mRNP Complexes Research Articles

A new view of mRNA export: separating the wheat from the chaff.

Current models for the export of messenger RNA share the notion that the highly abundant class of nuclear RNA-binding proteins--the hnRNP proteins--have a key role in exporting RNA. But recent studies have led to a new understanding of several non-hnRNP proteins, including SR proteins and the conserved mRNA export factor ALY, which are recruited to the mRNA during pre-mRNA splicing. These studies, together with older work on hnRNP particles and assembly of the spliceosome, lead us to a new view of mRNA export. In our model, the non-hnRNP factors form a splicing-dependent mRNP complex that specifically targets mature mRNA for export, while hnRNP proteins retain introns in the nucleus. A machinery that is conserved between yeast and higher eukaryotes functions to export the mRNA.

Isolation of proteins comprising native gene-specific messenger ribonucleoprotein particles using paramagnetic beads

A method for the rapid isolation of proteins comprising native messenger ribonucleoprotein particles (mRNPs), that utilises streptavidin-coated paramagnetic beads, coupled with biotinylated oligodeoxyribonucleotides, to pull down general and transcript-specific mRNP complexes from crude cell-free extracts, is described. Two biotinylated oligo probes were tested for their efficacy in isolating mRNPs from tobacco pollen extracts; a 25-mer oligo(dT) probe for the isolation of total mRNPs, and a transcript-specific oligo complementary to the pollen-specific ntp303 mRNA sequence. Following annealing to the target mRNAs, the oligo probes, with associated ribonucleoprotein particles, were captured by streptavidin-coated paramagnetic particles and pelleted in a magnetic field. After several washing steps, the bound RNPs were released and analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot immunodetection. This one-step mRNP enrichment method could be used either to enhance the detection of low-abundance mRNA-binding proteins in subsequent northwestern and RNA UV cross-linking experiments or the direct identification and biochemical characterisation of RNA-binding proteins.

A highly conserved protein family interacting with the fragile X mental retardation protein (FMRP) and displaying selective interactions with FMRP-related proteins FXR1P and FXR2P

The absence of the fragile X mental retardation protein (FMRP), encoded by the FMR1 gene, is responsible for pathologic manifestations in the Fragile X Syndrome, the most frequent cause of inherited mental retardation. FMRP is an RNA-binding protein associated with polysomes as part of a messenger ribonucleoprotein (mRNP) complex. Although its function is poorly understood, various observations suggest a role in local protein translation at neuronal dendrites and in dendritic spine maturation. We present here the identification of CYFIP1/2 (Cytoplasmic FMRP Interacting Proteins) as FMRP interactors. CYFIP1/2 share 88% amino acid sequence identity and represent the two members in humans of a highly conserved protein family. Remarkably, whereas CYFIP2 also interacts with the FMRP-related proteins FXR1P/2P, CYFIP1 interacts exclusively with FMRP. FMRP--CYFIP interaction involves the domain of FMRP also mediating homo- and heteromerization, thus suggesting a competition between interaction among the FXR proteins and interaction with CYFIP. CYFIP1/2 are proteins of unknown function, but CYFIP1 has recently been shown to interact with the small GTPase Rac1, which is implicated in development and maintenance of neuronal structures. Consistent with FMRP and Rac1 localization in dendritic fine structures, CYFIP1/2 are present in synaptosomal extracts.

The yeast hnRNP-Like proteins Yra1p and Yra2p participate in mRNA export through interaction with Mex67p.

Yra1p is an essential nuclear protein which belongs to the evolutionarily conserved REF (RNA and export factor binding proteins) family of hnRNP-like proteins. Yra1p contributes to mRNA export in vivo and directly interacts with RNA and the shuttling mRNP export receptor Mex67p in vitro. Here we describe a second nonessential Saccharomyces cerevisiae family member, called Yra2p, which is able to complement a YRA1 deletion when overexpressed. Like other REF proteins, Yra1p and Yra2p consist of two highly conserved N- and C-terminal boxes and a central RNP-like RNA-binding domain (RBD). These conserved regions are separated by two more variable regions, N-vr and C-vr. Surprisingly, the deletion of a single conserved box or the deletion of the RBD in Yra1p does not affect viability. Consistently, neither the conserved N and C boxes nor the RBD is required for Mex67p and RNA binding in vitro. Instead, the N-vr and C-vr regions both interact with Mex67p and RNA. We further show that Yra1 deletion mutants which poorly interact with Mex67p in vitro affect the association of Mex67p with mRNP complexes in vivo and are paralleled by poly(A)(+) RNA export defects. These observations support the idea that Yra1p promotes mRNA export by facilitating the recruitment of Mex67p to the mRNP.

Ribonucleoprotein infrastructure regulating the flow of genetic information between the genome and the proteome.

Following transcription and splicing, each mRNA of a mammalian cell passes into the cytoplasm where its fate is in the hands of a complex network of ribonucleoproteins (mRNPs). The success or failure of a gene to be expressed depends on the performance of this mRNP infrastructure. The entry, gating, processing, and transit of each mRNA through an mRNP network helps determine the composition of a cell's proteome. The machinery that regulates storage, turnover, and translational activation of mRNAs is not well understood, in part, because of the heterogeneous nature of mRNPs. Recently, subsets of cellular mRNAs clustered as members of mRNP complexes have been identified by using antibodies reactive with RNA-binding proteins, including ELAV/Hu, eIF-4E, and poly(A)-binding proteins. Cytoplasmic ELAV/Hu proteins are involved in the stability and translation of early response gene (ERG) transcripts and are expressed predominately in neurons. mRNAs recovered from ELAV/Hu mRNP complexes were found to have similar sequence elements, suggesting a common structural linkage among them. This approach opens the possibility of identifying transcripts physically clustered in vivo that may have similar fates or functions. Moreover, the proteins encoded by physically organized mRNAs may participate in the same biological process or structural outcome, not unlike operons and their polycistronic mRNAs do in prokaryotic organisms. Our goal is to understand the organization and flow of genetic information on an integrative systems level by analyzing the collective properties of proteins and mRNAs associated with mRNPs in vivo.

The brefeldin A resistance protein Bfr1p is a component of polyribosome-associated mRNP complexes in yeast.

The yeast gene BFR1 was originally isolated from a genetic screen for high-copy suppressors of brefeldin A-induced lethality in Saccharomyces cerevisiae. While this result suggested a possible role for the encoded protein, Bfr1p, in the secretory pathway, subsequent data have not fully supported this conclusion. Alternatively, Bfr1p has also been found by yeast two-hybrid analysis to interact with Bbp1p, a component of the spindle pole body. Finally, we have reported that Bfr1p associates with cytoplasmic mRNP complexes containing Scp160p, raising the possibility that Bfr1p may function in mRNA metabolism. Here, we have explored this possibility further. We report that Bfr1p associates with yeast polyribosomes and mRNP complexes even in the absence of Scp160p, and that its interaction with Scp160p-containing mRNP complexes is RNA-dependent. Furthermore, we have determined by fluorescence microscopy and subcellular fractionation that Bfr1p and Scp160p demonstrate similar cytoplasmic localization with enrichment around the nuclear envelope/endoplasmic reticulum. Finally, we report that loss of Bfr1p disrupts the interaction of Scp160p with polyribosomes, thereby demonstrating that the relationship between these two proteins is functional as well as physical. Considered together, these data raise the intriguing possibility that Bfr1p may provide a link between mRNA metabolism, the chromosomal segregation machinery and perhaps secretion in yeast.

Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays.

Genomic array technologies provide a means for profiling global changes in gene expression under a variety of conditions. However, it has been difficult to assess whether transcriptional or posttranscriptional regulation is responsible for these changes. Additionally, fluctuations in gene expression in a single cell type within a complex tissue like a tumor may be masked by overlapping profiles of all cell types in the population. In this paper, we describe the use of cDNA arrays to identify subsets of mRNAs contained in endogenous messenger ribonucleoprotein complexes (mRNPs) that are cell type specific. We identified mRNA subsets from P19 embryonal carcinoma stem cells by using mRNA-binding proteins HuB, eIF-4E, and PABP that are known to play a role in translation. The mRNA profiles associated with each of these mRNPs were unique and represented gene clusters that differed from total cellular RNA. Additionally, the composition of mRNAs detected in HuB-mRNP complexes changed dramatically after induction of neuronal differentiation with retinoic acid. We suggest that the association of structurally related mRNAs into mRNP complexes is dynamic and may help regulate posttranscriptional events such as mRNA turnover and translation. Recovering proteins specifically associated with mRNP complexes to identify and profile endogenously clustered mRNAs should provide insight into structural and functional relationships among gene transcripts and/or their protein products. We have termed this approach to functional genomics ribonomics and suggest that it will provide a useful paradigm for organizing genomic information in a biologically relevant manner.

Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules.

Mammalian stress granules (SGs) harbor untranslated mRNAs that accumulate in cells exposed to environmental stress. Drugs that stabilize polysomes (emetine) inhibit the assembly of SGs, whereas drugs that destabilize polysomes (puromycin) promote the assembly of SGs. Moreover, emetine dissolves preformed SGs as it promotes the assembly of polysomes, suggesting that these mRNP species (i.e., SGs and polysomes) exist in equilibrium. We used green flourescent protein-tagged SG-associated RNA-binding proteins (specifically, TIA-1 and poly[A] binding protein [PABP-I]) to monitor SG assembly, disassembly, and turnover in live cells. Fluorescence recovery after photobleaching shows that both TIA-1 and PABP-I rapidly and continuously shuttle in and out of SGs, indicating that the assembly of SGs is a highly dynamic process. This unexpected result leads us to propose that mammalian SGs are sites at which untranslated mRNAs are sorted and processed for either reinitiation, degradation, or packaging into stable nonpolysomal mRNP complexes. A truncation mutant of TIA-1 (TIA-1DeltaRRM), which acts as a transdominant inhibitor of SG assembly, promotes the expression of cotransfected reporter genes in COS transfectants, suggesting that this process of mRNA triage might, directly or indirectly, influence protein expression.

Identification of Mouse YB1/p50 as a Component of the FMRP-Associated mRNP Particle

Fragile X mental retardation is caused by the absence of FMRP, an RNA-binding protein found in a large mRNP complex. Although there is evidence that FMRP exists as a homo-multimer, additional proteins have been identified that associate with FMRP in the mRNP. The autosomal paralogs of FMRP, FXR1P, and FXR2P, associate with FMRP, as do nucleolin and NUFIP1, all RNA binding proteins. Using cell lines that were stably transfected with Flag-Fmr1, we identified an additional protein that coimmunoprecipitates with FMRP. The approximately 50 kDa protein was identified by mass spectrometry as mouse Y box-binding protein 1 (YB1), which is 97% identical to the core mRNP protein p50, an RNA-binding protein. An anti-p50 antiserum recognized the 50 kDa protein, confirming the identification. The association of the FMRP-mRNP with a Y box protein, the latter commonly found in mRNPs, further suggests the involvement of FMRP in translation modulation.

Human immunodeficiency virus type 1 Vif protein is an integral component of an mRNP complex of viral RNA and could be involved in the viral RNA folding and packaging process.

Virion infectivity factor (Vif) is a protein encoded by human immunodeficiency virus types 1 and 2 (HIV-1 and -2) and simian immunodeficiency virus, plus other lentiviruses, and is essential for viral replication either in vivo or in culture for nonpermissive cells such as peripheral blood lymphoid cells, macrophages, and H9 T cells. Defects in the vif gene affect virion morphology and reverse transcription but not the expression of viral components. It has been shown that Vif colocalizes with Gag in cells and Vif binds to the NCp7 domain of Gag in vitro. However, it seems that Vif is not specifically packaged into virions. The molecular mechanism(s) for Vif remains unknown. In this report, we demonstrate that HIV-1 Vif is an RNA-binding protein and specifically binds to HIV-1 genomic RNA in vitro. Further, Vif binds to HIV-1 RNA in the cytoplasm of virus-producing cells to form a 40S mRNP complex. Coimmunoprecipitation and in vivo UV cross-linking assays indicated that Vif directly interact with HIV-1 RNA in the virus-producing cells. Vif-RNA binding could be displaced by Gag-RNA binding, suggesting that Vif protein in the mRNP complex may mediate viral RNA interaction with HIV-1 Gag precursors. Furthermore, we have demonstrated that these Vif mutants that lose the RNA binding activity in vitro do not support vif-deficient HIV-1 replication in H9 T cells, suggesting that the RNA binding capacity of Vif is important for its function. Further studies regarding Vif-RNA interaction in virus-producing cells will be important for studying the function of Vif in the HIV-1 life cycle.

The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans.

In metazoans, most pre-messenger RNAs contain introns that are removed by splicing. The spliced mRNAs are then exported to the cytoplasm. Recent studies showed that splicing promotes efficient mRNA export, but the mechanism for coupling these two processes is not known. Here we show that Aly, the metazoan homologue of the yeast mRNA export factor Yralp (ref. 2), is recruited to messenger ribonucleoprotein (mRNP) complexes generated by splicing. In contrast, Aly does not associate with mRNPs assembled on identical mRNAs that already have no introns or with heterogenous nuclear RNP (hnRNP) complexes. Aly is recruited during spliceosome assembly, and then becomes tightly associated with the spliced mRNP. Aly shuttles between the nucleus and cytoplasm, and excess recombinant Aly increases both the rate and efficiency of mRNA export in vivo. Consistent with its splicing-dependent recruitment, Aly co-localizes with splicing factors in the nucleus. We conclude that splicing is required for efficient mRNA export as a result of coupling between the splicing and the mRNA export machineries.

RNA-dependent cytoplasmic anchoring of a transcription factor subunit during Xenopus development.

The CCAAT box transcription factor (CBTF) is a multimeric transcription factor that activates expression of the haematopoietic regulatory factor, GATA-2. The 122 kDa subunit of this complex, CBTF(122), is cytoplasmic in fertilized Xenopus eggs and subsequently translocates to the nucleus prior to activation of zygotic GATA-2 transcription at gastrulation. Here we present data suggesting both a role for CBTF(122) prior to its nuclear translocation and the mechanism that retains it in the cytoplasm before the midblastula transition (MBT). CBTF(122) and its variant CBTF(98) are associated with translationally quiescent mRNP complexes. We show that CBTF(122) RNA binding activity is both necessary and sufficient for its cytoplasmic retention during early development. The introduction of an additional nuclear localization signal to CBTF(122) is insufficient to overcome this retention, suggesting that RNA binding acts as a cytoplasmic anchor for CBTF(122). Destruction of endogenous RNA by microinjection of RNase promotes premature nuclear translocation of CBTF(122). Thus, the nuclear translocation of CBTF(122) at the MBT is likely to be coupled to the degradation of maternal mRNA that occurs at that stage.

Annexin II is associated with mRNAs which may constitute a distinct subpopulation

Protein-mRNA interactions affect mRNA transport, anchorage, stability and translatability in the cytoplasm. During the purification of three subpopulations of polysomes, it was observed that a 36-kDa protein, identified as annexin II, is associated with only one specific population of polysomes, namely cytoskeleton-associated polysomes. This association appears to be calcium-dependent since it was sensitive to EGTA and could be reconstituted in vitro. UV irradiation resulted in partial, EGTA-resistant cross-linking of annexin II to the polysomes. Binding of 32P-labelled total RNA to proteins isolated from the cytoskeleton-bound polysomes on a NorthWestern blot resulted in a radioactive band having the same mobility as annexin II and, most importantly, purified native annexin II immobilized on nitrocellulose specifically binds mRNA. The mRNA population isolated from cytoskeleton-bound polysomes binds to annexin II with the highest affinity as compared with those isolated from free or membrane-bound polysomes. Interestingly, the annexin II complex, isolated from porcine small intestinal microvilli was a far better substrate for mRNA binding than the complex derived from transformed Krebs II ascites cells. When cytoskeleton-associated polysomes were split into 60 S and 40 S ribosomal subunits, and a peak containing mRNA complexes, annexin II fractionated with the mRNAs. Finally, using affinity purification of mRNA on poly(A)+-coupled magnetic beads, annexin II was only detected in association with messenger ribonucleoproteins (mRNPs) present in the cytoskeletal fraction (non-polysomal mRNPs). These results, derived from both in vitro experiments and cell fractionation, suggest that annexin II binds directly to the RNA moiety of mRNP complexes containing a specific population of mRNAs.

Annexin II is associated with mRNAs which may constitute a distinct subpopulation

Protein-mRNA interactions affect mRNA transport, anchorage, stability and translatability in the cytoplasm. During the purification of three subpopulations of polysomes, it was observed that a 36-kDa protein, identified as annexin II, is associated with only one specific population of polysomes, namely cytoskeleton-associated polysomes. This association appears to be calcium-dependent since it was sensitive to EGTA and could be reconstituted in vitro. UV irradiation resulted in partial, EGTA-resistant cross-linking of annexin II to the polysomes. Binding of (32)P-labelled total RNA to proteins isolated from the cytoskeleton-bound polysomes on a NorthWestern blot resulted in a radioactive band having the same mobility as annexin II and, most importantly, purified native annexin II immobilized on nitrocellulose specifically binds mRNA. The mRNA population isolated from cytoskeleton-bound polysomes binds to annexin II with the highest affinity as compared with those isolated from free or membrane-bound polysomes. Interestingly, the annexin II complex, isolated from porcine small intestinal microvilli was a far better substrate for mRNA binding than the complex derived from transformed Krebs II ascites cells. When cytoskeleton-associated polysomes were split into 60 S and 40 S ribosomal subunits, and a peak containing mRNA complexes, annexin II fractionated with the mRNAs. Finally, using affinity purification of mRNA on poly(A)(+)-coupled magnetic beads, annexin II was only detected in association with messenger ribonucleoproteins (mRNPs) present in the cytoskeletal fraction (non-polysomal mRNPs). These results, derived from both in vitro experiments and cell fractionation, suggest that annexin II binds directly to the RNA moiety of mRNP complexes containing a specific population of mRNAs.

Specific types of prosomes distribute differentially between intermediate and actin filaments in epithelial, fibroblastic and muscle cells

First observed as components of non-translated mRNP complexes, prosomes harbour RNase and several proteinase activities; they are also the central constituent of the "Multicatalytic Proteinase (MCP) complexes" or "26S-proteasomes". In two recent publications (Arcangeletti et al., 1997b; De Conto et al., 1997) we have shown, by applying a new fixation technique, that these particles distribute differentially between the cytoskeletal networks of intermediate filament (IF) and actin types; previously they had been observed exclusively on the intermediate filaments. Here we further investigate the distribution of prosomes of several types, distinct by their subunit composition, between the IF of vimentin type and the actin network, as well as in the 3D space of the cell. It is shown that subtypes of prosomes occupy specific networks of the cytoskeleton, and that this pattern is specific for a given cell type. Confocal microscopy shows that prosome cytodistribution is not homogeneous in the 3D space: in the perinuclear area they colocalize most strongly with the IF, and more peripherally with the microfilament/stress fiber system; connections may exist between the two networks. Furthermore, new data indicate that the prosome-actin interaction may participate in the molecular structure of the stress fibers.

Nucleolin and YB-1 are required for JNK-mediated interleukin-2 mRNA stabilization during T-cell activation

Regulated mRNA turnover is a highly important process, but its mechanism is poorly understood. Using interleukin-2 (IL-2) mRNA as a model, we described a role for the JNK-signaling pathway in stabilization of IL-2 mRNA during T-cell activation, acting via a JNK response element (JRE) in the 5' untranslated region (UTR). We have now identified two major RNA-binding proteins, nucleolin and YB-1, that specifically bind to the JRE. Binding of both proteins is required for IL-2 mRNA stabilization induced by T-cell activation signals and for JNK-induced stabilization in a cell-free system that duplicates essential features of regulated mRNA decay. Nucleolin and YB-1 are required for formation of an IL-2 mRNP complex that responds to specific mRNA stabilizing signals.

Scp160p, a multiple KH-domain protein, is a component of mRNP complexes in yeast.

Scp160p is a 160 kDa protein in the yeast Saccharomyces cerevisiae that contains 14 repeats of the hnRNP K-homology (KH) domain, and demonstrates significant sequence homology to a family of proteins collectively known as vigilins. As a first step towards defining the function of Scp160p, we have characterized the subcellular distribution and in vivo interactions of this protein. Using sucrose gradient fractionation studies we have demonstrated that Scp160p in cytoplasmic lysates is predominantly associated with polyribosomes. Furthermore, we have found that Scp160p is released from polyribosomes by EDTA in the form of a large complex of> or =1300 kDa that is sensitive both to RNase and NaCl. Using affinity-chromatography to isolate these complexes, we have identified two protein components other than Scp160p: poly(A) binding protein, Pab1p, and Bfr1p. The presence of Pab1p confirms these complexes to be mRNPs. The presence of Bfr1p is intriguing because the null phenotype for this gene is essentially the same as that reported for scp160 -null cells: increased cell size and aberrant DNA content. These results demonstrate that Scp160p associates with polyribosome-bound mRNP complexes in vivo, implicating a role for this protein in one or more levels of mRNA metabolism in yeast.

Isolation of an FMRP-associated messenger ribonucleoprotein particle and identification of nucleolin and the fragile X-related proteins as components of the complex.

The loss of FMR1 expression due to trinucleotide repeat expansion leads to fragile X syndrome, a cause of mental retardation. The encoded protein, FMRP, is a member of a gene family that also contains the fragile X-related proteins, FXR1P and FXR2P. FMRP has been shown to be a nucleocytoplasmic shuttling protein that selectively binds a subset of mRNAs, forms messenger ribonucleoprotein (mRNP) complexes, and associates with translating ribosomes. Here we describe a cell culture system from which we can isolate epitope-tagged FMRP along with mRNA, including its own message, and at least six other proteins. We identify two of these proteins as FXR1P and FXR2P by using specific antisera and identify a third protein as nucleolin by using mass spectrometry. The presence of nucleolin is confirmed by both reactivity with a specific antiserum as well as reverse coimmunoprecipitation where antinucleolin antiserum immunoprecipitates endogenous FMRP from both cultured cells and mouse brain. The identification of nucleolin, a known component of other mRNPs, adds a new dimension to the analysis of FMRP function, and the approach described should also allow the identification of the remaining unknown proteins of this FMRP-associated mRNP as well as the other bound mRNAs.

Biology of the fragile X mental retardation protein, an RNA-binding protein

The fragile X syndrome, an X-linked disease, is the most frequent cause of inherited mental retardation. The syndrome results from the absence of expression of the FMR1 gene (fragile mental retardation 1) owing to the expansion of a CGG trinucleotide repeat located in the 5prime untranslated region of the gene and the subsequent methylation of its CpG island. The FMR1 gene product (FMRP) is a cytoplasmic protein that contains two KH domains and one RGG box, characteristics of RNA-binding proteins. FMRP is associated with mRNP complexes containing poly(A)+mRNA within actively translating polyribosomes and contains nuclear localization and export signals making it a putative transporter (chaperone) of mRNA from the nucleus to the cytoplasm. FMRP is the archetype of a novel family of cytoplasmic RNA-binding proteins that includes FXR1P and FXR2P. Both of these proteins are very similar in overall structure to FMRP and are also associated with cytoplasmic mRNPs. Members of the FMR family are widely expressed in mouse and human tissues, albeit at various levels, and seem to play a subtle choreography of expression. FMRP is most abundant in neurons and is absent in muscle. FXR1P is strongly expressed in muscle and low levels are detected in neurons. The complex expression patterns of the FMR1 gene family in different cells and tissues suggest that independent, however similar, functions for each of the three FMR-related proteins might be expected in the selection and metabolism of tissue-specific classes of mRNA. The molecular mechanisms altered in cells lacking FMRP still remain to be elucidated as well as the putative role(s) of FXR1P and FXR2P as compensatory molecules.Key words: RNA-binding proteins, polyribosomes, messenger ribonucleoprotein, messenger ribonucleoparticles, nucleocytoplasmic trafficking, mental retardation.

Why is Hu where? Shuttling of early-response-gene messenger RNA subsets.

Post-transcriptional regulation of gene expression involves multiple checkpoints at which decisions are made concerning the fate of each messenger RNA species. Primary transcripts assemble into multicomponent ribonucleoprotein complexes during pre-mRNA processing in the spliceosome, are transported out of the nucleus, and are either stored, degraded, or translated in the cytoplasm (1). Determining the outcome of this process for each individual mRNA species is complicated by the heterogeneity of the messenger ribonucleoprotein (mRNP) population, which contains various-size transcripts and diverse combinations of attached proteins. Although investigators have not yet devised methods to separate and characterize distinct cellular mRNPs, nature appears to distinguish each mRNP by using inherent signals in the RNAs and/or in the proteins in the mRNP complexes and tracking each mRNA to an appropriate functional outcome. For example, mRNAs may contain recognition sequences that can serve as “zip codes” for tracking or localization (2) whereas in other cases, proteins attached to the mRNPs contain localization signals that allow their movement out of, or into, the nucleus (reviewed in ref. 3). In a previous issue of the Proceedings , Fan and Steitz (4) define a novel nuclear shuttling sequence in the Hu RNA-binding protein HuR, which may transport a specific subset of cellular mRNAs containing AU-rich elements (ARE) from the nucleus to the cytoplasm (Fig. 1). They term this signal the “HNS” for HuR nuclear-cytoplasmic shuttling sequence because it contains both nuclear export and nuclear localization elements. This is particularly interesting because the ARE-containing mRNAs encode a functionally important subset of early-response gene (ERG) or immediate-early gene products, including protooncoproteins and cytokines. Figure 1 Nuclear-cytoplasmic shuttling of the ELAV/Hu protein HuR, which binds to a distinct subset of cellular messenger RNAs. ERG mRNAs encoding protooncoproteins and cytokines are bound to and stabilized by HuR, which contains an amino acid segment (HNS) with …