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.
Department of Microbiology and Immunology, Duke University Medical Center, Durham, NC 27710.
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Lutz-Freyermuth
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D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D015723:Gene Library; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D009154:Mutation; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes; D014176:Protein Biosynthesis; D012342:RNA, Small Nuclear / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear; D014158:Transcription, Genetic
D000939:Epitopes; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear