RNP Motif Research Articles

Two novel arginine/serine (SR) proteins in maize are differentially spliced and utilize non-canonical splice sites

The serine–arginine (SR)-rich splicing proteins are highly conserved RNA binding nuclear phosphor-proteins that play important roles in both regular and alternative splicing. Here we describe two novel putative SR genes from maize, designated zmRSp31A and zmRSp31B. Both genes contain characteristic RNA binding motifs RNP-1 and RNP-2, a serine/arginine-rich (RS) domain and share significant sequence similarity to the Arabidopsis atRSp31 family of SR proteins. Both zmRSp31A and zmRSp31B produce multiple transcripts by alternative splicing, of which majority of the alternatively spliced transcripts utilize non-canonical splice sites. zmRSp31A and zmRSp31B produce at least six and four transcripts, respectively, of which only one corresponds to the wild type proteins for each gene. All the alternatively spliced transcripts of both the genes, with one exception, are predicted to encode small truncated proteins containing only the RNP-2 domain of their first RNA recognition motif and completely lack the carboxyl terminal RS domain. We provide evidence that some of the alternatively spliced transcripts of both genes are associated with polysomes and interact with the translational machinery.

The second RNA-binding domain of the human splicing factor ASF/SF2 is the critical domain controlling adenovirus E1A alternative 5'-splice site selection.

The human splicing factor ASF/SF2 (alternative splicing factor/splicing factor 2) is modular in structure with two RNA-binding domains (RBD1 and RBD2) and a C-terminal domain rich in arginine-serine dipeptide repeats. ASF/SF2 is an essential splicing factor that also functions as an important regulator of alternative splicing. In adenovirus E1A (early region 1A) alternative pre-mRNA splicing, ASF/SF2 functions as a strong inducer of proximal 5'-splice-site selection, both in vitro and in vivo. In the present study, we tested the functional role of individual domains of ASF/SF2 in alternative splicing in vitro. We show that ASF/SF2-RBD2 is the critical domain controlling E1A alternative splicing. In fact, RBD2 alone is sufficient to mimic the activity of the full-length ASF/SF2 protein as an inducer of proximal 5'-splice-site selection in vitro. The RBD2 domain induces a switch to E1A-proximal 5'-splice-site usage by repressing distal 12 S splicing and simultaneously stimulates proximal 13 S splicing. In contrast, the ASF/SF2-RBD1 domain has a more general splicing enhancer phenotype and appears to stimulate preferentially cap-proximal 5'-splice-site selection. Furthermore, the SWQDLKD motif, which is conserved in all SR proteins (serine/arginine-rich proteins) containing two RBDs, and the ribonucleoprotein-1-type RNA recognition motif were both found to be necessary for the alternative splice-site-switching activity of ASF/SF2. The RNP-1 motif was necessary for efficient RNA binding, whereas the SWQDLKD motif most probably contributes by functioning as a surface-mediating critical protein-protein contact during spliceosome assembly.

A novel mode of RBD-protein recognition in the Y14-Mago complex.

Y14 and Mago are conserved eukaryotic proteins that associate with spliced mRNAs in the nucleus and remain associated at exon junctions during and after nuclear export. In the cytoplasm, Y14 is involved in mRNA quality control via the nonsense-mediated mRNA decay (NMD) pathway and, together with Mago, is involved in localization of osk (oskar) mRNA. We have determined the crystal structure of the complex between Drosophila melanogaster Y14 and Mago at a resolution of 2.5 A. The structure reveals an atypical mode of protein-protein recognition mediated by an RNA-binding domain (RBD). Instead of binding RNA, the RBD of Y14 engages its RNP1 and RNP2 motifs to bind Mago. Using structure-guided mutagenesis, we show that Mago is also a component of the NMD pathway, and that its association with Y14 is essential for function. Heterodimerization creates a single structural platform that interacts with the NMD machinery via phylogenetically conserved residues.

Structure of the Y14-Magoh Core of the Exon Junction Complex

Background: Splicing of pre-mRNA in eukaryotes imprints the resulting mRNA with a specific multiprotein complex, the exon-exon junction complex (EJC), at the sites of intron removal. The proteins of the EJC, Y14, Magoh, Aly/REF, RNPS1, Srm160, and Upf3, play critical roles in postsplicing processing, including nuclear export and cytoplasmic localization of the mRNA, and the nonsense-mediated mRNA decay (NMD) surveillance process. Y14 and Magoh are of particular interest because they remain associated with the mRNA in the same position after its export to the cytoplasm and require translation of the mRNA for removal. This tenacious, persistent, splicing-dependent, yet RNA sequence-independent, association suggests an important signaling function and must require distinct structural features for these proteins.Results: We describe the high-resolution structure and biochemical properties of the highly conserved human Y14 and Magoh proteins. Magoh has an unusual structure comprised of an extremely flat, six-stranded anti-parallel β sheet packed against two helices. Surprisingly, Magoh binds with high affinity to the RNP motif RNA binding domain (RBD) of Y14 and completely masks its RNA binding surface.Conclusions: The structure and properties of the Y14-Magoh complex suggest how the pre-mRNA splicing machinery might control the formation of a stable EJC-mRNA complex at splice junctions.

Molecular Sensing of Bacteria in Plants: THE HIGHLY CONSERVED RNA-BINDING MOTIF RNP-1 OF BACTERIAL COLD SHOCK PROTEINS IS RECOGNIZED AS AN ELICITOR SIGNAL IN TOBACCO

To detect microbial infection multicellular organisms have evolved sensing systems for pathogen-associated molecular patterns (PAMPs). Here, we identify bacterial cold shock protein (CSP) as a new such PAMP that acts as a highly active elicitor of defense responses in tobacco. Tobacco cells perceive a conserved domain of CSP and synthetic peptides representing 15 amino acids of this domain-induced responses at subnanomolar concentrations. Central to the elicitor-active domain is the RNP-1 motif KGFGFITP, a motif conserved also in many RNA- and DNA-binding proteins of eukaryotes. Csp15-Nsyl, a peptide representing the domain with highest homology to csp15 in a protein of Nicotiana sylvestris exhibited only weak activity in tobacco cells. Crystallographic and genetic data from the literature show that the RNP-1 domain of bacterial CSPs resides on a protruding loop and exposes a series of aromatic and basic side chains to the surface that are essential for the nucleotide-binding activity of CSPs. Similarly, these side chains were also essential for elicitor activity and replacement of single residues in csp15 with Ala strongly reduced or abolished activity. Most strikingly, csp15-Ala10, a peptide with the RNP-1 motif modified to KGAGFITP, lacked elicitor activity but acted as a competitive antagonist for CSP-related elicitors. Bacteria commonly have a small family of CSP-like proteins including both cold-inducible and noninducible members, and Csp-related elicitor activity was detected in extracts from all bacteria tested. Thus, the CSP domain containing the RNP-1 motif provides a structure characteristic for bacteria in general, and tobacco plants have evolved a highly sensitive chemoperception system to detect this bacterial PAMP.

Single-stranded DNA binding of the cold-shock protein CspB from Bacillus subtilis: NMR mapping and mutational characterization.

Cold-shock proteins (CSPs) bind to single-stranded nucleic acids, thereby acting as a "RNA chaperone." To gain deeper insights into the rather unspecific nature of ssDNA/RNA binding, we characterized the binding interface of CspB from Bacillus subtilis to a 25-mer of ssDNA (Y-Box25) using heteronuclear 2D NMR spectroscopy. Seventeen residues, including eight out of nine aromatic amino acids, are directly involved in the Y-Box25 interaction and were identified by extreme line broadening of their cross-peaks. Eight residues belong to the earlier proposed RNP binding motifs. A second set of seven backbone amides becomes evident by major chemical shift perturbations reporting remote conformational rearrangements upon binding. These residues are located in loop beta3-beta4 and loopbeta4-beta5, and include Ile18. The individual contributions of the so-identified residues were examined by fluorescence titration experiments of 15 CspB variants. Phenylalanine substitutions in- and outside the RNP motifs significantly reduce the binding affinity. Unrestricted possible backbone conformations of loop beta3-beta4 also markedly contribute to binding. Stopped-flow fluorescence kinetics revealed that the different binding affinities of CspB variants are determined by the dissociation rate, whereas the association rate remains unchanged. This might be of importance for the "RNA chaperone" activity of CspB.

Nucleolin associates with a subset of the human Ro ribonucleoprotein complexes.

Ro RNPs are evolutionarily conserved, small cytoplasmic RNA-protein complexes with an unknown function. In human cells, Ro RNPs consist of one of the four hY RNAs and two core proteins: Ro60 and La. Recently, the association of hnRNP I and hnRNP K with particles containing hY1 and hY3 RNAs has been described. The association of three other proteins, namely calreticulin, Ro52 and RoBPI, with (subsets of) the Ro RNPs is still controversial. To gain more insight into the composition and function of the Ro RNPs, we have immunopurified these particles from HeLa cell extracts using monoclonal antibodies against Ro60 and La. Using this approach, we have identified the RNA-binding protein nucleolin as a novel subunit of Ro RNP particles containing hY1 or hY3 RNA, but not hY4 and hY5 RNA. Using an in vitro hY RNA-binding assay we established that the internal pyrimidine-rich loop of hY1 and hY3 RNA is essential for the association of nucleolin. The binding is critically dependent on the presence of all four RNP motifs of nucleolin, but not of the C-terminal RGG-box. Moreover, we demonstrate that, in contrast to nucleolin and hnRNP K, nucleolin and hnRNP I can bind simultaneously to the internal pyrimidine-rich loop of hY1 RNA. We postulate that nucleolin functions in the biogenesis and/or trafficking of hY1 and hY3 RNPs through the nucleolus and subsequent transport to the cytoplasm.

Solution structure of protein SRP19 of Archaeoglobus fulgidus signal recognition particle

Protein SRP19 is an essential RNA-binding component of the signal recognition particle (SRP) in Archaea and Eucarya. A three-dimensional solution structure of the 104 residue SRP19 from the hyperthermophilic archaeon Archaeoglobus fulgidus, designated as Af19, was determined by NMR spectroscopy. Af19 contains three β-strands, two α-helical regions, arranged in a βαββα topology, a 3 10 helix, and a disordered C-terminal tail. This fold is similar to the βαββαβ RNP motif present in numerous other RNA-binding proteins, which engage their cognate RNAs using conserved sequence motifs present within β-strands 1 and 3. Mutagenesis studies of human SRP19, however, reveal the major contact sites with SRP RNA reside within loops 1, 3, and 4. These contacts were verified by the crystal structure of human SRP19 complexed to SRP RNA helix 6 reported subsequent to the submission of the manuscript. The crystal structure also reveals that, unlike canonical RNP motifs, SRP19 does not engage specific RNA bases through conserved sequence motifs present within β-strands 1 and 3. Instead, SRP19 uses residues both within and flanking β-strand 1 to stabilize the complex through direct and indirect contacts to the phosphate backbone of the tetraloop, leaving the bases of the tetraloop exposed. This, coupled with the fact that SRP19 appears relatively rigid and undergoes only minor changes in structure upon RNA binding, may underlie the molecular basis by which SRP19 functions to initiate SRP assembly.

Cloning and characterization of two subunits of Arabidopsisthaliana nuclear cap-binding complex

In this report we characterize two Arabidopsisthaliana proteins, named AtCBP20 and AtCBP80, that are homologues of human subunits of a nuclear cap-binding protein complex (CBC). AtCBP20 has a calculated molecular mass of 29.9 kDa, and AtCBP80 is a 96.5 kDa protein. AtCBP20 exhibits 68% identity and 82% similarity to human CBP20. Like its human homologue, AtCBP20 contains a canonical RNA binding domain (RBD) with single RNP2 and RNP1 motifs. In addition to the N-terminal part, which is similar to the human protein, AtCBP20 has a long C-terminus rich in arginine, glycine and aspartate residues. The second subunit of the Arabidopsis cap-binding complex, AtCBP80, shows 28% identity and 50% similarity to its homologue from HeLa cells. The protein contains a MIF4G domain at its N-terminus, the feature characteristic to all analyzed CBP80s. This domain, described also in eIF4G and NMD2 proteins, is thought to be involved in protein-protein and also in protein–RNA interactions. Both proteins AtCBP20 and AtCBP80 are encoded by single-copy genes in the A.thaliana genome. The AtCBP20 gene is located on chromosome V, and the AtCBP80 gene is encoded by chromosome II. Among introns identified in the AtCBP20 gene, we discovered an U12 type intervening sequence (an AT-AC intron). This intron is spliced out very efficiently in plants, but when isolated and tested for splicing in tobacco protoplasts, the efficiency of the U12 intron excision was low. Splicing efficiency of the U12 intron is improved by the addition of exon and intron sequences upstream or downstream of the U12 intron. AtCBP20 and AtCBP80 are constitutively expressed in all examined organs of A.thaliana, including roots, stems, leaves and flowers. Interestingly, the steady-state level of both transcripts seem to be very similar in all tissues analyzed.

High-affinity DNA binding of HU protein from the hyperthermophile Thermotoga maritima

Prokaryotic genomes are compacted by association with small basic proteins, generating what has been termed bacterial chromatin. The ubiquitous DNA-binding protein HU serves this function. DNA-binding properties of HU from the hyperthermophilic eubacterium Thermotoga maritima are shown here to differ significantly from those characteristic of previously described HU homologs. Electrophoretic mobility shift analyses show that T. maritima HU (TmHU) binds double-stranded DNA with high affinity (Kd=5.6(±0.7) nM for 37 bp DNA). Equivalent affinity is observed between 4°C and 45°C. TmHU has higher affinity for DNA containing a set of 4 nt loops separated by 9 bp (Kd=1.4(±0.3) nM), consistent with its introduction of two DNA kinks. Using DNA probes of varying length, the optimal binding site for TmHU is estimated at 37 bp, in sharp contrast to the 9–10 bp binding site reported for other HU homologs. Alignment of>60 HU sequences demonstrates significant sequence conservation: A DNA-intercalating proline residue is almost universally conserved, and it is preceded by arginine and asparagine in most sequences, generating a highly conserved RNP motif; V substitutes for R only in HU from Thermotoga, Thermus and Deinococcus. A fivefold increase in DNA-binding affinity is observed for TmHU in which V is replaced with R (TmHU-V61R; Kd=1.1(±0.2) nM), but a change in the trajectory of DNA flanking the sites of DNA intercalation is inferred from analysis of TmHU-V61R binding to DNA modified with 4 nt loops or with substitutions of 5-hydroxymethyluracil for thymine. Survival in extreme environments places unique demands on protection of genomic DNA from thermal destabilization and on access of DNA to the cellular machinery, demands that may be fulfilled by the specific DNA-binding properties of HU and by the fine structure of the bacterial chromatin.

Predominant Identification of RNA-binding Proteins in Fas-induced Apoptosis by Proteome Analysis

Proteome analysis of Jurkat T cells was performed in order to identify proteins that are modified during apoptosis. Subtractive analysis of two-dimensional gel patterns of apoptotic and nonapoptotic cells revealed differences in 45 protein spots. 37 protein spots of 21 different proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry. The hnRNPs A0, A2/B1, A3, K, and R; the splicing factors p54(nrb), SRp30c, ASF-2, and KH-type splicing regulatory protein (FUSE-binding protein 2); and alpha NAC, NS1-associated protein 1, and poly(A)-binding protein 4 were hitherto unknown to be involved in apoptosis. The putative cleavage sites of the majority of the proteins could be calculated by the molecular masses and isoelectric points in the two-dimensional electrophoresis gel, the peptide mass fingerprints, and after translation by treatment with recombinant caspase-3. Remarkably, 15 of the 21 identified proteins contained the RNP or KH motif, the best characterized RNA-binding motifs.

Mutational Definition of RNA-binding and Protein-Protein Interaction Domains of Heterogeneous Nuclear RNP C1

The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of approximately 80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.

The Splicing Factor U1C Represses EWS/FLI-mediated Transactivation

EWS is an RNA-binding protein involved in human tumor-specific chromosomal translocations. In approximately 85% of Ewing's sarcomas, such translocations give rise to the chimeric gene EWS/FLI. In the resulting fusion protein, the RNA binding domains from the C terminus of EWS are replaced by the DNA-binding domain of the ETS protein FLI-1. EWS/FLI can function as a transcription factor with the same DNA binding specificity as FLI-1. EWS and EWS/FLI can associate with the RNA polymerase II holoenzyme as well as with SF1, an essential splicing factor. Here we report that U1C, one of three human U1 small nuclear ribonucleoprotein-specific proteins, interacts in vitro and in vivo with both EWS and EWS/FLI. U1C interacts with other splicing factors and is important in the early stages of spliceosome formation. Importantly, co-expression of U1C represses EWS/FLI-mediated transactivation, demonstrating that this interaction can have functional ramifications. Our findings demonstrate that U1C, a well characterized splicing protein, can also function in transcriptional regulation. Furthermore, they suggest that EWS and EWS/FLI may function both in transcriptional and post-transcriptional processes.

A novel protein with RNA-binding motifs interacts with ataxin-2.

Spinocerebellar ataxia type 2 (SCA2) is caused by expansion of a polyglutamine tract in ataxin-2, a protein of unknown function. Using the yeast two-hybrid system, we identified a novel protein, A2BP1 (ataxin-2 binding protein 1) which binds to the C-terminus of ataxin-2. Northern blot analysis showed that A2BP1 was predominantly expressed in muscle and brain. By immunocfluorescent staining, A2BP1 and ataxin-2 were both localized to the trans -Golgi network. Immunocytochemistry showed that A2BP1 was expressed in the cytoplasm of Purkinje cells and dentate neurons in a pattern similar to that seen for ataxin-2 labeling. Western blot analysis of subcellular fractions indicated enrichment of A2BP1 in the same fractions as ataxin-2. Sequence analysis of the A2BP1 cDNA revealed an RNP motif that is highly conserved among RNA-binding proteins. A2BP1 had striking homology with a human cDNA clone, P83A20, of unknown function and at least two copies of A2BP1 homologs are found in the Caenorhabditis elegans genome database. A2BP1 and related proteins appear to form a novel gene family sharing RNA-binding motifs.

Temporal control of protein synthesis during spermatogenesis.

During oogenesis and spermatogenesis transcription ceases prior to the differentiation of the mature cells. To complete germ cell differentiation and initiate early embryogenesis, proteins are synthesized from pre-existing mRNAs that are stored for several days. It is well established that important regulatory elements functioning in spatial localization, temporal translation or messenger RNA stability are located in the 3' untranslated region (3' UTR) of mRNAs. During mammalian spermatogenesis temporal translational regulation of the protamine 1 (Prm1) mRNA is dependent on a highly conserved sequence located in the distal region of its 3' UTR. The 17-nucleotide translational control element (TCE) mediates translational repression of the Prm1 mRNA. Mutation of the TCE causes premature synthesis of protamine protein and sterility. The Prm1 mRNA is stored as a cytoplasmic ribonucleoprotein (mRNP) particle in spermatids. Contained within the particle are several members of the Y box family of nucleic acid binding proteins. In the yeast three-hybrid system the murine Y box proteins MSY1, MSY2 and MSY4 bind in a sequence-dependent manner to a conserved region in the proximal portion of the Prm1 3' UTR. Sequence-specific binding by MSY4 to the Y box recognition sequence (YRS) is dependent on the highly conserved cold shock domain, possibly through the RNP1 and RNP2 motifs present within it. The Y box proteins may function as translational repressors in vivo. Alternatively, their primary function may be to protect mRNAs from degradation during their extended period of storage. Translational activation of stored mRNAs is essential for the completion of gametogenesis. Proper translational activation of the Prm1 mRNA in elongated spermatids requires the cytoplasmic double-stranded RNA binding protein TARBP2. Tarbp2 is expressed at low levels in many cells but is expressed at robust levels in late stage meiotic cells and in postmeiotic spermatids. Mice mutant for Tarbp2 are defective in proper translational activation of the Prm1 and Prm2 mRNAs and are sterile. Current studies are designed to determine the mechanism by which proteins bound to the 3' UTR communicate with the 5' end of the message to control translational silencing and activation.

Spen encodes an RNP motif protein that interacts with Hox pathways to repress the development of head-like sclerites in the Drosophila trunk.

Drosophila has eight Hox proteins, and they require factors acting in parallel to regulate different segmental morphologies. Here we find that the Drosophila gene split ends (spen), has a homeotic mutant phenotype, and appears to encode such a parallel factor. Our results indicate that spen plays two important segment identity roles. One is to promote sclerite development in the head region, in parallel with Hox genes; the other is to cooperate with Antennapedia and teashirt to suppress head-like sclerite development in the thorax. Our results also indicate that without spen and teashirt functions, Antennapedia loses its ability to specify thoracic identity in the epidermis. spen transcripts encode extraordinarily large protein isoforms (approx. 5,500 amino acids), which are concentrated in embryonic nuclei. Both Spen protein isoforms and Spen-like proteins in other animals possess a clustered repeat of three RNP (or RRM) domains, as well as a conserved motif of 165 amino acids (SPOC domain) at their C-termini. Spen is the only known homeotic protein with RNP binding motifs, which indicates that splicing, transport, or other RNA regulatory steps are involved in the diversification of segmental morphology. Previous studies by Dickson and others (Dickson, B. J., Van Der Straten, A., Dominguez, M. and Hafen, E. (1996). Genetics 142, 163-171) identified spen as a gene that acts downstream of Raf to suppress Raf signaling in a manner similar to the ETS transcription factor Aop/Yan. This raises the intriguing possibility that the Spen RNP protein might integrate signals from both the Raf and Hox pathways.

Translational control by the La antigen. Structure requirements for rescue of the double-stranded RNA-mediated inhibition of protein synthesis.

The La antigen is a protein which can bind both single-stranded and double-stranded forms of RNA and has regulatory effects on gene expression at the levels of transcription and translation. It was previously shown to inhibit the activation of the dsRNA-dependent protein kinase PKR by sequestering and/or unwinding double-stranded RNA. Here, we demonstrate that, as predicted by these properties, the La antigen can rescue protein synthesis in the reticulocyte lysate system from inhibition by low concentrations of dsRNA. This effect is reversed by higher concentrations of dsRNA. Using a series of deletion mutants we have investigated the structural features of the La antigen that are required for these effects. The ability to bind dsRNA is influenced by regions within both the previously characterized N-terminal RNP motif and the C-terminal half of the protein. La mutants with either N-terminal or C-terminal deletions retain the ability to inhibit the protein kinase activity of PKR and to rescue protein synthesis from inhibition by dsRNA. It is notable that sequences in the C-terminal half of the La antigen, including a phosphorylation site at Ser366, which are needed for other regulatory effects of the protein on gene expression are dispensable for the effects of La on PKR. We suggest that La regulates PKR activity solely as a result of its ability to act as an RNA-binding protein that can compete with PKR for limiting amounts of dsRNA.

Identification and characterization of five cspA homologous genes from Myxococcus xanthus

Escherichia coli contains a large CspA family consisting of nine homologues, in which four are cold-shock inducible and one is stationary-phase inducible. Here, we demonstrate that Myxococcus xanthus possesses at least five CspA homologues, CspA to CspE. Hydrophobic residues forming a hydrophobic core, and aromatic residues, which are included in functional motifs RNP-1 and RNP-2 involved in binding to RNA and ssDNA, are well conserved. These facts suggest that M. xanthus CspA homologues have a similar structure and function as E. coli CspA. However, in contrast to the E. coli CspA family, the expression of M. xanthus csp genes as judged by primer extension analysis is not significantly regulated by temperature changes, except for cspB of which expression was reduced to less than 10% upon heat shock at 42°C. Such constitutive expression of the csp genes may be important for M. xanthus, a soil-dwelling bacterium, to survive under conditions of exposure to various environmental changes in nature.

Recognition of Polyadenylate RNA by the Poly(A)-Binding Protein

The cocrystal structure of human poly(A)-binding protein (PABP) has been determined at 2.6 Å resolution. PABP recognizes the 3′ mRNA poly(A) tail and plays critical roles in eukaryotic translation initiation and mRNA stabilization/degradation. The minimal PABP used in this study consists of the N-terminal two RRM-type RNA-binding domains connected by a short linker (RRM1/2). These two RRMs form a continuous RNA-binding trough, lined by an antiparallel β sheet backed by four α helices. The polyadenylate RNA adopts an extended conformation running the length of the molecular trough. Adenine recognition is primarily mediated by contacts with conserved residues found in the RNP motifs of the two RRMs. The convex dorsum of RRM1/2 displays a phylogenetically conserved hydrophobic/acidic portion, which may interact with translation initiation factors and regulatory proteins.

Purification of cold-shock-like proteins from Stigmatella aurantiaca - molecular cloning and characterization of the cspA gene.

Prominent low-molecular-weight proteins were isolated from vegetative cells of the myxobacterium Stigmatella aurantiaca and were found to be members of the cold-shock protein family. A first gene of this family (cspA) was cloned and sequenced. It encodes a protein of 68 amino acid residues that displays up to 71% sequence identity with other bacterial cold-shock(-like) proteins. A cysteine residue within the RNP-2 motif is a peculiarity of Stigmatella CspA. A cspA::(Deltatrp-lacZ) fusion gene construct was introduced into Stigmatella by electroporation, a method that has not been used previously for this strain. Analysis of the resultant transformants revealed that cspA transcription occurs at high levels during vegetative growth at 20 and 32 degrees C, and during fruiting body formation.