RNA recognition: towards identifying determinants of specificity.

Members of a family of proteins containing a conserved approximately 80-amino acid RNA recognition motif (RRM) bind specifically to a wide variety of RNA molecules. Structural studies, in combination with sequence alignments, indicate the structural context of both conserved and non-conserved elements in the motif. These analyses suggest that all RRM proteins share a common fold and a similar protein-RNA interface, and that non-conserved residues contribute additional contacts for sequence-specific RNA recognition.

A specific 31-nucleotide domain of U1 RNA directly interacts with the 70K small nuclear ribonucleoprotein component.

We have defined the nucleotide sequence of a protein-binding domain within U1 RNA that specifically recognizes and binds both to a U1 small nuclear ribonucleoprotein component (the 70K protein) and to the previously defined RNA-binding domain of the 70K protein. We have investigated direct interactions between purified U1 RNA and 70K protein by reconstitution in vitro. Thirty-one nucleotides of U1 RNA, corresponding to stem-loop I, were required for this interaction. Nucleotides at the 5' end of U1 RNA that are involved in base pairing with the 5' splice site of pre-mRNA were not required for binding. In contrast to other reports, these findings demonstrate that a specific domain of U1 RNA can bind directly to the 70K protein independently of any other snRNP-associated proteins.

A minimal spliceosomal complex A recognizes the branch site and polypyrimidine tract.

The association of U2 snRNP with the pre-mRNA branch region is a critical step in the assembly of spliceosomal complexes. We describe an assembly process that reveals both minimal requirements for formation of a U2 snRNP-substrate RNA complex, here designated the Amin complex, and specific interactions with the branch site adenosine. The substrate is a minimal RNA oligonucleotide, containing only a branch sequence and polypyrimidine tract. Interactions at the branch site adenosine and requirements for polypyrimidine tract-binding proteins for the Amin complex are the same as those of authentic prespliceosome complex A. Surprisingly, Amin complex formation does not require U1 snRNP or ATP, suggesting that these factors are not necessary for stable binding of U2 snRNP per se, but rather are necessary for accessibility of components on longer RNA substrates. Furthermore, there is an ATP-dependent activity that releases or destabilizes U2 snRNP from branch sequences. The simplicity of the Amin complex will facilitate a detailed understanding of the assembly of prespliceosomes.

A human autoimmune protein associated with U1 RNA contains a region of homology that is cross-reactive with retroviral p30gag antigen.

cDNA encoding a 70 kd protein (70K) associated with U1 small nuclear ribonucleoprotein (snRNP) was cloned from a human brain-stem library using autoantibodies from patients with connective tissue disease. The cDNA-derived amino acid sequence contains 23 residues homologous to a region of murine leukemia virus group-specific antigen p30gag. The homology residues in an antigenic portion of the 70K protein and is defined by a core consensus sequence, ETPEEREERRR, that occurs as a tandem repeat in p30gag of most mammalian type C retroviruses. Anti-p30gag antibodies recognized a recombinant 70K-LacZ fusion protein as well as U1 snRNPs. Using synthetic peptides as competitors, we demonstrated that the region of homology encompasses the site of immunological cross-reactivity. Thus autoantibodies against U1 snRNPs were elicited by immunization with p30gag. On the basis of these findings, we suggest a role for retroviruses in the initiation of autoimmunity.

The ATP requirement for U2 snRNP addition is linked to the pre-mRNA region 5' to the branch site.

Association of U2 snRNP with the pre-mRNA branch region is the first ATP-dependent step in spliceosome assembly. The basis of this energy dependence is not known. Previously, we identified minimal intron-derived substrates that form complexes with U2 independent of ATP. Here, we identify the intron region linked to the ATP dependence of this step by comparing these substrates to longer RNAs that recapitulate the ATP requirement. This region needed to impose ATP dependence lies immediately 5' to the branch site. Sequences ranging from 6 to 14 nt yield a near linear inhibitory effect on efficiency of complex formation with U2 snRNP, with 18 nt yielding near maximal ATP dependence. This region is not protected prior to U2 addition, and RNase H targeting of the region within nuclear extract converts an ATP-dependent substrate into an ATP-independent one. Within this region, there is no sequence specificity linked with the ATP requirement, as neither a specific sequence is needed, nor even nucleobases. These data and the results of other modifications suggest models in which the 18-nt region is a target for interactions with U2 snRNP in an ATP-bound or -activated conformation.

A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein.

We have defined the RNA binding domain of the 70K protein component of the U1 small nuclear ribonucleoprotein to a region of 111 amino acids. This domain encompasses an octamer sequence that has been observed in other proteins associated with RNA, but has not previously been shown to bind directly to a specific RNA sequence. Within the U1 RNA binding domain, an 80 amino acid consensus sequence that is conserved in many presumed RNA binding proteins was discerned. This sequence pattern appears to represent an RNA recognition motif (RRM) characteristic of a distinct family of proteins. By site-directed mutagenesis, we determined that the 70K protein consists of 437 amino acids (52 kd), and found that its aberrant electrophoretic migration is due to a carboxy-terminal charged domain structurally similar to two Drosophila proteins (su(wa) and tra) that may regulate alternative pre-messenger RNA splicing.

The branch site adenosine is recognized differently for the two steps of pre-mRNA splicing.

The functional groups responsible for branch site identity in the two steps of pre-mRNA splicing as well as for spliceosome assembly were tested by incorporation of site-specific modifications at the branch site of a pre-mRNA. These results show that recognition of the adenosine occurs early in complex formation and that the branch site adenosine is recognized differently before the first step and for the second step. Further, direct UV cross-linking with these modified RNAs was used to identify a factor whose interaction was dependent upon the identity of the branch site nucleotide.

Three recognition events at the branch-site adenine.

An adenosine at the branch site, the nucleophile for the first transesterification step of splicing, is nearly invariant in mammalian pre-mRNA introns. The chemical groups on the adenine base were varied systematically and assayed for formation of early spliceosome complexes and execution of the first and second steps of splicing. Recognition of constituents of the adenine is critical in formation of a U2 snRNP-containing complex on a minimal branch-site oligonucleotide. Furthermore, the efficiencies of the first and second chemical steps have different dependencies on the functional groups of the adenine. In total, the chemical groups on the adenine base at the branch site are differentially recognized during at least three different processes in the splicing of pre-mRNA. Moreover, a protein, p14, interacts with the adenine in a base-specific fashion and may mediate early recognition of this base.

Branch nucleophile selection in pre-mRNA splicing: evidence for the bulged duplex model.

Selection of the nucleophile for the first step of nuclear pre-mRNA splicing was probed by site-specific incorporation into splicing substrates of nucleotides modified at the 2' position. The differing abilities of ribose, 2'-deoxyribose, and arabinose nucleotides to base-pair within an RNA.RNA duplex and to contribute a nucleophilic 2'-OH group were exploited to analyze the paired/unpaired disposition of the branch site nucleotide. The results provide direct evidence for a bulged duplex model in which either of two adjacent purines within the consensus branch site sequence may shift into a bulged position and contribute the 2'-OH group for the first step of splicing. Furthermore, the presence of a consensus branch site that cannot present a reactive nucleophile suppresses splicing, including the use of cryptic branch sites elsewhere. We conclude that the branch site region base-pairing with U2 snRNA determines the first step nucleophile and persists at the time of the first transesterification reaction.

Quantitative determination that one of two potential RNA-binding domains of the A protein component of the U1 small nuclear ribonucleoprotein complex binds with high affinity to stem-loop II of U1 RNA.

Many RNA-associated proteins contain a ribonucleoprotein (RNP) consensus octamer encompassed by a conserved 80 amino acid sequence, which we have termed an RNA recognition motif (RRM). RRM family members contain either one (class I) or multiple (class II) copies of this motif. We report here that a class II component of the U1 small nuclear RNP (snRNP), the A protein of U1 snRNP (U1snRNP-A), contains two RRMs (RRM1 and -2), yet has only one binding domain (RRM1) that interacts specifically with stem-loop II of U1 RNA. Quantitative analysis of binding affinities of fragments of U1snRNP-A demonstrated that an 86-amino acid polypeptide was competent to bind to U1 RNA with an affinity comparable to that of the full-length protein (Kd approximately 80 nM). The carboxyl-terminal RRM2 of U1snRNP-A did not bind to U1 RNA and may recognize an unidentified heterologous RNA. We propose that class II proteins may function as bridges between RNA components of RNP complexes such as the spliceosome.

RNA-binding domain of the A protein component of the U1 small nuclear ribonucleoprotein analyzed by NMR spectroscopy is structurally similar to ribosomal proteins.

An RNA recognition motif (RRM) of approximately 80 amino acids constitutes the core of RNA-binding domains found in a large family of proteins involved in RNA processing. The U1 RNA-binding domain of the A protein component of the human U1 small nuclear ribonucleoprotein (RNP), which encompasses the RRM sequence, was analyzed by using NMR spectroscopy. The domain of the A protein is a highly stable monomer in solution consisting of four antiparallel beta-strands and two alpha-helices. The highly conserved RNP1 and RNP2 consensus sequences, containing residues previously suggested to be involved in nucleic acid binding, are juxtaposed in adjacent beta-strands. Conserved aromatic side chains that are critical for RNA binding are clustered on the surface of the molecule adjacent to a variable loop that influences recognition of specific RNA sequences. The secondary structure and topology of the RRM are similar to those of ribosomal proteins L12 and L30, suggesting a distant evolutionary relationship between these two types of RNA-associated proteins.

Dynamic association of proteins with the pre-mRNA branch region.

The association of proteins with the branch site region during pre-mRNA splicing was probed using a novel methodology to site-specifically modify the pre-mRNA with the photo-reagent benzophenone. Three sets of proteins were distinguished by the kinetics of their associations with pre-mRNAs, by their association with discrete splicing complexes, and by their differing factor requirements. An early U1 snRNP-dependent cross-link of the branch region to a p80 species was followed by cross-links to p14, p35, and p150 polypeptides associated with the U2 snRNP-pre-mRNA complex. Concomitant with formation of the spliceosome, a rearrangement of protein factors about the branch region occurred, in which the p35 and p150 cross-links were replaced by p220 and p70 species. These results establish that the branch region is recognized in a dynamic fashion by multiple distinct proteins during the course of spliceosomal assembly.

A novel U2 and U11/U12 snRNP protein that associates with the pre-mRNA branch site.

Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa protein (p14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this protein by microsequencing a 14 kDa protein isolated from U2-type spliceosomes. This protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded protein precipitated the 14 kDa protein cross-linked to the branch adenosine, confirming the identity of the p14 cDNA. A combination of immunoblotting, protein microsequencing and immunoprecipitation revealed that p14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2- and U12-type pre-mRNAs, respectively. p14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immuno precipitations indicated that p14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.

Expression of autoantibodies to recombinant (U1) RNP-associated 70K antigen in systemic lupus erythematosus.

To determine the specificity of antibodies to the (U1) ribonucleoprotein antigen in systemic lupus erythematosus (SLE), patient sera were tested for binding to a recombinant human 70K antigen. By solid-phase immunoassay, we detected anti-70K reactivity in sera from 31 of 96 patients with systemic lupus erythematosus (SLE), demonstrating that anti-70K antibodies may occur in patients with SLE as well as other clinical diagnoses. In sequential sera from 2 of these patients, we found that anti-70K binding varied dramatically over the course of disease. The changes in anti-70K antibody levels did not correlate with clinical events nor evolving antibody reactivity with the Sm-specific antigens.