NORMAL GENOMIC

• Leulliot N et al.
• A new alpha-helical extension promotes RNA binding by the dsRBD of Rnt1pRNAse III.
• EMBO J. 2004; 23: 2468-77
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• Rnt1 endoribonuclease, the yeast homolog of RNAse III, plays an importantrole in the maturation of a diverse set of RNAs. The enzymatic activityrequires a conserved catalytic domain, while RNA binding requires thedouble-stranded RNA-binding domain (dsRBD) at the C-terminus of theprotein. While bacterial RNAse III enzymes cleave double-stranded RNA,Rnt1p specifically cleaves RNAs that possess short irregular stem-loopscontaining 12-14 base pairs interrupted by internal loops and bulges andcapped by conserved AGNN tetraloops. Consistent with this substratespecificity, the isolated Rnt1p dsRBD and the 30-40 amino acids thatfollow bind to AGNN-containing stem-loops preferentially in vitro. Inorder to understand how Rnt1p recognizes its cognate processing sites, wehave defined its minimal RNA-binding domain and determined its structureby solution NMR spectroscopy and X-ray crystallography. We observe a newcarboxy-terminal helix following a canonical dsRBD structure. Removal ofthis helix reduces binding to Rnt1p substrates. The results suggest thatthis helix allows the Rnt1p dsRBD to bind to short RNA stem-loops bymodulating the conformation of helix alpha1, a key RNA-recognition elementof the dsRBD.

• Tremblay A et al.
• A physical interaction between Gar1p and Rnt1pi is required for thenuclear import of H/ACA small nucleolar RNA-associated proteins.
• Mol Cell Biol. 2002; 22: 4792-802
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• During rRNA biogenesis, multiple RNA and protein substrates are modifiedand assembled through the coordinated activity of many factors. InSaccharomyces cerevisiae, the double-stranded RNA nuclease Rnt1p and theH/ACA snoRNA pseudouridylase complex participate in the transformation ofthe nascent pre-rRNA transcript into 35S pre-rRNA. Here we demonstrate thebinding of a component of the H/ACA complex (Gar1p) to Rnt1p in vivo andin vitro in the absence of other factors. In vitro, Rnt1p binding to Gar1pis mutually exclusive of its RNA binding and cleavage activities.Mutations in Rnt1p that disrupt Gar1p binding do not inhibit RNA cleavagein vitro but slow RNA processing, prevent nucleolar localization of H/ACAsnoRNA-associated proteins, and reduce pre-rRNA pseudouridylation in vivo.These results demonstrate colocalization of various components of the rRNAmaturation complex and suggest a mechanism that links rRNApseudouridylation and cleavage factors.

• Shen EC, Henry MF, Weiss VH, Valentini SR, Silver PA, Lee MS
• Arginine methylation facilitates the nuclear export of hnRNP proteins.
• Genes Dev. 1998; 12: 679-91
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• Eukaryotic mRNA processing and export is mediated by various heterogeneousnuclear ribonucleoproteins (hnRNPs). Many of these hnRNPs are methylatedon arginine residues. In the yeast, Saccharomyces cerevisiae, thepredominant enzyme responsible for arginine methylation is Hmt1p. Hmt1pmethylates both Npl3p and Hrp1p, which are shuttling hnRNPs involved inmRNA processing and export. Here, we employ an in vivo nuclear exportassay to show that arginine methylation is important for the nuclearexport of these hnRNPs. Both Npl3p and Hrp1p fail to exit the nucleus incells lacking Hmt1p, and overexpression of Hmt1p enhances Npl3p export.The export of a novel hnRNP-like protein, Hrb1p, which does not bindpoly(A)+ RNA, however, is not affected by the lack of methylation.Furthermore, we find a genetic relationship between Hmt1p and cap-bindingprotein 80 (CBP80). Together, these findings establish that one biologicalrole for arginine methylation is in facilitating the export of certainhnRNPs out of the nucleus.