Entamoeba histolytica EhDEAD1 is a conserved DEAD-box RNA helicase with ATPase and ATP-dependent RNA unwinding activities

• Published in: Gene Vol. 414; no. 1; pp. 19 - 31
• Main Authors: López-Camarillo, César, de la Luz García-Hernández, María, Marchat, Laurence A., Luna-Arias, Juan P., Hernández de la Cruz, Olga, Mendoza, Leobardo, Orozco, Esther
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.05.2008
• Subjects: Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; Amino Acid Sequence; Amoebiasis; Animals; Conserved Sequence; DEAD-box RNA Helicases - genetics; DEAD-box RNA Helicases - metabolism; Electrophoretic Mobility Shift Assay; Entamoeba histolytica; Entamoeba histolytica - enzymology; Entamoeba histolytica - genetics; Entamoeba histolytica - growth & development; Genetic Complementation Test; Lasers; Molecular Sequence Data; Phylogeny; Posttranscriptional regulation; Protozoan parasite; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA metabolism; RNA, Messenger; RNA, Protozoan - genetics; RNA, Protozoan - metabolism; S Phase - physiology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae - metabolism; Sequence Homology, Amino Acid; aa; CE; nt; MESG; Posttranscriptional regulation; bp; PNP; Amoebiasis; NE; IPTG; BLAST; Protozoan parasite; RNA metabolism
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/j.gene.2008.01.024

RNA helicases are widely conserved key enzymes that perform multiple functions in RNA metabolism. Here, we present the cloning, expression and functional characterization of the EhDEAD1 RNA helicase in the protozoan parasite Entamoeba histolytica. According to its primary structure, EhDEAD1 is evolutionary related to yeast DED1 and human DDX3X RNA helicases, both involved in translation and cell cycle regulation. The EhDEAD1 predicted amino acid sequence exhibits the nine conserved motifs described for the DEAD-box SFII superfamily members reported in other organisms and it is evolutionary close to protozoan homologues. Purified recombinant EhDEAD1 protein presented ATPase activity and it was able to bind and unwind RNA in an ATPase-dependent manner in vitro. RT-PCR assays showed that EhDead1 gene is overtranscribed in the cell cycle S phase. Moreover, inhibition of EhDead1 gene expression by antisense RNA seemed to facilitate transition from S to G2/M phase. Intriguingly, our results showed that EhDEAD1 was unable to rescue two yeast Ded1 RNA helicase mutants affected in translation, in spite of the high sequence homology with yeast DED1.