A phosphate-binding pocket within the platform-PAZ-connector helix cassette of human Dicer.

We have solved two families of crystal structures of the human Dicer "platform-PAZ-connector helix" cassette in complex with small interfering RNAs (siRNAs). The structures possess two adjacently positioned pockets: a 2 nt 3'-overhang-binding pocket within the PAZ domain (3' pocket) and a phosphate-binding pocket within the platform domain (phosphate pocket). One family of complexes contains a knob-like α-helical protrusion, designated "hDicer-specific helix," that separates the two pockets and orients the bound siRNA away from the surface of Dicer, which could be indicative of a product release/transfer state. In the second complex, the helical protrusion is melted/disordered and the bound siRNA is aligned toward the surface of Dicer, suggestive of a cleavage-competent state. These structures allow us to propose that the transition from the cleavage-competent to the postulated product release/transfer state may involve release of the 5'-phosphate from the phosphate pocket while retaining the 3' overhang in the 3' pocket.

[1]  P. D. Adams,et al.  The Phenix refinement framework , 2022 .

[2]  V. Kim,et al.  The Drosha-DGCR8 complex in primary microRNA processing. , 2004, Genes & development.

[3]  V. Kim,et al.  Biogenesis of small RNAs in animals , 2009, Nature Reviews Molecular Cell Biology.

[4]  D. Patel,et al.  Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain , 2004, Nature.

[5]  I. MacRae,et al.  Structural determinants of RNA recognition and cleavage by Dicer , 2007, Nature Structural &Molecular Biology.

[6]  A. Caudy,et al.  Role for a bidentate ribonuclease in the initiation step of RNA interference , 2001 .

[7]  Ira M. Hall,et al.  Regulation of Heterochromatic Silencing and Histone H3 Lysine-9 Methylation by RNAi , 2002, Science.

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

[9]  Jack F Kirsch,et al.  Autoinhibition of human dicer by its internal helicase domain. , 2008, Journal of molecular biology.

[10]  Hyeshik Chang,et al.  Dicer recognizes the 5′ end of RNA for efficient and accurate processing , 2011, Nature.

[11]  V. Ambros,et al.  The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. , 1999, Developmental biology.

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

[13]  G. Hannon,et al.  Processing of primary microRNAs by the Microprocessor complex , 2004, Nature.

[14]  T. Tuschl,et al.  RNA interference is mediated by 21- and 22-nucleotide RNAs. , 2001, Genes & development.

[15]  Jennifer A. Doudna,et al.  Structural insights into RNA Processing by the Human RISC-Loading Complex , 2009, Nature Structural &Molecular Biology.

[16]  Amy A. Caudy,et al.  Post-transcriptional gene silencing by double-stranded RNA , 2001, Nature Reviews Genetics.

[17]  V. Kim.  MicroRNA biogenesis: coordinated cropping and dicing , 2005, Nature Reviews Molecular Cell Biology.

[18]  R. Shiekhattar,et al.  TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing , 2005, Nature.

[19]  Pick-Wei Lau,et al.  The Molecular Architecture of Human Dicer , 2012, Nature Structural &Molecular Biology.

[20]  G. Hannon,et al.  C . elegans involved in developmental timing in Dicer functions in RNA interference and in synthesis of small RNA , 2001 .

[21]  Anne Gatignol,et al.  TRBP, a regulator of cellular PKR and HIV‐1 virus expression, interacts with Dicer and functions in RNA silencing , 2005, EMBO reports.

[22]  A. Pasquinelli,et al.  Genes and Mechanisms Related to RNA Interference Regulate Expression of the Small Temporal RNAs that Control C. elegans Developmental Timing , 2001, Cell.

[23]  Jennifer A. Doudna,et al.  Substrate-specific structural rearrangements of human Dicer , 2013, Nature Structural &Molecular Biology.

[24]  R. Shiekhattar,et al.  The Microprocessor complex mediates the genesis of microRNAs , 2004, Nature.

[25]  A. Pasquinelli,et al.  A Cellular Function for the RNA-Interference Enzyme Dicer in the Maturation of the let-7 Small Temporal RNA , 2001, Science.

[26]  B. Bass,et al.  A Role for the RNase III Enzyme DCR-1 in RNA Interference and Germ Line Development in Caenorhabditis elegans , 2001, Science.

[27]  P. Katsamba,et al.  Kinetic studies of RNA-protein interactions using surface plasmon resonance. , 2002, Methods.

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

[29]  Hervé Seitz,et al.  Recognition of the pre-miRNA structure by Drosophila Dicer-1 , 2011, Nature Structural &Molecular Biology.

[30]  J. Doudna,et al.  siRNA repositioning for guide strand selection by human Dicer complexes. , 2011, Molecular cell.

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

[32]  Randy J Read,et al.  Electronic Reprint Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination , 2022 .

[33]  Kaihong Zhou,et al.  Coordinated activities of human dicer domains in regulatory RNA processing. , 2012, Journal of molecular biology.

[34]  Bo W. Han,et al.  Dicer Partner Proteins Tune the Length of Mature miRNAs in Flies and Mammals , 2012, Cell.

[35]  T. Tuschl,et al.  The Human DiGeorge Syndrome Critical Region Gene 8 and Its D. melanogaster Homolog Are Required for miRNA Biogenesis , 2004, Current Biology.

[36]  G. Hannon,et al.  RNase III enzymes and the initiation of gene silencing , 2004, Nature Structural &Molecular Biology.

[37]  Michael Sattler,et al.  Nucleic acid 3′-end recognition by the Argonaute2 PAZ domain , 2004, Nature Structural &Molecular Biology.

[38]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[39]  Byoung-Tak Zhang,et al.  Molecular Basis for the Recognition of Primary microRNAs by the Drosha-DGCR8 Complex , 2006, Cell.

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

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