Nuclear RNA-binding Protein Research Articles

FY Is an RNA 3′ End-Processing Factor that Interacts with FCA to Control the Arabidopsis Floral Transition

The nuclear RNA binding protein, FCA, promotes Arabidopsis reproductive development. FCA contains a WW protein interaction domain that is essential for FCA function. We have identified FY as a protein partner for this domain. FY belongs to a highly conserved group of eukaryotic proteins represented in Saccharomyces cerevisiae by the RNA 3′ end-processing factor, Pfs2p. FY regulates RNA 3′ end processing in Arabidopsis as evidenced through its role in FCA regulation. FCA expression is autoregulated through the use of different polyadenylation sites within the FCA pre-mRNA, and the FCA/FY interaction is required for efficient selection of the promoter-proximal polyadenylation site. The FCA/FY interaction is also required for the downregulation of the floral repressor FLC. We propose that FCA controls 3′ end formation of specific transcripts and that in higher eukaryotes, proteins homologous to FY may have evolved as sites of association for regulators of RNA 3′ end processing.

In vivo analysis of nucleolar proteins modified by the yeast arginine methyltransferase Hmt1/Rmt1p.

In this report, we have investigated the impact of arginine methylation on the Gar1, Nop1, and Nsr1 nucleolar proteins in Saccharomyces cerevisiae. Although previous reports have established that protein arginine methylation is important for nucleocytoplasmic shuttling, they have focused on the examination of heterogeneous nuclear ribonucleoproteins (hnRNPs). We have extended this analysis to several nucleolar proteins that represent a distinct functional class of arginine-methylated proteins. We first developed an in vivo assay to identify proteins methylated by the Hmt1 arginine methyltransferase. This assay is based on the fact that the Hmt1 enzyme utilizes S-Adenosyl-L-methionine as the methyl donor for protein arginine methylation. Following SDS polyacrylamide electrophoresis, 11 distinct proteins were identified as substrates for the Hmt1 methyltransferase. Hmt1p overexpression did not increase the methylation level on these proteins, suggesting they are fully methylated under the conditions examined. Three of the radiolabeled proteins were confirmed to be Gar1p, Nop1p, and Nsr1p. To monitor the cellular localization of these proteins, functional GFP fusion proteins were generated and found to be localized to the nucleolus. This localization was independent of arginine methylation. Furthermore, all three proteins examined did not export to the cytoplasm. In contrast, arginine methylation is required for the export of the nuclear RNA-binding proteins Npl3p, Hrp1p, and Nab2p. The observation that three nucleolar proteins are modified by Hmt1p but are not exported from the nucleolus implies an alternate role for arginine methylation.

P54 nrb acts as a transcriptional coactivator for activation function 1 of the human androgen receptor

The androgen receptor (AR) has two transactivation functions that have been mapped to the N- and C-terminal domains and designated as activation function-1 (AF-1) and AF-2, respectively. While the molecular basis for AF-2 function has been well studied, little is known about AF-1 coregulators. Therefore, we attempted to identify AF-1-interacting proteins from HEK293 cells by biochemical purification followed by mass fingerprinting by matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI–TOF MS). Purified AF-1 region-interacting proteins were found to contain nuclear RNA-binding protein p54 nrb, polypyrimidine tract-binding protein-associated splicing factor (PSF), paraspeckle protein 1 (PSP1), and PSP2, which are assumed to be involved in pre-mRNA processing. p54 nrb interacted with AR via the A/B domain in a ligand-dependent manner. Reflecting the physical interaction between p54 nrb and the AR A/B domain, AR AF-1 function was potentiated by p54 nrb. Our results suggest that p54 nrb functions as a coactivator of AR that potentiates transcription, and presumably splicing as well.

A functional deadenylation assay identifies human CUG-BP as a deadenylation factor

CUG-BP is a human nuclear and cytoplasmic RNA-binding protein. A role in the control of alternative splicing has been reported, but to date no cytoplasmic function for this protein has been demonstrated. A close sequence homolog of CUG-BP is EDEN-BP that is required for the specific cytoplasmic poly(A) tail shortening of certain mRNAs after fertilization of Xenopus eggs. Here, we show that human CUG-BP and Xenopus EDEN-BP have very similar RNA-binding specificities. In addition, we use a deadenylation assay to show that CUG-BP is able to act as a deadenylation factor. In contrast, a mutant form of CUG-BP, though still able to bind to RNA with a specificity similar to that of wild-type CUG-BP, does not act as a deadenylation factor. It is suggested that the CUG expansion associated with Type 1 myotonic dystrophy can affect the function or the activity of CUG-BP, leading to a trans-dominant effect on normal RNA processing. The results presented here identify CUG-BP-dependent deadenylation as a potential cytoplasmic target for this trans-dominant effect.

Identification of cellular mRNA targets for RNA-binding protein Sam68.

Sam68 (Src-associated in mitosis, 68 kDa), a nuclear RNA-binding protein, has been postulated to play a role in cell-growth control as a modulator of signal transduction and activation of RNA metabolism. Although Sam68 was demonstrated to bind to the UAAA sequences in synthetic oligoribonucleotides and poly(U) homopolymers in vitro, the legitimate cellular mRNA target remained unclear. By using the differential display and cDNA-representational difference analysis techniques, followed by reverse transcription polymerase chain reaction of RNAs co-immunoprecipitated with Sam68 from a HeLa cell lysate, we identified 10 mRNA species that bind in vivo to Sam68 in an RNA-binding domain-dependent manner. Among them, the mRNA species for hnRNP A2/B1 and beta-actin were found to bind prominently in vivo as well as in vitro, suggesting the possible involvement of Sam68 in the post- transcriptional regulation of these genes. Mapping of the Sam68-binding sequence revealed that Sam68 associates with these mRNAs through different nucleotide motifs, UAAA for hnRNP A2/B1 mRNA and UUUUUU for beta-actin mRNA, and that both binding sequences must reside in a loop structure for recognition by Sam68. The results indicated that Sam68 recognizes both the UAAA motif and poly(U) sequences in vivo for binding to cellular target mRNAs.

Signal-dependent regulation of splicing via phosphorylation of Sam68.

Evolution of human organismal complexity from a relatively small number of genes--only approximately twice that of worm or fly--is explained mainly by mechanisms generating multiple proteins from a single gene, the most prevalent of which is alternative pre-messenger-RNA splicing. Appropriate spatial and temporal generation of splice variants demands that alternative splicing be subject to extensive regulation, similar to transcriptional control. Activation by extracellular cues of several cellular signalling pathways can indeed regulate alternative splicing. Here we address the link between signal transduction and splice regulation. We show that the nuclear RNA-binding protein Sam68 is a new extracellular signal-regulated kinase (ERK) target. It binds exonic splice-regulatory elements of an alternatively spliced exon that is physiologically regulated by the Ras signalling pathway, namely exon v5 of CD44. Forced expression of Sam68 enhanced ERK-mediated inclusion of the v5-exon sequence in mRNA. This enhancement was impaired by mutation of ERK-phosphorylation sites in Sam68, whereas ERK phosphorylation of Sam68 stimulated splicing of the v5 exon in vitro. Finally, Ras-pathway-induced alternative splicing of the endogenous CD44-v5 exon was abolished by suppression of Sam68 expression. Our data define Sam68 as a prototype regulator of alternative splicing whose function depends on protein modification in response to extracellular cues.

Ready-mix protein cookery

Making a protein from scratch takes time. Not only must you produce messenger RNA and (usually) process it to remove introns, but you also have to export it to the cytoplasm and have it translated by a willing ribosome. Thus, one tends to think of a change in gene expression as being a relatively leisurely way of responding to an external signal. This, however, is not always true, as Li et al. describe [1xModulation of an RNA-binding protein by abscisic-acid-activated protein kinase. Li, J. et al. Nature. 2002; 418: 793–797CrossRef | PubMed | Scopus (112)See all References][1].Like ready-to-bake muffin mix, evolution has come up with a masterful shortcut to take the hassle out of protein cookery. And it seems that the first step is to stop throwing your mix away. Li et al. took abscisic-acid-activated protein kinase (AAPK) and screened a bean expression library for binding partners. This fishing expedition caught the humbly-named AAPK-interacting protein 1 (AKIP1)-a protein that is homologous to a class of single-stranded RNA-binding proteins termed hnRNPs (heterogeneous nuclear RNA-binding proteins). GFP-tagged AKIP1 localizes throughout the nucleus but, surprisingly, redistributes to nuclear speckles upon treatment with abscisic acid (ABA). This response to ABA is likely to be mediated by AAPK as this kinase can phosphorylate AKIP1 in vitro, but only if it has been isolated from cells treated with ABA.ABA is produced by plants in response to water shortage, making it likely that downstream targets of ABA are involved in drought resistance. Consequently, Li et al. asked whether AKIP1 could be involved in binding to RNA encoding dehydrins – stress-regulated proteins that stabilize enzymes and membranes. Sure enough, dehydrin mRNA could be bound by phosphorylated AKIP1 but not by inactive AKIP1. The authors point out that environmental stress has long been known to alter RNA longevity in plants. This suggests that RNA for proteins such as the dehydrins is transcribed constitutively, but degraded continuously before translation. During water shortage, ABA production would stimulate the phosphorylation of AKIP1 by AAPK. Active AKIP1 would then bind to dehydrin mRNA, stabilizing it and allowing translation to take place.This work demonstrates once again that posttranscriptional events are a major means of regulating gene expression, as well as hinting at another tool to add to the molecular biologist's toolkit. A quick squirt of ABA, and minutes later your transgenic Christmas tree starts to glow in the dark…patent application form, anyone?

Modulation of an RNA-binding protein by abscisic-acid-activated protein kinase.

Protein kinases are involved in stress signalling in both plant and animal systems. The hormone abscisic acid mediates the responses of plants to stresses such as drought, salinity and cold. Abscisic-acid-activated protein kinase (AAPK -- found in guard cells, which control stomatal pores -- has been shown to regulate plasma membrane ion channels. Here we show that AAPK-interacting protein 1 (AKIP1), with sequence homology to heterogeneous nuclear RNA-binding protein A/B, is a substrate of AAPK. AAPK-dependent phosphorylation is required for the interaction of AKIP1 with messenger RNA that encodes dehydrin, a protein implicated in cell protection under stress conditions. AAPK and AKIP1 are present in the guard-cell nucleus, and in vivo treatment of such cells with abscisic acid enhances the partitioning of AKIP1 into subnuclear foci which are reminiscent of nuclear speckles. These results show that phosphorylation-regulated RNA target discrimination by heterogeneous nuclear RNA-binding proteins may be a general phenomenon in eukaryotes, and implicate a plant hormone in the regulation of protein dynamics during rapid subnuclear reorganization.

Novel Binding of HuR and Poly(C)-binding Protein to a Conserved UC-rich Motif within the 3′-Untranslated Region of the Androgen Receptor Messenger RNA

The androgen receptor (AR) mediates androgen action and plays a central role in the proliferation of specific cancer cells. We demonstrated recently that AR mRNA stability is a major determinant of AR gene expression in prostate and breast cancer cells and that androgens differentially regulate AR mRNA decay dependent on cell type (Yeap, B. B., Kreuger, R. G., Leedman, P. J. (1999) Endocrinology 140, 3282-3291). Here, we have identified a highly conserved UC-rich region in the 3-untranslated region of AR mRNA that contains a 5'-C(U)(n)C motif and a 3'-CCCUCCC poly(C)-binding protein motif. In transfection studies with LNCaP human prostate cancer cells, the AR UC-rich region reduced expression of a luciferase reporter gene. The AR UC-rich region was a target for cytoplasmic and nuclear RNA-binding proteins from human prostate and breast cancer cells as well as human testicular and breast cancer tissue. One of these proteins is HuR, a ubiquitously expressed member of the Elav/Hu family of RNA-binding proteins involved in the stabilization of several mRNAs. Poly(C)-binding protein-1 and -2 (CP1 and CP2), previously implicated in the control of mRNA turnover and translation, also bound avidly to the UC-rich region. Mutational analysis of the UC-rich region identified specific binding motifs for both HuR and the CPs. HuR and CP1 bound simultaneously to the UC-rich RNA and in a cooperative manner. Immunoprecipitation studies confirmed that each of these proteins associated with AR mRNA in prostate cancer cells. In summary, we have identified and characterized a novel complex of AR mRNA-binding proteins that target the highly conserved UC-rich region. The binding of HuR, CP1, and CP2 to AR mRNA suggests a role for each of these proteins in the post-transcriptional regulation of AR expression in cancer cells.

Pasilla, the Drosophila Homologue of the Human Nova-1 and Nova-2 Proteins, Is Required for Normal Secretion in the Salivary Gland

From a screen for genes expressed and required in the Drosophila salivary gland, we identified pasilla (ps), which encodes a set of proteins most similar to human Nova-1 and Nova-2. Nova-1 and Nova-2 are nuclear RNA-binding proteins normally expressed in the CNS where they directly regulate splicing. In patients suffering from paraneoplastic opsoclonus myoclonus ataxia (POMA), Nova-1 and Nova-2 proteins are present as auto-antigens. Consistent with a role in splicing, PS is localized to nuclear puncta. The salivary glands of ps mutants internalize normally and maintain epithelial polarity. However, the mutant salivary glands develop irregularities in overall morphology and have defects in apical secretion. The secretory defects in ps mutants provide a potential mechanism for the loss of motor function observed in POMA patients.

RNP localization and transport in yeast.

The localization of mRNAs is used by various types of polarized cells to locally translate specific proteins, which restricts their distribution to a particular sub-region of the cytoplasm. This mechanism of protein sorting is involved in major biological processes such as asymmetric cell division, oogenesis, cellular motility, and synapse formation. With the finding of localized mRNAs in the yeast Saccharomyces cerevisiae, it is now possible to benefit from the powerful yeast laboratory tools to explore the molecular basis of RNA localization. Because mRNA transport and localization in yeast share many features with RNA localization in higher eukaryotes, including the formation of a large ribonucleoprotein (RNP) localization complex, the requirement of a polarized cytoskeleton and molecular motors, and the role of nuclear RNA-binding proteins in cytoplasmic localization, the yeast can be used as a paradigm for unraveling the molecular aspects of this process. This review summarizes the current knowledge on RNP transport and localization in yeast.

HUA1, a Regulator of Stamen and Carpel Identities in Arabidopsis, Codes for a Nuclear RNA Binding Protein

HUA1, a Regulator of Stamen and Carpel Identities in Arabidopsis, Codes for a Nuclear RNA Binding Protein

HUA1, a Regulator of Stamen and Carpel Identities in Arabidopsis, Codes for a Nuclear RNA Binding Protein

HUA1, a Regulator of Stamen and Carpel Identities in Arabidopsis, Codes for a Nuclear RNA Binding Protein

HUA1, a regulator of stamen and carpel identities in Arabidopsis, codes for a nuclear RNA binding protein.

Stamen and carpel identities are specified by the combinatorial activities of several floral homeotic genes, APETALA3, PISTILLATA, AGAMOUS (AG), SEPALLATA1 (SEP1), SEPALLATA2 (SEP2), and SEPALLATA3 (SEP3), all of which code for MADS domain DNA binding proteins. AG and the SEP genes also control floral determinacy. HUA1 and HUA2 were identified previously as regulators of stamen and carpel identities and floral determinacy because the recessive hua1-1 or hua2-1 allele affected these processes in plants with a lower dosage of functional AG (either homozygous for the weak ag-4 allele or heterozygous for the strong ag-1 allele). HUA2 was cloned previously and shown to code for a novel protein. We isolated the HUA1 gene using a map-based approach and show that it encodes a protein with six CCCH-type zinc finger motifs that is also found in yeast, Caenorhabditis elegans, Drosophila melanogaster, and mammalian proteins. Several such genes from invertebrates and mammals are known to play key regulatory roles in development. Therefore, HUA1 are another example of non-MADS domain proteins involved in organ identity specification. We demonstrated that HUA1 binds ribohomopolymers, preferentially poly rU and poly rG, but not double-stranded DNA in vitro. This finding suggests that HUA1, like several mammalian CCCH zinc finger proteins, is an RNA binding protein. Therefore, HUA1 likely participates in a new regulatory mechanism governing flower development.

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.

Male sterility associated with overexpression of the noncoding hsromega gene in cyst cells of testis of Drosophila melanogaster.

Of the several noncoding transcripts produced by the hsromega gene of Drosophila melanogaster, the nucleus-limited >10-kb hsromega-n transcript colocalizes with heterogeneous nuclear RNA binding proteins (hnRNPs) to form fine nucleoplasmic omega speckles. Our earlier studies suggested that the noncoding hsromega-n transcripts dynamically regulate the distribution of hnRNPs in active (chromatin bound) and inactive (in omega speckles) compartments. Here we show that a P transposon insertion in this gene's promoter (at -130 bp) in the hsromega05421; enhancer-trap line had no effect on viability or phenotype of males or females, but the insertion-homozygous males were sterile. Testes of hsromega05421; homozygous flies contained nonmotile sperms while their seminal vesicles were empty. RNA:RNA in situ hybridization showed that the somatic cyst cells in testes of the mutant male flies contained significantly higher amounts of hsromega-n transcripts, and unlike the characteristic fine omega speckles in other cell types they displayed large clusters of omega speckles as typically seen after heat shock. Two of the hnRNPs, viz. HRB87F and Hrb57A, which are expressed in cyst cells, also formed large clusters in these cells in parallel with the hsromega-n transcripts. A complete excision of the P transposon insertion restored male fertility as well as the fine-speckled pattern of omega speckles in the cyst cells. The in situ distribution patterns of these two hnRNPs and several other RNA-binding proteins (Hrp40, Hrb57A, S5, Sxl, SRp55 and Rb97D) were not affected by hsromega mutation in any of the meiotic stages in adult testes. The present studies, however, revealed an unexpected presence (in wild-type as well as mutant) of the functional form of Sxl in primary spermatocytes and an unusual distribution of HRB87F along the retracting spindle during anaphase telophase of the first meiotic division. It appears that the P transposon insertion in the promoter region causes a misregulated overexpression of hsromega in cyst cells, which in turn results in excessive sequestration of hnRNPs and formation of large clusters of omega speckles in these cell nuclei. The consequent limiting availability of hnRNPs is likely to trans-dominantly affect processing of other pre-mRNAs in cyst cells. We suggest that a compromise in the activity of cyst cells due to the aberrant hnRNP distribution is responsible for the failure of individualization of sperms in hsromega05421; mutant testes. These results further support a significant role of the noncoding hsromega-n transcripts in basic cellular activities, namely regulation of the availability of hnRNPs in active (chromatin bound) and inactive (in omega speckles) compartments.

An exclusively nuclear RNA-binding protein affects asymmetric localization of ASH1 mRNA and Ash1p in yeast.

The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae. A localization element that is predominantly in the 3'-untranslated region (UTR) can direct this mRNA to the bud. Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3'-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs. LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA. Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain. Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm. We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.

Characterization of hnRNP A2 and B1 using monoclonal antibodies: intracellular distribution and metabolism through cell cycle

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 are nuclear RNA binding proteins involved in pre-RNA processing. The alternative splicing of the second mini exon of A2/B1 gene produces A2 and less abundant B1. It has been reported that patients with autoimmune diseases frequently have blood autoantibody valence for A2/B1, and recently that the overexpression, especially of B1, is useful for detecting cancers in early stage. Three anti-A2/B1 monoclonal antibodies were developed using recombinant A2 protein and synthesized peptides around the second splicing site. Three antibodies could separately recognize A2 and B1, and their specificity made them useful in the study of the biochemical and functional properties of A2 and B1. These antibodies have demonstrated differences between A2 and B1 in the intracellular distribution and in the metabolism through cell cycle. They are valuable reagents to clarify the clinical significance of A2/B1 in autoimmune diseases and cancers.

Immune response to hn and snRNP in autoimmune mice. A model for the development of lupus autoimmunity by a single initiator T helper epitope?

Systemic lupus erythematosus is characterised by the presence of high titers of autoantibodies reacting with various components of the small and heterogeneous nuclear ribonucleoprotein particle. It has been suggested that these antibodies are produced by an antigen-driven mechanism under the dependence of antigen-specific T cells. To investigate the role of T cell help in this process, we sought with twenty overlapping peptides the Th epitopes on the U1-70K snRNP in unprimed H-2k MRL/lpr lupus mice and immunised CBA normal mice. The peptide 131-151 was recognized by both IgG autoantibodies and CD4+ T cells from 7-9 week-old MRL/lpr mice. In this test, APCs from MRL/lpr mice were required, APCs from naive CBA mice failed to stimulate CD4+ cells from MRL/lpr mice. Peptide 131-151 bound both I-Ak and I-Ek class II molecules and favoured an IL-2 positive T cell response but not IFN-γ, IL-6 and IL-10 secretion. Segment 131-151 is localised within the RNP80 motif and contains residues that are highly conserved in many nuclear, nucleolar and cytoplasmic RNA binding proteins. In parallel, we studied the Ab response to the A2/B1 hnRNP in different murine models of lupus, and found in residues 50-70 a major epitope recognized very early during the course of the disease by Abs from most of MRL/lpr mice. Peptide 50-70 generated in CBA/J mice an effective Th cell response with IL-2 and IFN-γ secretion. Interestingly, this peptide also contains the highly conserved sequence present in peptide 131-151 of the 70K protein. It is possible that starting from a single Th epitope, the sequence of which is repeated in several self-proteins involved in the same complex or close cellular components, a larger, diversified Th response is generated, which extends via intra-and inter-molecular spreading of the T and B cell responses.

Increased protein levels of heterogeneous nuclear ribonucleoprotein A2/B1 in fetal Down syndrome brains.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are predominantly nuclear RNA-binding proteins that form complexes with RNA polymerase II transcripts. These proteins play pivotal roles in transcription, pre-mRNA processing in the nucleus, cytoplasmic mRNA translation and its turnover. In addition, hnRNPs have been shown to be essential for embryonic development of Drosophila. Here we studied the protein levels of hnRNPs (A2/B1, H and H') in fetal brain with Down syndrome (DS; n = 5) compared to controls (n = 7). We used two-dimensional (2-D) gel electrophoresis, matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS) and specific software for quantification. hnRNP A2/B1 was significantly increased in fetal DS brain (13.52+/-4.50) compared to controls (9.16+/-1.35), but both hnRNP H and H' were unchanged. Increased hnRNP A2/B1 in fetal DS brain may represent high activity of RNA processing such as RNA trafficking and telomere protection, and/or it could contribute to abnormal development of DS brains. Furthermore, comparable expression of hnRNP H and H' suggest a specific upregulation of hnRNP A2/B.