Partial inhibition of two genes that encode spliceosomal proteins in Giardia intestinalis.
Giardia intestinalis is an early divergent organism that was recently shown to have introns. The machinery responsible for the removal of introns in higher eukaryotes is the spliceosome, which consists of five ribonucleoproteins. Each of these ribonucleoproteins has a small nuclear RNA, a set of seven Sm proteins (B, D1, D2, D3, E, F and G) and several specific proteins. Some genes that encode spliceosome proteins have been bioinformatically identified in the parasite genome. Although it is assumed that the spliceosome is responsible for splicing in this parasite, biochemical characterization is lacking. Objective. To inhibit two G. intestinalis spliceosome protein genes in order to determine whether this inhibition affects parasite growth or encystation. Materials and methods. Antisense sequences of the genes encoding the spliceosomal parasite proteins SmB and SmD3 were cloned into a specific G. intestinalis vector. G. intestinalis individuals were subsequently transfected with the recombinant vectors and those parasites that incorporated the vector were selected. A decrease in mRNA levels by real-time PCR was confirmed and the growth and encystation in wild and transfected parasites was assessed. Results. A decrease of 40% and 70% of SmB and SmD3 mRNA levels, respectively, was observed. Growth and encystation in these parasites were not affected. Conclusion. Decrease of SmB and SmD3 mRNA levels does not affect the parasite, indicating that the spliceosome remains functional or that splicing is not essential for parasite viability.
Laboratorio de Investigaciones Básicas en Bioquímica, Departamento de Química, Facultad de Ciencias, Universidad Nacional de Colombia, Bogotá, D.C., Colombia. mvgomezr@unal.edu.co.
Vanessa
V
Gómez
Moisés
M
Wasserman
D019295:Computational Biology; D016829:Giardia lamblia / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007438:Introns / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013045:Species Specificity; D017381:Spliceosomes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D012333:RNA, Messenger
Giardia lamblia; RNA splicing; parasites; spliceosomes; transfection; unicellular eukaryotic organisms