Crystal structure of UAP56, a DExD/H-box protein involved in pre-mRNA splicing and mRNA export.

UAP56 is an essential eukaryotic pre-mRNA splicing factor and mRNA export factor. The mechanisms of its functions are not well understood. We determined the crystal structures of the N- and C-terminal domains of human UAP56 (comprising 90% of the full-length UAP56) at 1.9 A resolution. The two domains each have a RecA-like fold and are connected by a flexible linker. The overall fold of each domain is highly similar to the corresponding domains of eIF4A (a prototypic DExD/H-box protein), with differences at the loops and termini. This structural similarity suggests that UAP56 is likely to possess ATPase and helicase activity similar to eIF4A. The NTP binding pocket of UAP56 is occupied by a citrate ion, mimicking the phosphates of NTP and retaining the P loop in an open conformation. The crystal structure of the N-terminal domain of UAP56 also reveals a dimer interface that is potentially important for UAP56's function.

[1]  D. Mckay,et al.  Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[2]  D. Mckay,et al.  Helicase structure and mechanism. , 2002, Current opinion in structural biology.

[3]  M. Green,et al.  Identification and characterization of yUAP/Sub2p, a yeast homolog of the essential human pre-mRNA splicing factor hUAP56. , 2001, Genes & development.

[4]  J. Valcárcel,et al.  U2AF65 recruits a novel human DEAD box protein required for the U2 snRNP-branchpoint interaction. , 1997, Genes & development.

[5]  R. Lührmann,et al.  The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[6]  C. Guthrie,et al.  The DEAH-box splicing factor Prp16 unwinds RNA duplexes in vitro , 1998, Current Biology.

[7]  Kevin Struhl,et al.  TREX is a conserved complex coupling transcription with messenger RNA export , 2002, Nature.

[8]  Haiyang Li,et al.  Crystal structure of a DEAD box protein from the hyperthermophile Methanococcus jannaschii. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[9]  U. Baumann,et al.  Crystal structure of the ATPase domain of translation initiation factor 4A from Saccharomyces cerevisiae--the prototype of the DEAD box protein family. , 1999, Structure.

[10]  N. Tanner,et al.  DExD/H box RNA helicases: from generic motors to specific dissociation functions. , 2001, Molecular cell.

[11]  J. Abelson,et al.  The Saccharomyces cerevisiae Prp5 Protein Has RNA-dependent ATPase Activity with Specificity for U2 Small Nuclear RNA* , 1996, The Journal of Biological Chemistry.

[12]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[13]  C. Guthrie,et al.  Mechanical Devices of the Spliceosome: Motors, Clocks, Springs, and Things , 1998, Cell.

[14]  E. Izaurralde,et al.  Genome‐wide analysis of nuclear mRNA export pathways in Drosophila , 2003, The EMBO journal.

[15]  W. Merrick,et al.  Biochemical and Kinetic Characterization of the RNA Helicase Activity of Eukaryotic Initiation Factor 4A* , 1999, The Journal of Biological Chemistry.

[16]  Roger E. Moore,et al.  Composition and functional characterization of the yeast spliceosomal penta-snRNP. , 2002, Molecular cell.

[17]  Anton J. Enright,et al.  References and Notes Materials and Methods Som Text Figs. S1 to S9 Tables S1 to S3 References and Notes Protein Displacement by Dexh/d " Rna Helicases " without Duplex Unwinding , 2022 .

[18]  D. Herschlag,et al.  The DEAD box protein eIF4A. 2. A cycle of nucleotide and RNA-dependent conformational changes. , 1998, Biochemistry.

[19]  M. Rosbash,et al.  The DECD box putative ATPase Sub2p is an early mRNA export factor , 2001, Current Biology.

[20]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[21]  S. Velankar,et al.  Crystal Structures of Complexes of PcrA DNA Helicase with a DNA Substrate Indicate an Inchworm Mechanism , 1999, Cell.

[22]  F. Corpet.  Multiple sequence alignment with hierarchical clustering. , 1988, Nucleic acids research.

[23]  Phillip A. Sharp,et al.  13 Splicing of Precursors to mRNA by the Spliceosome , 1993 .

[24]  J. Abelson,et al.  Four yeast spliceosomal proteins (PRP5, PRP9, PRP11, and PRP21) interact to promote U2 snRNP binding to pre-mRNA. , 1993, Genes & development.

[25]  Ed Hurt,et al.  Splicing factor Sub2p is required for nuclear mRNA export through its interaction with Yra1p , 2001, Nature.

[26]  B. Ganem.  RNA world , 1987, Nature.

[27]  D. Libri,et al.  Multiple roles for the yeast SUB2/yUAP56 gene in splicing. , 2001, Genes & development.

[28]  J P Griffith,et al.  Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. , 1998, Structure.

[29]  Ben Lehner,et al.  Analysis of a high-throughput yeast two-hybrid system and its use to predict the function of intracellular proteins encoded within the human MHC class III region. , 2004, Genomics.

[30]  C. Guthrie,et al.  Deletion of MUD2, the yeast homolog of U2AF65, can bypass the requirement for sub2, an essential spliceosomal ATPase. , 2001, Genes & development.

[31]  Gabriel Waksman,et al.  Major Domain Swiveling Revealed by the Crystal Structures of Complexes of E. coli Rep Helicase Bound to Single-Stranded DNA and ADP , 1997, Cell.

[32]  A. Krainer,et al.  Pre-mRNA splicing in the new millennium. , 2001, Current opinion in cell biology.

[33]  T. Blumenthal,et al.  UAP56 levels affect viability and mRNA export in Caenorhabditis elegans. , 2003, RNA.

[34]  C. Chothia,et al.  The structure of protein-protein recognition sites. , 1990, The Journal of biological chemistry.

[35]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[36]  D. Mckay,et al.  Crystallographic structure of the amino terminal domain of yeast initiation factor 4A, a representative DEAD-box RNA helicase. , 1999, RNA.

[37]  D. Gatfield,et al.  REF1/Aly and the additional exon junction complex proteins are dispensable for nuclear mRNA export , 2002, The Journal of cell biology.

[38]  M. Mann,et al.  Pre-mRNA splicing and mRNA export linked by direct interactions between UAP56 and Aly , 2001, Nature.

[39]  G. Ast,et al.  The U1 snRNP Base Pairs with the 5′ Splice Site within a Penta-snRNP Complex , 2003, Molecular and Cellular Biology.