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

Abstract

An RNA recognition motif (RRM) of '480 amino acids constitutes the core of RNA-binding domains found in a large family of proteins involved in RNA processing.The Ul RNA-binding domain ofthe A protein component ofthe human Ul 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 f-strands and two a-helices. The highly conserved RNP1 and RNP2 consensus sequences, containing residues previously suggested to be involved in nucleic acid binding, are juxtaposed in adjacent f-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.Over the past decade, the ways in which proteins interact specifically and nonspecifically with DNA have been revealed by biochemical and biophysical studies (reviewed in refs. 1 and 2). Much of this information has been the result of crystallographic analysis, but more recently, NMR techniques have been used (3). As regards RNA-binding proteins, however, there has been little structural information with the notable exception of the work by Steitz and coworkers (4) on the glutaminyl-tRNA synthetase/tRNAGIn complex.Sequence elements characteristic of a group of RNAassociated proteins began to emerge with the observation of four copies of an 80-amino acid repeat in poly(A)-binding protein (5). The most conserved region of 8 amino acids in these repeats was termed the ribonucleoprotein (RNP) consensus sequence, or octamer (RNP1) (6). Another 6-amino acid region was later termed RNP2 (7). It was subsequently shown by several groups that the sequence similarity appears in many other RNA-associated proteins (7-11) (recently reviewed in ref. 12), contains many conserved positions, and is notably rich in aromatic residues (8). Direct evidence of an interaction between this motif and RNA has led to its designation as an RNA recognition motif, or RRM (8). This motif has also been referred to as an RNP consensus sequence-type RNA binding domain (11) and as an RNP-80 motif (13). Members of the RRM-containing family of proteins include heterogeneous nuclear RNP proteins and U-class small nuclear RNP (snRNP) proteins, as well as poly(A)-binding protein, nucleolin, transcription termination factors La and rho, eukaryotic initiation factor 4B, and sexdetermination proteins in Drosophila. The sources of these proteins range from Escherichia coli to man; thus, the RRM is clearly an ancient protein structure.The RRM can be viewed as analogous to the homeobox domain (5) or to the zinc-binding finger motif (14) of DNAbinding proteins. In many cases, the RRM is a modular component ofa larger protein; other m...

The hnRNP F protein: unique primary structure, nucleic acid-binding properties, and subcellular localization

“…NMR and crystallography studies have shown that the RNP-CS RBD has a (31-aI-32-33-a2--34 (3 = 3-sheet; ai = a-helix) structure (18,21,22). In addition to RNP1 and RNP2, these structural studies have revealed a number of other conserved residues that contribute to the hydrophobic core of the RBD (16).…”

“…A common feature of most of the hnRNP proteins cloned and sequenced to date (with the exceptions of hnRNP K and hnRNP U) is an evolutionarily conserved 80-100 amino acid RNA-binding domain (RBD), the RNP-consensus sequence (RNP-CS), also referred to as RNP motif and RNA-recognition motif (14)(15)(16). The structure of the RBD, and experimental evidence that it functions as an RNA-binding domain, has been presented for the hnRNP Cl protein (17,18) and for the Ul snRNP A protein (19)(20)(21)(22). Significantly, RNA appears to bind to the surface of the RBD and, therefore, remains exposed and accessible for interaction with other factors (17).…”

Target discrimination by RNA-binding proteins: Role of the ancillary protein U2A′ and a critical leucine residue in differentiating the RNA-binding specificity of spliceosomal proteins U1A and U2B′′

“…RNA-binding proteins (RNA-BPs) play a key role in a variety of cellular regulatory processes+ Crucial to the function of this important class of proteins is their RNA target specificity+ Only through their ability to distinguish between closely related RNA elements can these proteins act with the requisite selectivity+ Most RNA-BPs can be categorized into families on the basis of shared sequence motifs in their RNAbinding domains (reviewed by Mattaj, 1993;Burd & Dreyfuss, 1994)+ The largest of these is the RRM (RNA recognition motif) family of RNA-BPs, also referred to as the RNP family+ RRM proteins are characterized by the presence of one or more structurally related RNAbinding domains, each comprising 90-100 amino acid residues and containing two conserved sequence motifs (RNP-1 and RNP-2)+ Members of this protein family occur in all types of organisms and bind to RNA targets that vary in sequence and secondary structure+ A paradigm for the RRM family is the spliceosomal protein U1A, a component of the U1 small nuclear ribonucleoprotein complex (U1 snRNP)+ U1A uses its amino-terminal RRM domain to bind hairpin II of U1 snRNA (U1hpII) (Scherly et al+, 1989;Lutz-Freyermuth et al+, 1990)+ The three-dimensional structure of this U1A domain has been solved in both the absence and presence of bound RNA (Nagai et al+, 1990;Hoffman et al+, 1991;Howe et al+, 1994;Oubridge et al+, 1994)+ It consists of a four-stranded b-sheet (the RNA-binding surface) supported on one side by two a-helices+ The conserved RNP motifs lie on the two central b-strands (b1 and b3; see Fig+ 1)+ It has been proposed that these conserved sequences provide basal RNA-binding activity, while the variable regions surrounding them are thought to determine binding specificity (Scherly et al+, 1990a;Bentley & Keene, 1991)+ Much has been learned about the specificity of RRM proteins by comparing U1A to the closely related spliceosomal protein U2B0, a U2 snRNP component that binds hairpin IV of U2 snRNA (U2hpIV)+ Despite being 75% identical and 94% similar in sequence (Fig+ 1), the amino-terminal RRM domains of U1A and U2B0 bind their respective RNA targets selectively and with little cross-reactivity (Scherly et al+, 1990a;Bentley & Keene, 1991;Scherly et al+, 1991)+ However, in contrast to the high intrinsic affinity of U1A for U1hpII, U2B0 requires help from an ancillary snRNP protein, U2A9, to bind tightly to U2hpIV (Scherly et al+, 1990a;Scherly et al+, 1990b;Bentley & Keene, 1991;Boelens et al+, 1991)+ The target RNAs U1hpII and U2hpIV also resemble one another (Fig+ 1), but important sequence differences in both their loop and stem regions allow these hairpins to be distinguished by U1A and U2B0 (Scherly et al+, 1990a;<...>…”

Molecular cloning of a RNA binding protein, S1-1

“…Core amino acid residues of the consensus RNP motif as well as the positions of the submotifs (RNP1 and RNP2) are listed above and below. The secondary structures in the RNP motifs (α-helices, β-sheets, loops and tight turns TI-1 and TI-2) have been characterized (40)(41)(42) and reviewed (25,26,43). These regions were cited for reference.…”