ABSTRACT
The spliceosomal protein p14, a component of the SF3b complex in the U2 small nuclear ribonucleoprotein (snRNP), is essential for the U2 snRNP to recognize the branch site adenosine. The elucidation of the dynamic process of the splicing machinery rearrangement awaited the solution structural information. We identified a suitable complex of human p14 and the SF3b155 fragment for the determination of its solution structure by NMR. In addition to the overall structure of the complex, which was recently reported in a crystallographic study (typical RNA recognition motif fold beta1-alpha1-beta2-beta3-alpha2-beta4 of p14, and alphaA-betaA fold of the SF3b155 fragment), we identified three important features revealed by the NMR solution structure. First, the C-terminal extension and the nuclear localization signal of p14 (alpha3 and alpha4 in the crystal structure, respectively) were dispensable for the complex formation. Second, the proline-rich segment of SF3b155, following betaA, closely approaches p14. Third, interestingly, the beta1-alpha1 loop and the alpha2-beta4 beta-hairpin form a positively charged groove. Extensive mutagenesis analyses revealed the functional relevance of the residues involved in the protein-protein interactions: two aromatic residues of SF3b155 (Phe408 and Tyr412) play crucial roles in the complex formation, and two hydrophobic residues (Val414 and Leu415) in SF3b 155 serve as an anchor for the complex formation, by cooperating with the aromatic residues. These findings clearly led to the conclusion that SFb155 binds to p14 with three contact points, involving Phe408, Tyr412, and Val414/Leu415. Furthermore, to dissect the interactions between p14 and the branch site RNA, we performed chemical-shift-perturbation experiments, not only for the main-chain but also for the side-chain resonances, for several p14-SF3b155 complex constructs upon binding to RNA. These analyses identified a positively charged groove and the C-terminal extension of p14 as RNA-binding sites. Strikingly, an aromatic residue in the beta1-alpha1 loop, Tyr28, and a positively charged residue in the alpha2-beta4 beta-hairpin, Agr85, are critical for the RNA-binding activity of the positively charged groove. The Tyr28Ala and Arg85Ala point mutants and a deletion mutant of the C-terminal extension clearly revealed that their RNA binding activities were independent of each other. Collectively, this study provides details for the protein-recognition mode of p14 and insight into the branch site recognition.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Ribonucleoprotein, U2 Small Nuclear/chemistry , Ribonucleoprotein, U2 Small Nuclear/metabolism , Ribonucleoproteins/chemistry , Ribonucleoproteins/metabolism , Spliceosomes/chemistry , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Cell Nucleus/chemistry , DNA, Complementary/chemistry , Glutathione Transferase/metabolism , Histidine/metabolism , Humans , Hydrolysis , Hydrophobic and Hydrophilic Interactions , Leucine/chemistry , Models, Molecular , Molecular Sequence Data , Nuclear Localization Signals/chemistry , Phenylalanine/metabolism , Phosphoproteins/genetics , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , RNA Splicing , RNA Splicing Factors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ribonucleoprotein, U2 Small Nuclear/genetics , Ribonucleoproteins/genetics , Sequence Homology, Amino Acid , Spliceosomes/metabolism , Trypsin/pharmacology , Tyrosine/metabolism , Valine/chemistryABSTRACT
None of the mammalian splicing factors that have been cloned corresponds to the yeast pre-messenger RNA splicing factors, the PRP proteins. Here, a generalizable strategy was used to isolate a complementary DNA encoding the mammalian spliceosome-associated protein (SAP) SAP 62. It is demonstrated that SAP 62 is the likely functional homolog of the yeast PRP11 protein. Both PRP11 and SAP 62 associate stably with the spliceosome, contain a single zinc finger, and display significant amino acid sequence similarity. Unlike PRP11, SAP 62 contains 22 proline-rich heptapeptide repeats at the carboxyl-terminus.