title: A new alpha-helical extension promotes RNA binding by the dsRBD of Rnt1p RNAse III.
abstract: Rnt1 endoribonuclease, the yeast homolog of RNAse III, plays an important role in the maturation of a diverse set of RNAs. The enzymatic activity requires a conserved catalytic domain, while RNA binding requires the double-stranded RNA-binding domain (dsRBD) at the C-terminus of the protein. While bacterial RNAse III enzymes cleave double-stranded RNA, Rnt1p specifically cleaves RNAs that possess short irregular stem-loops containing 12-14 base pairs interrupted by internal loops and bulges and capped by conserved AGNN tetraloops. Consistent with this substrate specificity, the isolated Rnt1p dsRBD and the 30-40 amino acids that follow bind to AGNN-containing stem-loops preferentially in vitro. In order to understand how Rnt1p recognizes its cognate processing sites, we have defined its minimal RNA-binding domain and determined its structure by solution NMR spectroscopy and X-ray crystallography. We observe a new carboxy-terminal helix following a canonical dsRBD structure. Removal of this helix reduces binding to Rnt1p substrates. The results suggest that this helix allows the Rnt1p dsRBD to bind to short RNA stem-loops by modulating the conformation of helix alpha1, a key RNA-recognition element of the dsRBD.
authors.affiliation: Institut de Biochimie et de Biophysique Moléculaire et Cellulaire (CNRS-UMR 8619), Université Paris-Sud, Orsay, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Leulliot
authors.forename: Sophie
authors.initials: S
authors.lastname: Quevillon-Cheruel
authors.forename: Marc
authors.initials: M
authors.lastname: Graille
authors.forename: Herman
authors.initials: H
authors.lastname: van Tilbeurgh
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Leeper
authors.forename: Katherine S
authors.initials: KS
authors.lastname: Godin
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Edwards
authors.forename: Snorri T L
authors.initials: ST
authors.lastname: Sigurdsson
authors.forename: Natasha
authors.initials: N
authors.lastname: Rozenkrants
authors.forename: Roland J
authors.initials: RJ
authors.lastname: Nagel
authors.forename: Manuel
authors.initials: M
authors.lastname: Ares
authors.forename: Gabriele
authors.initials: G
authors.lastname: Varani
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D012330:RNA, Double-Stranded; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; C468367:RNT1 protein, S cerevisiae; D043244:Ribonuclease III