title: Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF.
abstract: Many cellular functions involve multi-domain proteins, which are composed of structurally independent modules connected by flexible linkers. Although it is often well understood how a given domain recognizes a cognate oligonucleotide or peptide motif, the dynamic interaction of multiple domains in the recognition of these ligands remains to be characterized. Here we have studied the molecular mechanisms of the recognition of the 3'-splice-site-associated polypyrimidine tract RNA by the large subunit of the human U2 snRNP auxiliary factor (U2AF65) as a key early step in pre-mRNA splicing. We show that the tandem RNA recognition motif domains of U2AF65 adopt two remarkably distinct domain arrangements in the absence or presence of a strong (that is, high affinity) polypyrimidine tract. Recognition of sequence variations in the polypyrimidine tract RNA involves a population shift between these closed and open conformations. The equilibrium between the two conformations functions as a molecular rheostat that quantitatively correlates the natural variations in polypyrimidine tract nucleotide composition, length and functional strength to the efficiency to recruit U2 snRNP to the intron during spliceosome assembly. Mutations that shift the conformational equilibrium without directly affecting RNA binding modulate splicing activity accordingly. Similar mechanisms of cooperative multi-domain conformational selection may operate more generally in the recognition of degenerate nucleotide or amino acid motifs by multi-domain proteins.
authors.affiliation: Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany.
authors.forename: Cameron D
authors.initials: CD
authors.lastname: Mackereth
authors.forename: Tobias
authors.initials: T
authors.lastname: Madl
authors.forename: Sophie
authors.initials: S
authors.lastname: Bonnal
authors.forename: Bernd
authors.initials: B
authors.lastname: Simon
authors.forename: Katia
authors.initials: K
authors.lastname: Zanier
authors.forename: Alexander
authors.initials: A
authors.lastname: Gasch
authors.forename: Vladimir
authors.initials: V
authors.lastname: Rybin
authors.forename: Juan
authors.initials: J
authors.lastname: Valcárcel
authors.forename: Michael
authors.initials: M
authors.lastname: Sattler
mesh_terms: D020816:Amino Acid Motifs; D001483:Base Sequence; D006801:Humans; D007438:Introns / Q000235:genetics; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011743:Pyrimidines / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism*; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism*; D017381:Spliceosomes / Q000737:chemistry / Q000378:metabolism; D000072668:Splicing Factor U2AF; D013379:Substrate Specificity
chemical_list: D008024:Ligands; D009687:Nuclear Proteins; D011743:Pyrimidines; D012322:RNA Precursors; D022821:RNA Splice Sites; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human