelope-bound fraction of Dbp5 has been localized to the cytoplasmic fibrils of the nuclear pore complex (NPC). Consistent with this localization, we show that both the human and yeast proteins directly interact with an N-terminal region of the nucleoporins CAN/Nup159p. In a conditional yeast strain in which Nup159p is degraded when shifted to the nonpermissive temperature, yDbp5p dissociates from the NPC and localizes to the cytoplasm. Thus, Dbp5 is recruited to the NPC via a conserved interaction with CAN/Nup159p. To investigate its function, we generated defective hDbp5 mutants and analysed their effects in RNA export by microinjection in Xenopus oocytes. A mutant protein containing a Glu-->Gln change in the conserved DEAD-box inhibited the nuclear exit of mRNAs. Together, our data indicate that Dbp5 is a conserved RNA-dependent ATPase which is recruited to the cytoplasmic fibrils of the NPC where it participates in the export of mRNAs out of the nucleus.
Adenosine Triphosphatases↗