Amino acid residues Leu135 and Tyr236 are required for RNA binding activity of CFIm25 in Entamoeba histolytica.
Pre-mRNA 3' end processing in the nucleus is essential for mRNA stability, efficient nuclear transport, and translation in eukaryotic cells. In Human, the cleavage/polyadenylation machinery contains the 25 kDa subunit of the Cleavage Factor Im (CFIm25), which specifically recognizes two UGUA elements and regulates the assembly of polyadenylation factors, poly(A) site selection and polyadenylation. In Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, EhCFIm25 has been reported as a RNA binding protein that interacts with the Poly(A) Polymerase. Here, we follow-up with the study of EhCFIm25 to characterize its interaction with RNA. Using in silico strategy, we identified Leu135 and Tyr236 in EhCFIm25 as conserved amino acids among CFIm25 homologues. We therefore generated mutant EhCFIm25 proteins to investigate the role of these residues for RNA interaction. Results showed that RNA binding activity was totally abrogated when Leu135 and Tyr236 were replaced with Ala residue, and Tyr236 was changed for Phe. In contrast, RNA binding activity was less affected when Leu135 was substituted by Thr. Our data revealed for the first time -until we know-the functional relevance of the conserved Leu135 and Tyr236 in EhCFIm25 for RNA binding activity. They also gave some insights about the possible chemical groups that could be interacting with the RNA molecule.
Biotechnology Program, ENMH-IPN, Mexico City, Mexico.
Juan David
JD
Ospina-Villa
Biotechnology Program, ENMH-IPN, Mexico City, Mexico; Molecular Biomedicine Program, ENMH-IPN, Mexico City, Mexico.
Absalom
A
Zamorano-Carrillo
Genomics Sciences Program, UACM, Mexico City, Mexico.
Cesar
C
Lopez-Camarillo
Subdirección de Enseñanza e Investigación, Hospital Regional de Alta Especialidad de Oaxaca, Oaxaca, Mexico.
Carlos A
CA
Castañon-Sanchez
Molecular Biomedicine Program, ENMH-IPN, Mexico City, Mexico.
Jacqueline
J
Soto-Sanchez
Biotechnology Program, ENMH-IPN, Mexico City, Mexico; Molecular Biomedicine Program, ENMH-IPN, Mexico City, Mexico.
Esther
E
Ramirez-Moreno
Biotechnology Program, ENMH-IPN, Mexico City, Mexico; Molecular Biomedicine Program, ENMH-IPN, Mexico City, Mexico. Electronic address: lmarchat@ipn.mx.
Laurence A
LA
Marchat
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003198:Computer Simulation; D017124:Conserved Sequence; D004748:Entamoeba histolytica; D006801:Humans; D007930:Leucine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D014443:Tyrosine
D015800:Protozoan Proteins; D014443:Tyrosine; D012313:RNA; D007930:Leucine
Cleavage factor Im; Entamoeba histolytica; Mutagenesis; Polyadenylation; RNA binding