TY - JOUR
T1 - Entamoeba histolytica EhDEAD1 is a conserved DEAD-box RNA helicase with ATPase and ATP-dependent RNA unwinding activities
AU - López-Camarillo, César
AU - de la Luz García-Hernández, María
AU - Marchat, Laurence A.
AU - Luna-Arias, Juan P.
AU - Hernández de la Cruz, Olga
AU - Mendoza, Leobardo
AU - Orozco, Esther
N1 - Funding Information:
The EhDead1 gene sequence was obtained from TIGR and Sanger E. histolytica genome sequence databases. We thank Dr. Jean-Leon Chong from the Department of Molecular Genetics at the Ohio State University for generously providing the yeast Ded1 mutants. We are grateful to M. en C. Victor Rosales from the Central Laboratory of CINVESTAV-IPN for helping us with the acquisition of the data on CompuCyte. Our thanks are also to Alfredo Padilla and Sollange Archer for their help in the artwork. This work was supported by UACM (Mexico), CONACyT (Mexico), COFAA-IPN (Mexico) and SIP-IPN (Mexico) grants.
PY - 2008/5/15
Y1 - 2008/5/15
N2 - 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.
AB - 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.
KW - Amoebiasis
KW - Posttranscriptional regulation
KW - Protozoan parasite
KW - RNA metabolism
UR - http://www.scopus.com/inward/record.url?scp=41949129684&partnerID=8YFLogxK
U2 - 10.1016/j.gene.2008.01.024
DO - 10.1016/j.gene.2008.01.024
M3 - Artículo
SN - 0378-1119
VL - 414
SP - 19
EP - 31
JO - Gene
JF - Gene
IS - 1-2
ER -