An mRNA-Binding Protein of The Ancient Protist Trichomonas Vaginalis |Biomedgrid

An in vivo UV-crosslinking assay using lysates of Trichomonas vaginalis was used to identify a 21.5-kDa protein called RNA-binding protein 1 (RBP1) that interacts with mRNA. A single transcript was detected using Northern blots that showed RBP1 mRNA was expressed in parasites. Nitrocellulose with RBP1 blotted after electrophoresis of total proteins from high versus low-iron parasites and probed with MAb CF130C3 to RBP1 were identical. A cDNA expression library screened with CF130C3 yielded a cDNA encoding for RBP1 of 189 amino acids with Mr 21.5-kDa and pI 11.01. The nucleotide sequence of the 3'-end revealed the presence of the ATTTA (AUUUA for mRNA) putative transcript destabilizing element and poly(A)-tail. Importantly, the amino acid sequence of RBP1 disclosed the presence of ribonucleoprotein (RNP) elements found within the functional domains of known RNP RNA-binding proteins. Furthermore, RBP1 was found to have two distinct RNP RNA-binding domains (RNP-1 and RNP-2) found among eukaryotic RNA-binding proteins. Southern analysis indicated a single copy gene. In vitro RNA-binding assays using the mRNA subclones without 5'- and 3'-untranslated regions (UTRs) of the ap51-1 adhesin of T. vaginalis showed the binding of RBP1 to the 3'-UTR. Finally, in addition to T. vaginalis, RBP1 was detected among three different members of the family Trichomonidae. This is the first demonstration of a protein that binds to the 3'-UTR of transcripts identified for this ancient protist.