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".
The spliceosomal proteins U1A and U2B" each use a homologous RRM domain to bind specifically to their respective snRNA targets, U1hpll and U2hpIV, two stem-loops that are similar yet distinct in sequence. Previous studies have shown that binding of U2B" to U2hpIV is facilitated by the ancillary protein U2A', whereas specific binding of U1A to U1hpll requires no cofactor. Here we report that U2A' enables U2B" to distinguish the loop sequence of U2hpIV from that of U1hpll but plays no role in stem sequence discrimination. Although U2A' can also promote heterospecific binding of U1A to U2hpIV, a much higher concentration of the ancillary protein is required due to the approximately 500-fold greater affinity of U2A' for U2B". Additional experiments have identified a single leucine residue in U1A(Leu-44) that is critical for the intrinsic specificity of this protein for the loop sequence of U1 hpll in preference to that of U2hpIV. Our data suggest that most of the difference in RNA-binding specificity between U1A and U2B" can be accounted for by this leucine residue and by the contribution of the ancillary protein U2A' to the specificity of U2B".
Rimmele
M E
ME
Skirball Institute of Biomolecular Medicine and Department of Microbiology, New York University School of Medicine, New York 10016, USA.
Belasco
J G
JG
D000595:Amino Acid Sequence; D001324:Autoantigens; D001483:Base Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007930:Leucine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010641:Phenotype; D012342:RNA, Small Nuclear / Q000096:biosynthesis / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology / Q000502:physiology*; D017411:Ribonucleoproteins, Small Nuclear; D017381:Spliceosomes / Q000378:metabolism*; D013816:Thermodynamics; D055517:snRNP Core Proteins
D001324:Autoantigens; D007930:Leucine; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D017413:Ribonucleoprotein, U2 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D055517:snRNP Core Proteins; C526408:SNRPB2 protein, human; C075339:U1A protein; C484318:SNRPA1 protein, human