Arginine methylation of the nuclear poly(a) binding protein weakens the interaction with its nuclear import receptor, transportin

The nuclear poly(A) binding protein, PABPN1, promotes mRNA polyadenylation in the cell nucleus by increasing the processivity of poly(A) polymerase and contributing to poly(A) tail length control. In its C-terminal domain, the protein carries 13 arginine residues that are all asymmetrically dimethylated. The function of this modification in PABPN1 has been unknown. Part of the methylated domain serves as nuclear localization signal, binding the import receptor transportin. Here we report that arginine methylation weakens the affinity of PABPN1 for transportin. Recombinant, unmethylated PABPN1 binds more strongly to transportin than its methylated counterpart from mammalian tissue, and in vitro methylation reduces the affinity. Transportin and RNA compete for binding to PABPN1. Methylation favors RNA binding. Transportin also inhibits in vitro methylation of the protein. Finally, a peptide corresponding to the nuclear localization signal of PABPN1 competes with transportin-dependent nuclear import of the protein in a permeabilized cell assay and does so less efficiently when it is methylated. We hypothesize that transportin binding might delay methylation of PABPN1 until after nuclear import. In the nucleus, arginine methylation may favor the transition of PABPN1 to the competing ligand RNA and serve to reduce the risk of the protein being reexported to the cytoplasm by transportin.     

Stimulation of poly(A) polymerase through a direct interaction with the nuclear poly(A) binding protein allosterically regulated by RNA

During polyadenylation of mRNA precursors in metazoan cells, poly(A) polymerase is stimulated by the nuclear poly(A) binding protein PABPN1. We report that stimulation depends on binding of PABPN1 to the substrate RNA directly adjacent to poly(A) polymerase and results in an approximately 80-fold increase in the apparent affinity of poly(A) polymerase for RNA without significant effect on catalytic efficiency. PABPN1 associates directly with poly(A) polymerase either upon allosteric activation by oligo(A) or, in the absence of RNA, upon deletion of its N-terminal domain. The N-terminal domain of PABPN1 may function to inhibit undesirable interactions of the protein; the inhibition is relieved upon RNA binding. Tethering of poly(A) polymerase is mediated largely by the C-terminal domain of PABPN1 and is necessary but not sufficient for stimulation of the enzyme; an additional interaction dependent on a coiled-coil structure located within the N-terminal domain of PABPN1 is required for a productive interaction.     

The domain of the Bacillus subtilis DEAD-box helicase YxiN that is responsible for specific binding of 23S rRNA has an RNA recognition motif fold

The YxiN protein of Bacillus subtilis is a member of the DbpA subfamily of prokaryotic DEAD-box RNA helicases. Like DbpA, it binds with high affinity and specificity to segments of 23S ribosomal RNA as short as 32 nucleotides (nt) that include hairpin 92. Several experiments have shown that the 76-residue carboxy-terminal domain of YxiN is responsible for the high-affinity RNA binding. The domain has been crystallized and its structure has been solved to 1.7 Angstroms resolution. The structure reveals an RNA recognition motif (RRM) fold that is found in many eukaryotic RNA binding proteins; the RRM fold was not apparent from the amino acid sequence. The domain has two solvent exposed aromatic residues at sites that correspond to the aromatic residues of the ribonucleoprotein (RNP) motifs RNP1 and RNP2 that are essential for RNA binding in many RRMs. However, mutagenesis of these residues (Tyr404 and Tyr447) to alanine has little effect on RNA affinity, suggesting that the YxiN domain binds target RNAs in a manner that differs from the binding mode commonly found in many eukaryotic RRMs.     

Conformational stability of the RNP domain controls fibril formation of PABPN1

The disease oculopharyngeal muscular dystrophy is caused by alanine codon trinucleotide expansions in the N‐terminal segment of the nuclear poly(A) binding protein PABPN1. As histochemical features of the disease, intranuclear inclusions of PABPN1 have been reported. Whereas the purified N‐terminal domain of PABPN1 forms fibrils in an alanine‐dependent way, fibril formation of the full‐length protein occurs also in the absence of alanines. Here, we addressed the question whether the stability of the RNP domain or domain swapping within the RNP domain may add to fibril formation. A variant of full‐length PABPN1 with a stabilizing disulfide bond at position 185/201 in the RNP domain fibrillized in a redox‐sensitive manner suggesting that the integrity of the RNP domain may contribute to fibril formation. Thermodynamic analysis of the isolated wild‐type and the disulfide‐linked RNP domain showed two state unfolding/refolding characteristics without detectable intermediates. Quantification of the thermodynamic stability of the mutant RNP domain pointed to an inverse correlation between fibril formation of full‐length PABPN1 and the stability of the RNP domain.     

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.     

Structure of the Shq1-Cbf5-Nop10-Gar1 complex and implications for H/ACA RNP biogenesis and dyskeratosis congenita

Shq1 is a conserved protein required for the biogenesis of eukaryotic H/ACA ribonucleoproteins (RNPs), including human telomerase. We report the structure of the Shq1-specific domain alone and in complex with H/ACA RNP proteins Cbf5, Nop10 and Gar1. The Shq1-specific domain adopts a novel helical fold and primarily contacts the PUA domain and the otherwise disordered C-terminal extension (CTE) of Cbf5. The structure shows that dyskeratosis congenita mutations found in the CTE of human Cbf5 likely interfere with Shq1 binding. However, most mutations in the PUA domain are not located at the Shq1-binding surface and also have little effect on the yeast Cbf5-Shq1 interaction. Shq1 binds Cbf5 independently of the H/ACA RNP proteins Nop10, Gar1 and Nhp2 and the assembly factor Naf1, but shares an overlapping binding surface with H/ACA RNA. Shq1 point mutations that disrupt Cbf5 interaction suppress yeast growth particularly at elevated temperatures. Our results suggest that Shq1 functions as an assembly chaperone that protects the Cbf5 protein complexes from non-specific RNA binding and aggregation before assembly of H/ACA RNA.     

Equilibrium studies on the association of the nuclear poly(A) binding protein with poly(A) of different lengths

The nuclear poly(A) binding protein (PABPN1) binds the growing poly(A) tail during pre-mRNA 3'-end processing, stimulating its elongation and controlling its final length. Here we report binding studies of PABPN1 to poly(A) in solution. Quantitative fluorescence titration was used to determine the stoichiometry, intrinsic affinity, and cooperativity of binding to a series of size-fractionated poly(A). The intrinsic association constant K(i) was about 2 x 10(6) M(-1) for oligo(A) and all size classes of poly(A). The binding of PABPN1 to poly(A) was enhanced by protein-protein interactions which were, however, weak (cooperativity parameter omega < 50). No significant change of cooperativity could be detected with increasing polynucleotide length in the range of 140-450 nucleotides. An average binding site size n of 11-14 was found for all poly(A) lengths, which is close to the minimal site size m found for binding to oligo(A). The data are discussed with respect to the previous observation of two different forms of the poly(A)-PABPN1 complex.     

Isolation of genomic and cDNA clones encoding bovine poly(A) binding protein II.

cDNA clones for bovine poly(A) binding protein II (PAB II) were isolated. Their sequence predicts a protein of 32.8 kDa, revising earlier estimates of molecular mass. The protein contains one putative RNA-binding domain of the RNP type, an acidic N-terminal and a basic C-terminal domain. Analyses of authentic PAB II were in good agreement with all predictions from the cDNA sequence except that a number of arginine residues appeared to be post-translationally modified. Poly(A) binding protein II expressed in Escherichia coli was active in poly(A) binding and reconstitution of processive polyadenylation, including poly(A) tail length control. The cDNA clones showed a number of potential PAB II binding sites in the 3' untranslated sequence. Bovine poly(A)+RNA contained two mRNAs hybridizing to a PAB II-specific probe. Analysis of a genomic clone revealed six introns in the coding sequence. The revised molecular mass led to a demonstration of PAB II oligomer formation and a reinterpretation of earlier data concerning the protein's binding to poly(A).     

RNA-binding activities of the different domains of a spinach chloroplast ribonucleoprotein.

An RNA-binding protein of 28 kD (28RNP) has been previously isolated from spinach chloroplasts and was found to be required for 3' end processing of chloroplast mRNAs. The amino acid sequence of 28RNP revealed two approximately 80 amino-acid RNA-binding domains, as well as an acidic and glycine-rich amino terminal domain. Each domain by itself, as well as in combination with other domains, was expressed in bacterial cells and the polypeptides were purified to homogeneity. We have investigated the RNA-binding properties of the different structural domains using UV-crosslinking, saturation binding and competition between the different domains on RNA-binding. It was found that the acidic domain does not bind RNA, but that each of the RNA-binding domains, expressed either individually or together, do bind RNA, although with differing affinities. When either the first or second RNA-binding domain was coupled to the acidic domain, the affinity for RNA was greatly reduced. However, the acidic domain has a positive effect on the binding of the full-length protein to RNA, because the mature protein binds RNA with a better affinity than the truncated protein which lacks the acidic domain. In addition, it was found that a stretch of two or three G residues is enough to mediate binding of the 28RNP, whereas four U residues were insufficient. The implications of the RNA-binding properties of 28RNP to its possible function in the processing of chloroplast RNA is discussed.     

Structural basis of the RNA-binding specificity of human U1A protein.

The RNP domain is a very common eukaryotic protein domain involved in recognition of a wide range of RNA structures and sequences. Two structures of human U1A in complex with distinct RNA substrates have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning the origin of binding specificity. The beta-sheet of the domain provides an extensive RNA-binding platform for packing aromatic RNA bases and hydrophobic protein side chains. However, many interactions between functional groups on the single-stranded nucleotides and residues on the beta-sheet surface are potentially common to RNP proteins with diverse specificity and therefore make only limited contribution to molecular discrimination. The refined structure of the U1A complex with the RNA polyadenylation inhibition element reported here clarifies the role of the RNP domain principal specificity determinants (the variable loops) in molecular recognition. The most variable region of RNP proteins, loop 3, plays a crucial role in defining the global geometry of the intermolecular interface. Electrostatic interactions with the RNA phosphodiester backbone involve protein side chains that are unique to U1A and are likely to be important for discrimination. This analysis provides a novel picture of RNA-protein recognition, much closer to our current understanding of protein-protein recognition than that of DNA-protein recognition.     

The existence of eukaryotic ribonucleoprotein consensus sequence-type RNA-binding proteins in a prokaryote, Synechococcus 6301.

A group of proteins containing a conserved ribonucleoprotein consensus sequence (RNP-CS)-type RNA-binding domain (CS-RBD) of approximately 80 amino acids is present in eukaryotic cells and binds specifically to a wide variety of RNA molecules. We have isolated 12 kDa single-stranded DNA binding proteins from the unicellular cyanobacterium Synechococcus 6301. The amino-terminal sequence was determined and two distinct genomic clones were isolated from a Synechococcus 6301 genomic library. Sequence analysis revealed that two closely related proteins contain a single CS-RBD of 82 amino acids and are named as 12RNP1 and 12RNP2. Both of the CS-RBDs share the highest amino acid identity with those of chloroplast ribonucleoproteins (40-51%). The 12RNP proteins were expressed in Escherichia coli bearing plasmids encoding glutathione S-transferase/12RNP fusion proteins and subjected to in vitro nucleic acid-binding assay. Both 12RNP1 and 12RNP2 bind to RNA homopolymers poly(U) and poly(G), indicating that they might be RNA-binding proteins. This is the first example of such proteins in prokaryotes. The 12RNP1 and 12RNP2 genes are transcribed as monocistronic mRNAs and the steady-state mRNA level of 12RNP1 is over 20-fold than that of 12RNP2. Due to the easiness of genetic manipulations the cyanobacterium will provide an excellent system to analyze the function of not only cyanobacterial but also plant RNA-binding proteins.     

The Elav-like proteins bind to AU-rich elements and to the poly(A) tail of mRNA.

The Elav-like proteins are specific mRNA binding proteins which are required for cellular differentiation. They contain three characteristic RNP2/RNP1-type RNA binding motifs. Previously we have shown that the first and second RNA binding domains bind to AU-rich elements in the 3'-UTR of mRNA. In this paper we show that the Elav-like proteins exhibit poly(A) binding activity. This activity is distinct from poly(A) binding activities that have been previously described. The Elav-like proteins specifically bind to long chain poly(A) tails. We have shown that the third RNA binding domain encompasses this poly(A) binding activity. Using poly(A)-Sepharose beads in a 'sandwich' assay we have shown that the Elav-like proteins can bind simultaneously to the AU-rich element and to the poly(A) tail.     

Contribution of the C-terminal tail of U1A RBD1 to RNA recognition and protein stability.

The N-terminal RNA binding domain (RBD1) of the human U1A protein interacts specifically with a short RNA hairpin containing the U1 snRNA stem/loop II sequence. Previous RNA binding studies have suggested that the C-terminal tail of RBD1 contributes to RNA recognition in addition to interactions on the beta-sheet surface of the protein. To evaluate the contributions of these C-terminal residues in RBD1 to RNA binding affinity and specificity, as well as to study the thermodynamic stability of RBDs, a number of RBD1 mutants with truncated tails, with single amino acid substitutions, and with both a truncation and an amino acid substitution, have been constructed. The thermodynamic stabilities of these mutants have been measured and compared by GdnHCI unfolding experiments. The RNA binding affinity and specificity of these mutant proteins have been assessed by measuring the binding of each protein to the wild-type RNA hairpin and to selected RNA mutants with nucleotide substitutions in the RNA loop. The results demonstrate first that, although the C-terminal tail of RBD1 makes significant contributions to RNA binding affinity, it is not required for RNA binding, and second, its contributions to binding specificity are mediated only through selected nucleotides in the RNA loop, for in the absence of the tail, the protein continues to use other nucleotides to discriminate among RNAs. In these truncated proteins, the secondary structure intrinsic to the C-terminal tail is absent, yet their affinity and discrimination for RNAs are not lost. Thus, a structured tail is not required for RNA recognition.     

Promiscuous modification of the nuclear poly(A)-binding protein by multiple protein-arginine methyltransferases does not affect the aggregation behavior

The mammalian nuclear poly(A)-binding protein, PABPN1, carries 13 asymmetrically dimethylated arginine residues in its C-terminal domain. By fractionation of cell extracts, we found that protein-arginine methyltransferases (PRMTs)-1, -3, and -6 are responsible for the modification of PABPN1. Recombinant PRMT1, -3, and -6 also methylated PABPN1. Our data suggest that these enzymes act on their own, and additional polypeptides are not involved in recognizing PABPN1 as a substrate. PRMT1 is the predominant methyltransferase acting on PABPN1. Nevertheless, PABPN1 was almost fully methylated in a Prmt1(-/-) cell line; thus, PRMT3 and -6 suffice for methylation. In contrast to PABPN1, the heterogeneous nuclear ribonucleoprotein (hnRNP) K is selectively methylated only by PRMT1. Efficient methylation of synthetic peptides derived from PABPN1 or hnRNP K suggested that PRMT1, -3, and -6 recognize their substrates by interacting with local amino acid sequences and not with additional domains of the substrates. However, the use of fusion proteins suggested that the inability of PRMT3 and -6 to modify hnRNP K is because of structural masking of the methyl-accepting amino acid sequences by neighboring domains. Mutations leading to intracellular aggregation of PABPN1 cause the disease oculopharyngeal muscular dystrophy. The C-terminal domain containing the methylated arginine residues is known to promote PAPBN1 self-association, and arginine methylation has been reported to inhibit self-association of an orthologous protein. Thus, arginine methylation might be relevant for oculopharyngeal muscular dystrophy. However, in two different types of assays we have been unable to detect any effect of arginine methylation on the aggregation of bovine PABPN1.     

Critical residues for RNA discrimination of the histone hairpin binding protein (HBP) investigated by the yeast three-hybrid system

The histone hairpin binding protein (HBP, also called SLBP, which stands for stem-loop binding protein) binds specifically to a highly conserved hairpin structure located in the 3' UTR of the cell-cycle-dependent histone mRNAs. HBP consists of a minimal central RNA binding domain (RBD) flanked by an N- and C-terminal domain. The yeast three-hybrid system has been used to investigate the critical residues of the human HBP involved in the binding of its target hairpin structure. By means of negative selections followed by positive selections, we isolated mutant HBP species. Our results indicate tight relationships between the RBD and the N- and C-terminal domains.     

Functional significance of the interaction between the mRNA-binding protein, Nab2, and the nuclear pore-associated protein, Mlp1, in mRNA export

Nuclear export of mRNA requires several key mRNA-binding proteins that recognize and remodel the mRNA and target it for export via interactions with the nuclear pore complex. In Saccharomyces cerevisiae, the shuttling heterogeneous nuclear ribonucleoprotein, Nab2, which is essential for mRNA export, specifically recognizes poly(A) RNA and binds to the nuclear pore-associated protein, myosin-like protein 1 (Mlp1), which functions in mRNA export and quality control. Specifically, the N-terminal domain of Nab2 (Nab2-N; residues 1-97) interacts directly with the C-terminal globular domain of Mlp1 (CT-Mlp1: residues 1490-1875). Recent structural and binding studies focused on Nab2-N have shown that Nab2-N contains a hydrophobic patch centered on Phe(73) that is critical for interaction with Mlp1. Engineered amino acid changes within this patch disrupt the Nab2/Mlp1 interaction in vitro. Given the importance of Nab2 and Mlp1 to mRNA export, we have examined the Nab2/Mlp1 interaction in greater detail and analyzed the functional consequences of disrupting the interaction in vivo. We find that the Nab2-binding domain of Mlp1 (Mlp1-NBD) maps to a 183-residue region (residues 1586-1768) within CT-Mlp1, binds directly to Nab2 with micromolar affinity, and confers nuclear accumulation of poly(A) RNA. Furthermore, we show that cells expressing a Nab2 F73D mutant that cannot interact with Mlp1 exhibit nuclear accumulation of poly(A) RNA and that this nab2 F73D mutant genetically interacts with alleles of two essential mRNA export genes, MEX67 and YRA1. These data provide in vivo evidence for a model of mRNA export in which Nab2 is important for targeting mRNAs to the nuclear pore for export.     

Dissecting FMR1, the protein responsible for fragile X syndrome, in its structural and functional domains.

FMR1 is an RNA-binding protein that is either absent or mutated in patients affected by the fragile X syndrome, the most common inherited cause of mental retardation in humans. Sequence analysis of the FMR1 protein has suggested that RNA binding is related to the presence of two K-homologous (KH) modules and an RGG box. However, no attempt has been so far made to map the RNA-binding sites along the protein sequence and to identify possible differential RNA-sequence specificity. In the present article, we describe work done to dissect FMR1 into regions with structurally and functionally distinct properties. A semirational approach was followed to identify four regions: an N-terminal stretch of 200 amino acids, the two KH regions, and a C-terminal stretch. Each region was produced as a recombinant protein, purified, and probed for its state of folding by spectroscopical techniques. Circular dichroism and NMR spectra of the N-terminus show formation of secondary structure with a strong tendency to aggregate. Of the two homologous KH motifs, only the first one is folded whereas the second remains unfolded even when it is extended both N- and C-terminally. The C-terminus is, as expected from its amino acid composition, nonglobular. Binding assays were then performed using the 4-nt homopolymers. Our results show that only the first KH domain but not the second binds to RNA, and provide the first direct evidence for RNA binding of both the N-terminal and the C-terminal regions. RNA binding for the N-terminus could not be predicted from sequence analysis because no known RNA-binding motif is identifiable in this region. Different sequence specificity was observed for the fragments: both the N-terminus of the protein and KH1 bind preferentially to poly-(rG). The C-terminal region, which contains the RGG box, is nonspecific, as it recognizes the bases with comparable affinity. We therefore conclude that FMR1 is a protein with multiple sites of interaction with RNA: sequence specificity is most likely achieved by the whole block that comprises the first approximately 400 residues, whereas the C-terminus provides a nonspecific binding surface.