Putative DEAD and DExH-box RNA helicases families in Entamoeba histolytica.

RNA helicases catalyze the unwinding of double-stranded RNA structures to perform numerous genetic processes. These enzymes are characterized by the presence of a conserved helicase domain with specific helicases motifs whose amino acid sequence allows the differentiation between DEAD and DExH-box RNA helicase families. Taking advantage of the availability of the complete genome sequence of Entamoeba histolytica, the protozoan responsible for human amoebiasis, we have performed a genomic survey for DEAD and DExH-box RNA helicases encoding genes in this organism. By extensive in silico analysis, we identified 20 EhDEAD and 13 EhDExH-box RNA helicases, which contain almost all the conserved helicase motifs. Additionally, several EhDEAD and EhDExH proteins present specific N- and C-terminal domains that could be related to subcellular localization or function. Phylogenetic studies and sequences analysis suggested that this large EhDEAD/DExH-box RNA helicases family has been generated by gene or internal regions duplication, mutation events, introns formation and motif deletions. Interestingly, EhDexh1 and EhDeaxh10 genes seem to be formed by gene fusion of two ancestral bacterial genes, a mechanism that appears to be evolutionary conserved in the eukaryotic lineage of orthologous proteins. Finally, RT-PCR assays, microarrays and proteomics data analysis showed that several EhDead are differentially expressed in relation to distinct culture conditions. These computational and experimental data provide new information on the evolution of EhDEAD/EhDExH-box RNA helicases and their potential relevance for RNA metabolism in E. histolytica.

[1]  T. Sugiura,et al.  DDX39, upregulated in lung squamous cell cancer, displays RNA helicase activities and promotes cancer cell growth , 2007, Cancer biology & therapy.

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

[3]  P. Linder,et al.  Are DEAD-box proteins becoming respectable helicases? , 2000, Nature Structural Biology.

[4]  Y. Shimura,et al.  A human RNA helicase-like protein, HRH1, facilitates nuclear export of spliced mRNA by releasing the RNA from the spliceosome. , 1996, Genes & development.

[5]  E. Koonin,et al.  Evolution of gene fusions: horizontal transfer versus independent events , 2002, Genome Biology.

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

[7]  C. López-Camarillo,et al.  Entamoeba histolytica: cloning and expression of the poly(A) polymerase EhPAP. , 2005, Experimental parasitology.

[8]  K. Bjornson,et al.  Mechanisms of helicase-catalyzed DNA unwinding. , 1996, Annual review of biochemistry.

[9]  N. Tanner The Newly Identified Q Motif of DEAD Box Heicases IS Involved in Adenine Recognition , 2003, Cell cycle.

[10]  N. Tanner,et al.  The newly discovered Q motif of DEAD‐box RNA helicases regulates RNA‐binding and helicase activity , 2004, The EMBO journal.

[11]  P. Linder,et al.  DEAD-box proteins: the driving forces behind RNA metabolism , 2004, Nature Reviews Molecular Cell Biology.

[12]  A. Pradhan,et al.  Unraveling the ‘DEAD-box’ helicases of Plasmodium falciparum , 2006, Gene.

[13]  M. Mann,et al.  AU Binding Proteins Recruit the Exosome to Degrade ARE-Containing mRNAs , 2001, Cell.

[14]  A. Kornblihtt,et al.  mRNA Encoding a Putative RNA Helicase of the DEAD-Box Gene Family is Up-Regulated in Trypomastigotes of Trypanosoma cruzi , 2000, The Journal of eukaryotic microbiology.

[15]  C. Guthrie,et al.  RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2 , 1998, Current Biology.

[16]  L. Lemaire,et al.  High-level expression in male germ cells of murine P68 RNA helicase mRNA. , 1993, Life sciences.

[17]  Bernard B. Suh,et al.  The genome of the protist parasite Entamoeba histolytica , 2005, Nature.

[18]  Esther Orozco,et al.  Entamoeba histolytica EhDEAD1 is a conserved DEAD-box RNA helicase with ATPase and ATP-dependent RNA unwinding activities. , 2008, Gene.

[19]  P. Leroy,et al.  The protein encoded by a murine male germ cell-specific transcript is a putative ATP-dependent RNA helicase , 1989, Cell.

[20]  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.

[21]  C. Foresta,et al.  Y chromosome microdeletions and alterations of spermatogenesis. , 2001, Endocrine reviews.

[22]  L. Johnson,et al.  Growth-regulated expression and G0-specific turnover of the mRNA that encodes URH49, a mammalian DExH/D box protein that is highly related to the mRNA export protein UAP56. , 2004, Nucleic acids research.

[23]  N. Tanner,et al.  The Q motif: a newly identified motif in DEAD box helicases may regulate ATP binding and hydrolysis. , 2003, Molecular cell.

[24]  S. Aubourg,et al.  The DEAD box RNA helicase family in Arabidopsis thaliana. , 1999, Nucleic acids research.

[25]  J. Luna-Arias,et al.  EhPgp5 mRNA Stability Is a Regulatory Event in theEntamoeba histolytica Multidrug Resistance Phenotype* , 2003, The Journal of Biological Chemistry.

[26]  B. Schwer A new twist on RNA helicases: DExH/D box proteins as RNPases , 2001, Nature Structural Biology.

[27]  L. Maltais,et al.  The human DDX and DHX gene families of putative RNA helicases. , 2003, Genomics.

[28]  Eugene V. Koonin,et al.  Helicases: amino acid sequence comparisons and structure-function relationships , 1993 .

[29]  F. Stutz,et al.  mRNA export: Travelling with DEAD box proteins , 2001, Current Biology.

[30]  C. Davis,et al.  Functional Characterization of Spliceosomal Introns and Identification of U2, U4, and U5 snRNAs in the Deep-Branching Eukaryote Entamoeba histolytica , 2007, Eukaryotic Cell.

[31]  S. Aubourg,et al.  Evolution of intron/exon structure of DEAD helicase family genes in Arabidopsis, Caenorhabditis, and Drosophila. , 2001, Genome research.

[32]  D. Eisenberg,et al.  Detecting protein function and protein-protein interactions from genome sequences. , 1999, Science.

[33]  G. J. Graham,et al.  Tandem genes and clustered genes. , 1995, Journal of theoretical biology.

[34]  Sudhir Kumar,et al.  MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment , 2004, Briefings Bioinform..

[35]  T. Nakagawa,et al.  The Saccharomyces cerevisiae MER3 gene, encoding a novel helicase‐like protein, is required for crossover control in meiosis , 1999, The EMBO journal.

[36]  Zhangjun Fei,et al.  Impact of intestinal colonization and invasion on the Entamoeba histolytica transcriptome. , 2006, Molecular and biochemical parasitology.

[37]  P Linder,et al.  D‐E‐A‐D protein family of putative RNA helicases , 1992, Molecular microbiology.

[38]  C. López-Camarillo,et al.  Entamoeba histolytica: comparative genomics of the pre-mRNA 3' end processing machinery. , 2005, Experimental parasitology.

[39]  A. Aguilera,et al.  Molecular Evidence That the Eukaryotic THO/TREX Complex Is Required for Efficient Transcription Elongation* , 2003, Journal of Biological Chemistry.

[40]  Christiane Bouchier,et al.  Stress by Heat Shock Induces Massive Down Regulation of Genes and Allows Differential Allelic Expression of the Gal/GalNAc Lectin in Entamoeba histolytica , 2006, Eukaryotic Cell.

[41]  C. Ponting Proteins of the endoplasmic-reticulum-associated degradation pathway: domain detection and function prediction. , 2000, The Biochemical journal.

[42]  N. Guillén,et al.  Signalization and cytoskeleton activity through myosin IB during the early steps of phagocytosis in Entamoeba histolytica: a proteomic approach , 2005, Cellular microbiology.

[43]  M. Nei,et al.  The neighbor-joining method , 1987 .

[44]  N. Tanner,et al.  From RNA helicases to RNPases. , 2001, Trends in biochemical sciences.

[45]  N. Sonenberg,et al.  Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF‐4A. , 1992, The EMBO journal.

[46]  Y. Sheng,et al.  Cell-specific and Hormone-regulated Expression of Gonadotropin-regulated Testicular RNA Helicase Gene (GRTH/Ddx25) Resulting from Alternative Utilization of Translation Initiation Codons in the Rat Testis* , 2003, Journal of Biological Chemistry.

[47]  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.

[48]  Barry P. Young,et al.  Sec63p and Kar2p are required for the translocation of SRP‐dependent precursors into the yeast endoplasmic reticulum in vivo , 2001, The EMBO journal.

[49]  Tobias Mourier,et al.  Eukaryotic Intron Loss , 2003, Science.

[50]  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.

[51]  J. Grönroos,et al.  mRNA differential display of gene expression in colonic carcinoma , 2002, Electrophoresis.

[52]  J. de la Cruz,et al.  Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families. , 1999, Trends in biochemical sciences.

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