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.     

Altering the RNA-binding mode of the U1A RBD1 protein

The N-terminal RNA-binding domain (RBD1) of the human U1A protein is evolutionarily designed to bind its RNA targets with great affinity and specificity. The physical mechanisms that modulate the coupling (local cooperativity) among amino acid residues on the extensive binding surface of RBD1 are investigated here, using mutants that replace a highly conserved glycine residue. This glycine residue, at the strand/loop junction of beta3/loop3, is found in U1A RBD1, and in most RBD domains, suggesting it has a specific role in modulation of RNA binding. Here, two RBD1 proteins are constructed in which that residue (Gly53) is replaced by either alanine or valine. These new proteins are shown by NMR methods and molecular dynamics simulations to be very similar to the wild-type RBD1, both in structure and in their backbone dynamics. However, RNA-binding assays show that affinity for the U1 snRNA stem-loop II RNA target is reduced by nearly 200-fold for the RBD1-G53A protein, and by 1.6 x 10(4)-fold for RBD1-G53V. The mode of RNA binding by RBD1-G53A is similar to that of RBD1-WT, displaying its characteristic non-additive free energies of base recognition and its salt-dependence. The binding mode of RBD1-G53V is altered, having lost its salt-dependence and displaying site-independence of base recognition. The molecular basis for this alteration in RNA-binding properties is proposed to result from the inability of the RNA to induce a change in the structure of the free protein to produce a high-affinity complex.     

Selective binding by the RNA binding domain of PKR revealed by affinity cleavage

The RNA-dependent protein kinase (PKR) is regulated by the binding of double-stranded RNA (dsRNA) or single-stranded RNAs with extensive duplex secondary structure. PKR has an RNA binding domain (RBD) composed of two copies of the dsRNA binding motif (dsRBM). The dsRBM is an alpha-beta-beta-beta-alpha structure present in a number of proteins that bind RNA, and the selectivity demonstrated by these proteins is currently not well understood. We have used affinity cleavage to study the binding of PKR's RBD to RNA. In this study, we site-specifically modified the first dsRBM of PKR's RBD at two different amino acid positions with the hydroxyl radical generator EDTA.Fe. Cleavage by these proteins of a synthetic stem-loop ligand of PKR indicates that PKR's dsRBMI binds the RNA in a preferred orientation, placing the loop between strands beta1 and beta2 near the single-stranded RNA loop. Additional cleavage experiments demonstrated that defects in the RNA stem, such as an A bulge and two GA mismatches, do not dictate dsRBMI's binding orientation preference. Cleavage of VA(I) RNA, an adenoviral RNA inhibitor of PKR, indicates that dsRBMI is bound near the loop of the apical stem of this RNA in the same orientation as observed with the synthetic stem-loop RNA ligands. This work, along with an NMR study of the binding of a dsRBM derived from the Drosophila protein Staufen, indicates that dsRBMs can bind stem-loop RNAs in distinct ways. In addition, the successful application of the affinity cleavage technique to localizing dsRBMI of PKR on stem-loop RNAs and defining its orientation suggests this approach could be applied to dsRBMs found in other proteins.     

Solution structure of the two N-terminal RNA-binding domains of nucleolin and NMR study of the interaction with its RNA target

Nucleolin is an abundant 70 kDa nucleolar protein involved in many aspects of ribosomal RNA biogenesis. The central region of nucleolin contains four tandem consensus RNA-binding domains (RBD). The two most N-terminal domains (RBD12) bind with nanomolar affinity to an RNA stem-loop containing the consensus sequence UCCCGA in the loop. We have determined the solution structure of nucleolin RBD12 in its free form and have studied its interaction with a 22 nt RNA stem-loop using multidimensional NMR spectroscopy. The two RBDs adopt the expected beta alpha beta beta alpha beta fold, but the position of the beta 2 strand in both domains differs from what was predicted from sequence alignments. RBD1 and RBD2 are significantly different from each others and this is likely important in their sequence specific recognition of the RNA. RBD1 has a longer alpha-helix 1 and a shorter beta 2-beta 3 loop than RBD2, and differs from most other RBDs in these respects. The two RBDs are separated by a 12 amino acid flexible linker and do not interact with one another in the free protein. This linker becomes ordered when RBD12 binds to the RNA. Analysis of the observed NOEs between the protein and the RNA indicates that both RBDs interact with the RNA loop via their beta-sheet. Each domain binds residues on one side of the loop; specifically, RBD2 contacts the 5' side and RBD1 contacts the 3'.     

Recognition of pre-formed and flexible elements of an RNA stem-loop by nucleolin

Nucleolin is an abundant nucleolar protein which is essential for ribosome biogenesis. The first two of its four tandem RNA-binding domains (RBD12) specifically recognize a stem-loop structure containing a conserved UCCCGA sequence in the loop called the nucleolin-recognition element (NRE). We have determined the structure of the consensus SELEX NRE (sNRE) by NMR spectroscopy. In both the free and bound RNA the top part of the stem forms a loop E (or S-turn) motif. In the absence of protein, the structure of the hairpin loop is not well defined due to conformational heterogeneity, and appears to be in equilibrium between two families of conformations. Titrations of RBD1, RBD2, and RBD12 with the sNRE show that specific binding requires RBD12. In complex with RBD12, the hairpin loop interacts specifically with the protein and adopts a well-defined structure which shares some of the features of the free form. The loop E motif also has specific interactions with the protein. Implications of these findings for the mechanism of recognition of RNA structures by modular proteins are discussed.     

Crosslinking of an iodo-uridine-RNA hairpin to a single site on the human U1A N-terminal RNA binding domain.

The N-terminal RNA binding domain (RBD) of the human U1A snRNP protein binds tightly and specifically to an RNA hairpin that contains a 10-nucleotide loop. The protein is one of a class of RNA binding proteins that adopts a beta alpha beta beta alpha beta global fold, which in turn forms a four-stranded antiparallel beta-sheet. This sheet forms the primary binding surface for the RNA, as shown by the crosslinking results described here, and in more detail by a recently described co-crystal of this RBD with an RNA hairpin (Oubridge C, et al., 1994, Nature 372:432-438). The RNA hairpin sequence used in the crosslinking experiments, containing 5-iodo-uridine, is a variant of the normal U1 snRNA sequence which is able to form a crosslink with the protein, in contrast to the wild-type sequence, which does not. This single uridine substitution in the 10-nucleotide loop is the site of cross-linking to one tyrosine (Tyr 13) in the beta 1 strand of the U1A N-terminal RBD. This same uridine is also crosslinked to a mutant Tyr 13 Phe RBD, at this Phe 13 substitution.     

Structure of Musashi1 in a complex with target RNA: the role of aromatic stacking interactions

Mammalian Musashi1 (Msi1) is an RNA-binding protein that regulates the translation of target mRNAs, and participates in the maintenance of cell 'stemness' and tumorigenesis. Msi1 reportedly binds to the 3'-untranslated region of mRNA of Numb, which encodes Notch inhibitor, and impedes initiation of its translation by competing with eIF4G for PABP binding, resulting in triggering of Notch signaling. Here, the mechanism by which Msi1 recognizes the target RNA sequence using its Ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2 has been revealed on identification of the minimal binding RNA for each RBD and determination of the three-dimensional structure of the RBD1:RNA complex. Unique interactions were found for the recognition of the target sequence by Msi1 RBD1: adenine is sandwiched by two phenylalanines and guanine is stacked on the tryptophan in the loop between β1 and α1. The minimal recognition sequences that we have defined for Msi1 RBD1 and RBD2 have actually been found in many Msi1 target mRNAs reported to date. The present study provides molecular clues for understanding the biology involving Musashi family proteins.     

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.     

Site-specific modification and RNA crosslinking of the RNA-binding domain of PKR

RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates and inhibits the function of the translation initiation factor eIF-2. PKR is activated in vitro by binding RNA molecules with extensive duplex structure. To further define the nature of the RNA regulation of PKR, we have prepared and characterized site-specifically modified proteins consisting of the PKR 20 kDa RNA-binding domain (RBD). Here we show that the two cysteines found naturally in this domain can be altered by site-directed mutagenesis without loss of RNA binding affinity or the RNA-regulated kinase activity. Introduction of cysteine residues at other sites in the PKR RBD allows for site-specific modification with thiol-selective reagents. PKR RBD mutants reacted selectively with a maleimide to introduce a photoactivatable crosslinking aryl azide at three different positions in the protein. RNA crosslinking efficiency was found to be dependent on the amino acid modified, suggesting differences in access to the RNA from these positions in the protein. One of the amino acid modifications that led to crosslinking of the RNA is located at a residue known to be an autophosphorylation site, suggesting that autophosphorylation at this site could influence the RNA binding properties of PKR. The PKR RBD conjugates described here and other similar reagents prepared via these methods are applicable to future studies of PKR–RNA complexes using techniques such as photocrosslinking, fluorescence resonance energy transfer and affinity cleaving.     

Molecular basis of RNA recognition by the human alternative splicing factor Fox-1

The Fox-1 protein regulates alternative splicing of tissue-specific exons by binding to GCAUG elements. Here, we report the solution structure of the Fox-1 RNA binding domain (RBD) in complex with UGCAUGU. The last three nucleotides, UGU, are recognized in a canonical way by the four-stranded beta-sheet of the RBD. In contrast, the first four nucleotides, UGCA, are bound by two loops of the protein in an unprecedented manner. Nucleotides U1, G2, and C3 are wrapped around a single phenylalanine, while G2 and A4 form a base-pair. This novel RNA binding site is independent from the beta-sheet binding interface. Surface plasmon resonance analyses were used to quantify the energetic contributions of electrostatic and hydrogen bond interactions to complex formation and support our structural findings. These results demonstrate the unusual molecular mechanism of sequence-specific RNA recognition by Fox-1, which is exceptional in its high affinity for a defined but short sequence element.     

Essential amino acids of the hantaan virus N protein in its interaction with RNA

The nucleocapsid (N) protein of hantavirus encapsidates viral genomic and antigenomic RNAs. Previously, deletion mapping identified a central, conserved region (amino acids 175 to 217) within the Hantaan virus (HTNV) N protein that interacts with a high affinity with these viral RNAs (vRNAs). To further define the boundaries of the RNA binding domain (RBD), several peptides were synthesized and examined for the ability to bind full-length S-segment vRNA. Peptide 195-217 retained 94% of the vRNA bound by the HTNV N protein, while peptides 175-186 and 205-217 bound only 1% of the vRNA. To further explore which residues were essential for binding vRNA, we performed a comprehensive mutational analysis of the amino acids in the RBD. Single and double Ala substitutions were constructed for 18 amino acids from amino acids 175 to 217 in the full-length N protein. In addition, Ala substitutions were made for the three R residues in peptide 185-217. An analysis of protein-RNA interactions by electrophoretic mobility shift assays implicated E192, Y206, and S217 as important for binding. Chemical modification experiments showed that lysine residues, but not arginine or cysteine residues, contribute to RNA binding, which agreed with bioinformatic predictions. Overall, these data implicate lysine residues dispersed from amino acids 175 to 429 of the protein and three amino acids located in the RBD as essential for RNA binding.     

Recognition of two distinct elements in the RNA substrate by the RNA-binding domain of the T. thermophilus DEAD box helicase Hera

DEAD box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the RNA substrate. The Thermus thermophilus DEAD-box helicase Hera (for “heat-resistant RNA-binding ATPase”) contains a C-terminal RNA-binding domain (RBD). We have analyzed RNA binding to the Hera RBD by a combination of mutational analyses, nuclear magnetic resonance and X-ray crystallography, and identify residues on helix α1 and the C-terminus as the main determinants for high-affinity RNA binding. A crystal structure of the RBD in complex with a single-stranded RNA resolves the RNA–protein interactions in the RBD core region around helix α1. Differences in RNA binding to the Hera RBD and to the structurally similar RBD of the Bacillus subtilis DEAD box helicase YxiN illustrate the versatility of RNA recognition motifs as RNA-binding platforms. Comparison of chemical shift perturbation patterns elicited by different RNAs, and the effect of sequence changes in the RNA on binding and unwinding show that the RBD binds a single-stranded RNA region at the core and simultaneously contacts double-stranded RNA through its C-terminal tail. The helicase core then unwinds an adjacent RNA duplex. Overall, the mode of RNA binding by Hera is consistent with a possible function as a general RNA chaperone.     

Interaction of the RNA-binding domain of the hnRNP C proteins with RNA.

The hnRNP C proteins are among the most abundant and avid pre-mRNA-binding proteins and they contain a consensus sequence RNA-binding domain (RBD) that is found in a large number of RNA-binding proteins. The interaction of the RBD of the hnRNP C proteins with an RNA oligonucleotide [r(U)8] was monitored by nuclear magnetic resonance (NMR). 15N and 13C/15N-labelled hnRNP C protein RBD was mixed with r(U)8 and one- and two-dimensional (1D and 2D) NMR spectra were recorded in a titration experiment. NMR studies of the uncomplexed 93 amino acid hnRNP C RBD (Wittekind et al., 1992) have shown that it has a compact folded structure (beta alpha beta beta alpha beta), which is typical for the RBD of this family of proteins and which is comprised of a four-stranded antiparallel beta-sheet, two alpha-helices and relatively unstructured amino- and carboxy-terminal regions. Sequential assignments of the polypeptide main-chain atoms of the hnRNP C RBD-r(U)8 complex revealed that these typical structural features are maintained in the complex, but significant perturbations of the chemical shifts of amide group atoms occur in a large number of residues. Most of these residues are in the beta-sheet region and especially in the terminal regions of the RBD. In contrast; chemical shifts of the residues of the well conserved alpha-helices, with the exception of Lys30, are not significantly perturbed. These observations localize the candidate residues of the RBD that are involved in the interaction with the RNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Defining the orientation of the human U1A RBD1 on its UTR by tethered-EDTA(Fe) cleavage.

The N-terminal RNA binding domain of the human U1A protein (RBD1) specifically binds an RNA hairpin of U1 snRNA as well as two internal loops in the 3' UTR of its own mRNA. Here, a single cysteine has been introduced into Loop 1 of RBD1, which is subsequently used to attach (EDTA-2-aminoethyl) 2-pyridyl disulfide-Fe3+ (EPD-Fe). This EDTA-Fe derivative is used to generate hydroxyl radicals to cleave the proximal RNA sugar-phosphate backbone in the RNA-RBD complexes. RBD1(K20C)-EPD-Fe cleaves the 5' strand of the RNA hairpin stem, centered four base pairs away from the base of the loop, and cleaves the UTR in two places, again centered on the 5' side of the fourth base pair from each internal loop. These data, extrapolated to the position of Lys 20 in RBD1, orient the two proteins bound to the UTR, and provide direct biochemical evidence for the proposed model of the RBD1:UTR complex.     

Functional implications for a prototypical K-turn binding protein from structural and dynamical studies of 15.5K

The kink-turn (K-turn) motif is recognized and bound by a family of proteins that act as nucleation factors for ribonucleoparticle assembly. The binding of various proteins to a conserved RNA structural motif known as the K-turn has been shown to be an important component of regulation in the ribosome, in the spliceosome, and in RNA modification. 15.5K is a prototypical example of a K-turn binding protein, which has been shown to bind the 5'-U4 stem-loop of the spliceosome and the box C/D motif. We describe the solution NMR structure of free 15.5K, as well as studies of conformational flexibility from 15N NMR relaxation and H/D exchange experiments. The protein appears well-structured aside from conformational fluctuation in alpha3. Flexibility in fast time scale motions and the observation of limited intermediate and slow motions further characterize the free protein and may suggest local contributions to recognition and binding.     

Identification of Sam68 arginine glycine-rich sequences capable of conferring nonspecific RNA binding to the GSG domain

Sam68 is an RNA-binding protein that contains a heterogeneous nuclear ribonucleoprotein K homology domain embedded in a larger RNA binding domain called the GSG (GRP33, Sam68, GLD-1) domain. This family of proteins is often referred to as the STAR (signal transduction and activators of RNA metabolism) proteins. It is not known whether Sam68 is a general nonspecific RNA-binding protein or whether it recognizes specific response elements in mRNAs with high affinity. Sam68 has been shown to bind homopolymeric RNA and a synthetic RNA sequence called G8-5 that has a core UAAA motif. Here we performed a structure function analysis of Sam68 and identified two arginine glycine (RG)-rich regions that confer nonspecific RNA binding to the Sam68 GSG domain. In addition, by using chimeric proteins between Sam68 and QKI-7, we demonstrated that one of the Sam68 RG-rich sequences of 26 amino acids was sufficient to confer homopolymeric RNA binding to the GSG domain of QKI-7, another STAR protein. Furthermore, that minimal sequence can also give QKI-7 the ability (as Sam68) to functionally substitute for HIV-1 REV to facilitate the nuclear export of RNAs. Our studies suggest that neighboring RG-rich sequences may impose nonspecific RNA binding to GSG domains. Because the Sam68 RNA binding activity is negatively regulated by tyrosine phosphorylation, our data lead us to propose that Sam68 might be a specific RNA-binding protein when tyrosine phosphorylated.     

Covariance analysis of RNA recognition motifs identifies functionally linked amino acids.

The RNA recognition motif (RRM) is one of the most common eukaryotic protein motifs. RRM sequences form a conserved globular structure known as the RNA-binding domain (RBD) or the ribonucleoprotein domain. Many proteins that contain RRM sequences bind RNA in a sequence-specific manner. To investigate the basis for the RNA-binding specificity of RRMs, we subjected 330 aligned RRM sequences to covariance analysis. The analysis revealed a single network of covariant amino acid pairs comprising the buried core of the RBD and a surface patch. Structural studies have implicated a subset of these residues in RNA binding. The covariance linkages identify a larger set of amino acid residues, including some not directly in contact with bound RNA, that may influence RNA-binding specificity.     

Characterization of multimeric complexes formed by the human PTB1 protein on RNA

Polypyrimidine tract binding protein (PTB or hnRNP I) has several known functions in eukaryotic cells, including exon exclusion during alternative splicing events, mRNA stabilization, and regulation of viral translation and replication. PTB contains four RNA Binding Domains (RBDs, or RRMs), all of which can potentially bind RNA, but their roles in the various biological functions of PTB are not clear. We investigate the properties of the complexes formed by human PTB1 on two target RNAs: the rat GABAA receptor gamma2 subunit pre-mRNA and the Hepatitis C Virus 3' NonTranslated RNA. The GABA RNA contains four polypyrimidine tracts in the intron and exon, while the HCV NTR contains a 75-nt U-rich tract and a highly structured 3'-terminus. Electrophoretic mobility shift assays show that PTB1 protein first binds to both RNAs with nanomolar affinities, but subsequent protein addition leads to formation of higher-order complexes. Stoichiometry experiments show that the ultimate complexes contain up to eight PTB1 proteins per RNA strand. Protein constructs containing two tandem RBDs also bind the two RNAs, but with different affinities and stoichiometries. Nuclease protection assays show that PTB1 protects the polypyrimidine tracts in the GABA RNA, as does a construct consisting of RBD3 and RBD4; however, a construct containing RBD1 and RBD2 enhances cleavage of bound RNA. The binding mechanisms of PTB1 are unique to the full-length protein; these modes appear to include direct association with the RNA as well as weaker intermolecular protein associations.     

Massive sequence perturbation of the Raf ras binding domain reveals relationships between sequence conservation, secondary structure propensity, hydrophobic core organization and stability

The contributions of specific residues to the delicate balance between function, stability and folding rates could be determined, in part by [corrected] comparing the sequences of structures having identical folds, but insignificant sequence homology. Recently, we have devised an experimental strategy to thoroughly explore residue substitutions consistent with a specific class of structure. Using this approach, the amino acids tolerated at virtually all residues of the c-Raf/Raf1 ras binding domain (Raf RBD), an exemplar of the common beta-grasp ubiquitin-like topology, were obtained and used to define the sequence determinants of this fold. Herein, we present analyses suggesting that more subtle sequence selection pressure, including propensity for secondary structure, the hydrophobic core organization and charge distribution are imposed on the Raf RBD sequence. Secondly, using the Gibbs free energies (DeltaG(F-U)) obtained for 51 mutants of Raf RBD, we demonstrate a strong correlation between amino acid conservation and the destabilization induced by truncating mutants. In addition, four mutants are shown to significantly stabilize Raf RBD native structure. Two of these mutations, including the well-studied R89L, are known to severely compromise binding affinity for ras. Another stabilized mutant consisted of a deletion of amino acid residues E104-K106. This deletion naturally occurs in the homologues a-Raf and b-Raf and could indicate functional divergence. Finally, the combination of mutations affecting five of 78 residues of Raf RBD results in stabilization of the structure by approximately 12 kJ mol(-1) (DeltaG(F-U) is -22 and -34 kJ mol(-1) for wt and mutant, respectively). The sequence perturbation approach combined with sequence/structure analysis of the ubiquitin-like fold provide a basis for the identification of sequence-specific requirements for function, stability and folding rate of the Raf RBD and structural analogues, highlighting the utility of conservation profiles as predictive tools of structural organization.     

RNA binding specificity of hnRNP A1: significance of hnRNP A1 high-affinity binding sites in pre-mRNA splicing.

Pre-mRNA is processed as a large complex of pre-mRNA, snRNPs and pre-mRNA binding proteins (hnRNP proteins). The significance of hnRNP proteins in mRNA biogenesis is likely to be reflected in their RNA binding properties. We have determined the RNA binding specificity of hnRNP A1 and of each of its two RNA binding domains (RBDs), by selection/amplification from pools of random sequence RNA. Unique RNA molecules were selected by hnRNP A1 and each individual RBD, suggesting that the RNA binding specificity of hnRNP A1 is the result of both RBDs acting as a single RNA binding composite. Interestingly, the consensus high-affinity hnRNP A1 binding site, UAGGGA/U, resembles the consensus sequences of vertebrate 5' and 3' splice sites. The highest affinity 'winner' sequence for hnRNP A1 contained a duplication of this sequence separated by two nucleotides, and was bound by hnRNP A1 with an apparent dissociation constant of 1 x 10(-9) M. hnRNP A1 also bound other RNA sequences, including pre-mRNA splice sites and an intron-derived sequence, but with reduced affinities, demonstrating that hnRNP A1 binds different RNA sequences with a > 100-fold range of affinities. These experiments demonstrate that hnRNP A1 is a sequence-specific RNA binding protein. UV light-induced protein-RNA crosslinking in nuclear extracts demonstrated that an oligoribonucleotide containing the A1 winner sequence can be used as a specific affinity reagent for hnRNP A1 and an unidentified 50 kDa protein. We also show that this oligoribonucleotide, as well as two others containing 5' and 3' pre-mRNA splice sites, are potent inhibitors of in vitro pre-mRNA splicing.