{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T10:53:22Z","timestamp":1790074402077,"version":"4.0.1"},"reference-count":55,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2007,12,12]],"date-time":"2007-12-12T00:00:00Z","timestamp":1197417600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Proteins"],"published-print":{"date-parts":[[2008,6]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>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 \u03b21\u2010\u03b11\u2010\u03b22\u2010\u03b23\u2010\u03b12\u2010\u03b24 of p14, and \u03b1A\u2010\u03b2A fold of the SF3b155 fragment), we identified three important features revealed by the NMR solution structure. First, the C\u2010terminal extension and the nuclear localization signal of p14 (\u03b13 and \u03b14 in the crystal structure, respectively) were dispensable for the complex formation. Second, the proline\u2010rich segment of SF3b155, following \u03b2A, closely approaches p14. Third, interestingly, the \u03b21\u2010\u03b11 loop and the \u03b12\u2010\u03b24 \u03b2\u2010hairpin form a positively charged groove. Extensive mutagenesis analyses revealed the functional relevance of the residues involved in the protein\u2013protein 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\u2010shift\u2010perturbation experiments, not only for the main\u2010chain but also for the side\u2010chain resonances, for several p14\u2010SF3b155 complex constructs upon binding to RNA. These analyses identified a positively charged groove and the C\u2010terminal extension of p14 as RNA\u2010binding sites. Strikingly, an aromatic residue in the \u03b21\u2010\u03b11 loop, Tyr28, and a positively charged residue in the \u03b12\u2010\u03b24 \u03b2\u2010hairpin, Agr85, are critical for the RNA\u2010binding activity of the positively charged groove. The Tyr28Ala and Arg85Ala point mutants and a deletion mutant of the C\u2010terminal extension clearly revealed that their RNA binding activities were independent of each other. Collectively, this study provides details for the protein\u2010recognition mode of p14 and insight into the branch site recognition. Proteins 2008. \u00a9 2007 Wiley\u2010Liss, Inc.<\/jats:p>","DOI":"10.1002\/prot.21839","type":"journal-article","created":{"date-parts":[[2007,12,12]],"date-time":"2007-12-12T18:17:37Z","timestamp":1197483457000},"page":"1617-1636","source":"Crossref","is-referenced-by-count":16,"title":["Complex assembly mechanism and an RNA\u2010binding mode of the human p14\u2010SF3b155 spliceosomal protein complex identified by NMR solution structure and functional analyses"],"prefix":"10.1002","volume":"71","author":[{"given":"Kanako","family":"Kuwasako","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naoshi","family":"Dohmae","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mio","family":"Inoue","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mikako","family":"Shirouzu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seiichi","family":"Taguchi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter","family":"G\u00fcntert","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bertrand","family":"S\u00e9raphin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yutaka","family":"Muto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shigeyuki","family":"Yokoyama","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2007,12,12]]},"reference":[{"key":"e_1_2_7_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)80925-3"},{"key":"e_1_2_7_3_2","first-page":"525","volume-title":"The 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