Structure of a eukaryotic RNase III postcleavage complex reveals a double-ruler mechanism for substrate selection.

Ribonuclease III (RNase III) enzymes are a family of double-stranded RNA (dsRNA)-specific endoribonucleases required for RNA maturation and gene regulation. Prokaryotic RNase III enzymes have been well characterized, but how eukaryotic RNase IIIs work is less clear. Here, we describe the structure of the Saccharomyces cerevisiae RNase III (Rnt1p) postcleavage complex and explain why Rnt1p binds to RNA stems capped with an NGNN tetraloop. The structure shows specific interactions between a structural motif located at the end of the Rnt1p dsRNA-binding domain (dsRBD) and the guanine nucleotide in the second position of the loop. Strikingly, structural and biochemical analyses indicate that the dsRBD and N-terminal domains (NTDs) of Rnt1p function as two rulers that measure the distance between the tetraloop and the cleavage site. These findings provide a framework for understanding eukaryotic RNase IIIs.

[1]  R. Hannoush,et al.  Molecular requirements for duplex recognition and cleavage by eukaryotic RNase III: discovery of an RNA-dependent DNA cleavage activity of yeast Rnt1p. , 2004, Journal of molecular biology.

[2]  A. Nicholson,et al.  Intrinsic double-stranded-RNA processing activity of Escherichia coli ribonuclease III lacking the dsRNA-binding domain. , 2001, Biochemistry.

[3]  Phillip D Zamore,et al.  RNAi: nature abhors a double-strand. , 2002, Current opinion in genetics & development.

[4]  Jules Gagnon,et al.  Reporter mRNAs cleaved by Rnt1p are exported and degraded in the cytoplasm , 2011, Nucleic acids research.

[5]  Sherif Abou Elela,et al.  RNase III Cleaves Eukaryotic Preribosomal RNA at a U3 snoRNP-Dependent Site , 1996, Cell.

[6]  Zhihua Du,et al.  Structural and biochemical insights into the dicing mechanism of mouse Dicer: A conserved lysine is critical for dsRNA cleavage , 2008, Proceedings of the National Academy of Sciences.

[7]  Sherif Abou Elela,et al.  Sequence dependence of substrate recognition and cleavage by yeast RNase III. , 2003, Journal of molecular biology.

[8]  Sherif Abou Elela,et al.  The RNase III family: a conserved structure and expanding functions in eukaryotic dsRNA metabolism. , 2001, Current issues in molecular biology.

[9]  S. Abou Elela,et al.  Yeast ribonuclease III uses a network of multiple hydrogen bonds for RNA binding and cleavage. , 2008, Biochemistry.

[10]  D. Court,et al.  Posttranscriptional control of the lysogenic pathway in bacteriophage lambda. , 1993, Progress in nucleic acid research and molecular biology.

[11]  D. Court,et al.  Intermediate states of ribonuclease III in complex with double-stranded RNA. , 2005, Structure.

[12]  M. Buckle,et al.  Recognition of a conserved class of RNA tetraloops by Saccharomyces cerevisiae RNase III. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[13]  D. Bartel,et al.  The Inside-Out Mechanism of Dicers from Budding Yeasts , 2011, Cell.

[14]  V. Kim,et al.  The nuclear RNase III Drosha initiates microRNA processing , 2003, Nature.

[15]  Eric Westhof,et al.  Single Processing Center Models for Human Dicer and Bacterial RNase III , 2004, Cell.

[16]  S. Abou Elela,et al.  Deletion of Rnt1p Alters the Proportion of Open versus Closed rRNA Gene Repeats in Yeast , 2007, Molecular and Cellular Biology.

[17]  Sherif Abou Elela,et al.  Evaluation of the RNA Determinants for Bacterial and Yeast RNase III Binding and Cleavage* , 2004, Journal of Biological Chemistry.

[18]  D. Bechhofer,et al.  Mini‐III, an unusual member of the RNase III family of enzymes, catalyses 23S ribosomal RNA maturation in B. subtilis , 2008, Molecular microbiology.

[19]  J. Feigon,et al.  Intrinsic dynamics of an extended hydrophobic core in the S. cerevisiae RNase III dsRBD contributes to recognition of specific RNA binding sites. , 2013, Journal of molecular biology.

[20]  A. Nicholson.  Ribonuclease III and the Role of Double-Stranded RNA Processing in Bacterial Systems , 2011 .

[21]  D. Court,et al.  Structural Insight into the Mechanism of Double-Stranded RNA Processing by Ribonuclease III , 2006, Cell.

[22]  W. Filipowicz,et al.  Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP , 2002, The EMBO journal.

[23]  Gabriele Varani,et al.  RNA recognition by a Staufen double‐stranded RNA‐binding domain , 2000, The EMBO journal.

[24]  Chow H Lee,et al.  Endoribonucleases – enzymes gaining spotlight in mRNA metabolism , 2010, The FEBS journal.

[25]  Sherif Abou Elela,et al.  The N-Terminal Domain That Distinguishes Yeast from Bacterial RNase III Contains a Dimerization Signal Required for Efficient Double-Stranded RNA Cleavage , 2000, Molecular and Cellular Biology.

[26]  P. Legrain,et al.  Yeast RNase III as a key processing enzyme in small nucleolar RNAs metabolism. , 1998, Journal of molecular biology.

[27]  Sherif Abou Elela,et al.  Characterization of the reactivity determinants of a novel hairpin substrate of yeast RNase III. , 2006, Journal of molecular biology.

[28]  R. Garrett,et al.  RNA–protein interactions of an archaeal homotetrameric splicing endoribonuclease with an exceptional evolutionary history , 1997, The EMBO journal.

[29]  A. Nicholson.  Function, mechanism and regulation of bacterial ribonucleases. , 1999, FEMS microbiology reviews.

[30]  J. Feigon,et al.  Structure of a yeast RNase III dsRBD complex with a noncanonical RNA substrate provides new insights into binding specificity of dsRBDs. , 2011, Structure.

[31]  H D Robertson,et al.  Purification and properties of ribonuclease III from Escherichia coli. , 1968, The Journal of biological chemistry.

[32]  Koji Nagata,et al.  Homodimeric structure and double-stranded RNA cleavage activity of the C-terminal RNase III domain of human dicer. , 2007, Journal of molecular biology.

[33]  M. Ares,et al.  Substrate recognition by a eukaryotic RNase III: the double-stranded RNA-binding domain of Rnt1p selectively binds RNA containing a 5'-AGNN-3' tetraloop. , 2000, RNA.

[34]  G. Chanfreau,et al.  RNAse III-mediated degradation of unspliced pre-mRNAs and lariat introns. , 2003, Molecular cell.

[35]  Sherif Abou Elela,et al.  Purification and characterization of Saccharomyces cerevisiae Rnt1p nuclease. , 2001, Methods in enzymology.

[36]  I. MacRae,et al.  Ribonuclease revisited: structural insights into ribonuclease III family enzymes. , 2007, Current opinion in structural biology.

[37]  Yue Zhang,et al.  The Loop Position of shRNAs and Pre-miRNAs Is Critical for the Accuracy of Dicer Processing In Vivo , 2012, Cell.

[38]  Sherif Abou Elela,et al.  Solution structure of conserved AGNN tetraloops: insights into Rnt1p RNA processing , 2001, The EMBO journal.

[39]  Anthony Henras,et al.  Structural basis for recognition of the AGNN tetraloop RNA fold by the double-stranded RNA-binding domain of Rnt1p RNase III. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[40]  N. Costantino,et al.  RNase III: Genetics and function; structure and mechanism. , 2013, Annual review of genetics.

[41]  Angela N. Brooks,et al.  Structural Basis for Double-Stranded RNA Processing by Dicer , 2006, Science.

[42]  D. Court,et al.  A stepwise model for double‐stranded RNA processing by ribonuclease III , 2007, Molecular microbiology.

[43]  Donald Court,et al.  5 – RNA Processing and Degradation by RNase III , 1993 .

[44]  S. Abou Elela,et al.  Short RNA Guides Cleavage by Eukaryotic RNase III , 2007, PloS one.

[45]  M. Ares,et al.  Depletion of yeast RNase III blocks correct U2 3′ end formation and results in polyadenylated but functional U2 snRNA , 1998, The EMBO journal.