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

Abstract

We have defined the nucleotide sequence of a protein-binding domain within Ul RNA that specifically recognizes and binds both to a Ul 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 Ul RNA and 70K protein by reconstitution in vitro. Thirty-one nucleotides of Ul RNA, corresponding to stem-loop I, were required for this interaction. Nucleotides at the 5' end of Ul 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 Ul RNA can bind directly to the 70K protein independently of any other snRNP-associated proteins.The 52-kilodalton Ul RNA-associated protein (the 70K protein) is a component of the Ul small nuclear ribonucleoprotein (snRNP) complex. Several snRNPs have been implicated in the removal of intervening sequences during precursor mRNA splicing (see references 11, 25, and 41 for reviews). The Ul snRNP, in particular, has been shown to interact with the 5' splice site, at least in part by base pairing of this site with the 5'-terminal 10 nucleotides (nt) of Ul RNA (1,5,18,29,45). The human Ul snRNP is composed of 165 nt of Ul RNA and two classes of proteins: Ul-specific proteins (70K, A, and C) and the Sm complex (consisting of six U snRNP-common proteins) (9, 22). Although these proteins probably play important structural roles in snRNPs and at least some are required for the interaction of Ul snRNP with 5' splice sites (29), none have been assigned specific functions.Previous studies of the RNA-binding properties of the Ul snRNP-specific proteins have involved indirect methods including immunoprecipitation of nuclease-treated cell extracts (34) and microinjection of RNA into Xenopus oocytes (12). These studies suggested that the 70K protein, as well as the A protein and perhaps the C protein, associates with the 5' half of Ul RNA, in particular, stem-loop I. In addition, weak requirements were implicated for regions of stemloops II, III, and IV (12,34 purified human Ul RNA and 70K protein, we have investigated the direct interaction of these two components of the Ul snRNP. Using various binding methods, we maintained the specificity of the Ul RNA-70K reconstitution through progressive deletion of both components. We previously defined the RNA-binding domain of the 70K protein required for this interaction (35). In this study, we report that both the 70K protein and the RNA-binding domain alone are directly recognized by 31 nt of Ul RNA that make up the 5'-most stem-loop structure. The A, C, and Sm proteins were not required for this recognition and binding. Further deletion of the 31-nt structure resulted in reduced efficiency of reconstitution, thus defining the minimal RNA domain needed for recognition of the 70K protein. MATERIALS AND METHODS

Systematic analysis and evolution of 5S ribosomal DNA in metazoans

“…Green motifs were used as additional filters in some species (H. sapiens, P. pygmaeus, M. mulatta, B. taurus, P. vampyrus and S. kowalevskii) because the number of candidates was too large. Whereas function of U1 boxes are diverse (Box A recognizes 5 0 -splice site in mRNA precursors (Zhuang and Weiner, 1986, and references therein); Box B is part of the U1-70K protein-binding site (Query et al, 1989); Box C is part of the U1-A protein-binding region (Scherly et al, 1989) and the Sm protein-binding region, named 'domain A' (Branlant et al, 1982)), those 5S rRNA boxes are essential for transcription of 5S rRNA itself Bogenhagen, 1993;Hall, 2005). A full color version of this figure is available at the Heredity journal online.…”

Structure and expression of the Drosophila melanogaster gene for the U1 small nuclear ribonucleoprotein particle 70K protein.

“…protein makes contacts with Ul snRNA at the first stem-loop (20,41) in a sequence-specific manner (46,56). The carboxyterminal portion of the protein is particularly rich in arginine residues and contains repetitions of arginine-glutamic acid, arginine-aspartic acid, and arginine-serine.…”

“…We have followed others (15,46) in using Ul 70K or 70K as a name for this protein, although this name says nothing about the size of the protein. A conserved region of amino acid sequence found in a number of RNAbinding proteins (1,3,33,57) occurs in the amino-terminal half of the human Ul 70K protein at amino acids 104 to 183, and this region of the protein together with a minimum of flanking amino acids (amino acids 92 to 202) is capable of binding Ul snRNA specifically (45).…”

“…We have followed others (15, 46) in using Ul 70K or 70K as a name for this protein, although this name says nothing about the size of the protein. A conserved region of amino acid sequence found in a number of RNAbinding proteins (1,3,33,57) (46,56). The carboxyterminal portion of the protein is particularly rich in arginine residues and contains repetitions of arginine-glutamic acid, arginine-aspartic acid, and arginine-serine.…”

Autoantigenic epitopes of the b polypeptide of SM small nuclear RNP particles

“…In contrast, other regions such as the epitope BSm-' are exposed on all Sm snRNPs. Since the U1 and U2 snRNPs contain unique proteins which have different binding sites on their RNAs (17)(18)(19), and since the other abundant Sm snRNPs also contain unique proteins (5,6), it would not be surprising that differences in the array of these proteins could render one or more of the epitopes of B available for antibody binding only within the Ul snRNP.…”