{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,17]],"date-time":"2026-01-17T10:54:47Z","timestamp":1768647287572,"version":"3.49.0"},"reference-count":45,"publisher":"Public Library of Science (PLoS)","issue":"11","license":[{"start":{"date-parts":[[2020,11,30]],"date-time":"2020-11-30T00:00:00Z","timestamp":1606694400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000048","name":"American Cancer Society","doi-asserted-by":"publisher","award":["RSG-0-222-01"],"award-info":[{"award-number":["RSG-0-222-01"]}],"id":[{"id":"10.13039\/100000048","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["1S10RR023748-01"],"award-info":[{"award-number":["1S10RR023748-01"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the <jats:italic>AdML<\/jats:italic> 3' splice site, <jats:italic>AdML<\/jats:italic>3\u2019). We found that the PUF60\u2013<jats:italic>AdML<\/jats:italic>3\u2019 dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60\u2019s two central RNA recognition motifs (RRMs). A 1.95 \u00c5 crystal structure of the two PUF60 RRMs in complex with <jats:italic>AdML<\/jats:italic>3\u2019 revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV\/Vis spectroscopy suggested a potential 2:1 (PUF60<jats:sub>2<\/jats:sub>:<jats:italic>AdML<\/jats:italic>3\u2019) stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3\u2019 splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3\u2019 splice sites.<\/jats:p>","DOI":"10.1371\/journal.pone.0242725","type":"journal-article","created":{"date-parts":[[2020,11,30]],"date-time":"2020-11-30T13:43:17Z","timestamp":1606743797000},"page":"e0242725","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":6,"title":["Unraveling the mechanism of recognition of the 3\u2019 splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60"],"prefix":"10.1371","volume":"15","author":[{"given":"Hsin-hao T.","family":"Hsiao","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gregg V.","family":"Crichlow","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"James W.","family":"Murphy","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ewa J.","family":"Folta-Stogniew","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elias J.","family":"Lolis","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0541-4689","authenticated-orcid":true,"given":"Demetrios T.","family":"Braddock","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2020,11,30]]},"reference":[{"issue":"1","key":"pone.0242725.ref001","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1038\/nrg2673","article-title":"RNA processing and its regulation: global insights into biological networks","volume":"11","author":"DD Licatalosi","year":"2010","journal-title":"Nat Rev Genet"},{"issue":"8","key":"pone.0242725.ref002","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1016\/S0968-0004(00)01604-2","article-title":"Alternative pre-mRNA splicing: the logic of combinatorial control","volume":"25","author":"CW Smith","year":"2000","journal-title":"Trends Biochem Sci"},{"key":"pone.0242725.ref003","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1146\/annurev.biochem.72.121801.161720","article-title":"Mechanisms of alternative pre-messenger RNA splicing","volume":"72","author":"DL Black","year":"2003","journal-title":"Annu Rev Biochem"},{"issue":"11","key":"pone.0242725.ref004","doi-asserted-by":"crossref","first-page":"741","DOI":"10.1038\/nrm2777","article-title":"Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches","volume":"10","author":"M Chen","year":"2009","journal-title":"Nat Rev Mol Cell Biol"},{"issue":"8","key":"pone.0242725.ref005","doi-asserted-by":"crossref","first-page":"810","DOI":"10.1038\/embor.2009.170","article-title":"Missed threads. The impact of pre-mRNA splicing defects on clinical practice","volume":"10","author":"D Baralle","year":"2009","journal-title":"EMBO Rep"},{"issue":"2","key":"pone.0242725.ref006","first-page":"108","article-title":"Splicing therapeutics in SMN2 and APOB","volume":"11","author":"B Khoo","year":"2009","journal-title":"Curr Opin Mol Ther"},{"issue":"6361","key":"pone.0242725.ref007","doi-asserted-by":"crossref","first-page":"609","DOI":"10.1038\/355609a0","article-title":"Cloning and domain structure of the mammalian splicing factor U2AF","volume":"355","author":"PD Zamore","year":"1992","journal-title":"Nature"},{"issue":"6","key":"pone.0242725.ref008","doi-asserted-by":"crossref","first-page":"858","DOI":"10.1101\/gad.12.6.858","article-title":"A cooperative interaction between U2AF65 and mBBP\/SF1 facilitates branchpoint region recognition","volume":"12","author":"JA Berglund","year":"1998","journal-title":"Genes Dev"},{"issue":"50","key":"pone.0242725.ref009","doi-asserted-by":"crossref","first-page":"50572","DOI":"10.1074\/jbc.M307976200","article-title":"Structuring of the 3' splice site by U2AF65","volume":"278","author":"OA Kent","year":"2003","journal-title":"J Biol Chem"},{"issue":"22","key":"pone.0242725.ref010","doi-asserted-by":"crossref","first-page":"7673","DOI":"10.1128\/MCB.21.22.7673-7681.2001","article-title":"Dual function for U2AF(35) in AG-dependent pre-mRNA splicing","volume":"21","author":"S Guth","year":"2001","journal-title":"Mol Cell Biol"},{"issue":"1","key":"pone.0242725.ref011","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.molcel.2006.05.025","article-title":"Structural basis for polypyrimidine tract recognition by the essential pre-mRNA splicing factor U2AF65","volume":"23","author":"EA Sickmier","year":"2006","journal-title":"Mol Cell"},{"issue":"12","key":"pone.0242725.ref012","doi-asserted-by":"crossref","first-page":"1548","DOI":"10.1017\/S1355838299991938","article-title":"PUF60: a novel U2AF65-related splicing activity","volume":"5","author":"PS Page-McCaw","year":"1999","journal-title":"RNA"},{"issue":"13","key":"pone.0242725.ref013","doi-asserted-by":"crossref","first-page":"4002","DOI":"10.1046\/j.1432-1327.2000.01431.x","article-title":"A trans-acting factor, isolated by the three-hybrid system, that influences alternative splicing of the amyloid precursor protein minigene","volume":"267","author":"A Poleev","year":"2000","journal-title":"Eur J Biochem"},{"issue":"6","key":"pone.0242725.ref014","doi-asserted-by":"crossref","first-page":"e538","DOI":"10.1371\/journal.pone.0000538","article-title":"Control of pre-mRNA splicing by the general splicing factors PUF60 and U2AF(65)","volume":"2","author":"ML Hastings","year":"2007","journal-title":"PLoS One"},{"issue":"3","key":"pone.0242725.ref015","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1016\/S1534-5807(02)00128-4","article-title":"Half pint regulates alternative splice site selection in Drosophila","volume":"2","author":"C Van Buskirk","year":"2002","journal-title":"Dev Cell"},{"issue":"5","key":"pone.0242725.ref016","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1016\/j.ejmg.2009.05.006","article-title":"An 8.35 Mb overlapping interstitial deletion of 8q24 in two patients with coloboma, congenital heart defect, limb abnormalities, psychomotor retardation and convulsions","volume":"52","author":"JB Verheij","year":"2009","journal-title":"Eur J Med Genet"},{"issue":"5","key":"pone.0242725.ref017","doi-asserted-by":"crossref","first-page":"798","DOI":"10.1016\/j.ajhg.2013.09.010","article-title":"SCRIB and PUF60 are primary drivers of the multisystemic phenotypes of the 8q24.3 copy-number variant","volume":"93","author":"A Dauber","year":"2013","journal-title":"Am J Hum Genet"},{"issue":"1","key":"pone.0242725.ref018","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1002\/ajmg.a.37411","article-title":"First fetal case of the 8q24.3 contiguous genes syndrome","volume":"170A","author":"C Wells","year":"2016","journal-title":"Am J Med Genet A"},{"key":"pone.0242725.ref019","article-title":"Eye coloboma and complex cardiac malformations belong to the clinical spectrum of PUF60 variants","author":"F Santos-Simarro","year":"2017","journal-title":"Clin Genet"},{"key":"pone.0242725.ref020","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1038\/ejhg.2016.133","article-title":"Dominant variants in the splicing factor PUF60 cause a recognizable syndrome with intellectual disability, heart defects and short stature","volume":"25","author":"S El Chehadeh","year":"2017","journal-title":"Eur J Hum Genet"},{"issue":"5","key":"pone.0242725.ref021","doi-asserted-by":"crossref","first-page":"552","DOI":"10.1038\/ejhg.2017.27","article-title":"PUF60 variants cause a syndrome of ID, short stature, microcephaly, coloboma, craniofacial, cardiac, renal and spinal features","volume":"25","author":"KJ Low","year":"2017","journal-title":"Eur J Hum Genet"},{"issue":"12","key":"pone.0242725.ref022","doi-asserted-by":"crossref","first-page":"2657","DOI":"10.1261\/rna.1312808","article-title":"Flexibility in the site of exon junction complex deposition revealed by functional group and RNA secondary structure alterations in the splicing substrate","volume":"14","author":"DM Mishler","year":"2008","journal-title":"RNA"},{"issue":"2","key":"pone.0242725.ref023","first-page":"51","article-title":"Determination of molecular masses of proteins in solution: Implementation of an HPLC size exclusion chromatography and laser light scattering service in a core laboratory","volume":"10","author":"E Folta-Stogniew","year":"1999","journal-title":"J Biomol Tech"},{"key":"pone.0242725.ref024","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1016\/S0076-6879(97)76066-X","article-title":"Processing of X-ray diffraction data collected in oscillation mode","volume":"276","author":"Z Otwinowski","year":"1997","journal-title":"Methods Enzymol"},{"issue":"9","key":"pone.0242725.ref025","doi-asserted-by":"crossref","first-page":"1865","DOI":"10.1110\/ps.0350503","article-title":"Matthews coefficient probabilities: Improved estimates for unit cell contents of proteins, DNA, and protein-nucleic acid complex crystals","volume":"12","author":"KA Kantardjieff","year":"2003","journal-title":"Protein Sci"},{"issue":"2","key":"pone.0242725.ref026","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1016\/0022-2836(68)90205-2","article-title":"Solvent content of protein crystals","volume":"33","author":"BW Matthews","year":"1968","journal-title":"J Mol Biol"},{"issue":"Pt 4","key":"pone.0242725.ref027","doi-asserted-by":"crossref","first-page":"658","DOI":"10.1107\/S0021889807021206","article-title":"Phaser crystallographic software","volume":"40","author":"AJ McCoy","year":"2007","journal-title":"J Appl Crystallogr"},{"issue":"Pt 4","key":"pone.0242725.ref028","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1107\/S0907444910045749","article-title":"Overview of the CCP4 suite and current developments","volume":"67","author":"MD Winn","year":"2011","journal-title":"Acta Crystallogr D Biol Crystallogr"},{"key":"pone.0242725.ref029","doi-asserted-by":"crossref","first-page":"1022","DOI":"10.1107\/S0021889897006766","article-title":"MOLREP: an automated program for molecular replacement","volume":"30","author":"A Vagin","year":"1997","journal-title":"Journal of Applied Crystallography"},{"issue":"Pt 5","key":"pone.0242725.ref030","doi-asserted-by":"crossref","first-page":"905","DOI":"10.1107\/S0907444998003254","article-title":"Crystallography & NMR system: A new software suite for macromolecular structure determination","volume":"54","author":"AT Brunger","year":"1998","journal-title":"Acta Crystallogr D Biol Crystallogr"},{"issue":"11","key":"pone.0242725.ref031","doi-asserted-by":"crossref","first-page":"2728","DOI":"10.1038\/nprot.2007.406","article-title":"Version 1.2 of the Crystallography and NMR system","volume":"2","author":"AT Brunger","year":"2007","journal-title":"Nat Protoc"},{"issue":"Pt 3","key":"pone.0242725.ref032","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1107\/S0907444996012255","article-title":"Refinement of macromolecular structures by the maximum-likelihood method","volume":"53","author":"GN Murshudov","year":"1997","journal-title":"Acta Crystallogr D Biol Crystallogr"},{"issue":"Pt 2","key":"pone.0242725.ref033","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1107\/S0108767390010224","article-title":"Improved methods for building protein models in electron density maps and the location of errors in these models","volume":"47","author":"TA Jones","year":"1991","journal-title":"Acta Crystallogr A"},{"issue":"Pt 4","key":"pone.0242725.ref034","doi-asserted-by":"crossref","first-page":"486","DOI":"10.1107\/S0907444910007493","article-title":"Features and development of Coot","volume":"66","author":"P Emsley","year":"2010","journal-title":"Acta Crystallogr D Biol Crystallogr"},{"key":"pone.0242725.ref035","doi-asserted-by":"crossref","first-page":"e01456","DOI":"10.7554\/eLife.01456","article-title":"Collaboration gets the most out of software","volume":"2","author":"A Morin","year":"2013","journal-title":"Elife"},{"issue":"1","key":"pone.0242725.ref036","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1038\/sj.emboj.7601936","article-title":"Dimerization of FIR upon FUSE DNA binding suggests a mechanism of c-myc inhibition","volume":"27","author":"GV Crichlow","year":"2008","journal-title":"EMBO J"},{"issue":"3","key":"pone.0242725.ref037","doi-asserted-by":"crossref","first-page":"703","DOI":"10.1016\/j.jmb.2006.11.077","article-title":"Alternative conformations at the RNA-binding surface of the N-terminal U2AF(65) RNA recognition motif","volume":"366","author":"KR Thickman","year":"2007","journal-title":"J Mol Biol"},{"issue":"48","key":"pone.0242725.ref038","doi-asserted-by":"crossref","first-page":"33641","DOI":"10.1074\/jbc.M806297200","article-title":"Solution conformation and thermodynamic characteristics of RNA binding by the splicing factor U2AF65","volume":"283","author":"JL Jenkins","year":"2008","journal-title":"J Biol Chem"},{"issue":"3","key":"pone.0242725.ref039","doi-asserted-by":"crossref","first-page":"290","DOI":"10.1016\/j.sbi.2008.04.002","article-title":"RNA recognition motifs: boring? Not quite","volume":"18","author":"A Clery","year":"2008","journal-title":"Curr Opin Struct Biol"},{"issue":"5214","key":"pone.0242725.ref040","doi-asserted-by":"crossref","first-page":"1173","DOI":"10.1126\/science.7761834","article-title":"Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins","volume":"268","author":"R Singh","year":"1995","journal-title":"Science"},{"issue":"5","key":"pone.0242725.ref041","doi-asserted-by":"crossref","first-page":"2970","DOI":"10.1128\/MCB.17.5.2970","article-title":"Conserved intron elements repress splicing of a neuron-specific c-src exon in vitro","volume":"17","author":"RC Chan","year":"1997","journal-title":"Mol Cell Biol"},{"issue":"4","key":"pone.0242725.ref042","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1261\/rna.2191903","article-title":"Antagonistic regulation of alpha-actinin alternative splicing by CELF proteins and polypyrimidine tract binding protein","volume":"9","author":"N Gromak","year":"2003","journal-title":"RNA"},{"issue":"10","key":"pone.0242725.ref043","doi-asserted-by":"crossref","first-page":"1268","DOI":"10.1101\/gad.2.10.1268","article-title":"The role of the mammalian branchpoint sequence in pre-mRNA splicing","volume":"2","author":"R Reed","year":"1988","journal-title":"Genes Dev"},{"issue":"8","key":"pone.0242725.ref044","doi-asserted-by":"crossref","first-page":"998","DOI":"10.1017\/S1355838298980499","article-title":"The KH domain of the branchpoint sequence binding protein determines specificity for the pre-mRNA branchpoint sequence","volume":"4","author":"JA Berglund","year":"1998","journal-title":"RNA"},{"issue":"4","key":"pone.0242725.ref045","doi-asserted-by":"crossref","first-page":"965","DOI":"10.1016\/S1097-2765(03)00115-1","article-title":"Structural basis for the molecular recognition between human splicing factors U2AF65 and SF1\/mBBP","volume":"11","author":"P Selenko","year":"2003","journal-title":"Mol Cell"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0242725","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2020,11,30]],"date-time":"2020-11-30T13:43:55Z","timestamp":1606743835000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0242725"}},"subtitle":[],"editor":[{"given":"Muhammad Aamer","family":"Mehmood","sequence":"first","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2020,11,30]]},"references-count":45,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2020,11,30]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0242725","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,11,30]]}}}