RNA interference in schistosomes: machinery and methodology

Abstract

SUMMARYRNA interference (RNAi) is a potent gene silencing process that is playing an increasingly important role in investigations of gene function in schistosomes. Here we review what is known about the process in these parasites and provide an update on the methodology and machinery of RNAi. Data are presented to demonstrate that: (1) not all schistosome genes can be suppressed to the same extent, using the methods employed here; (2) while there is variation in the level of suppression achieved for one target gene (SmAP) in adult parasites, all individuals exhibit robust (>80%) suppression; (3) short interfering RNAs (siRNAs) can effect suppression when delivered by soaking (and not just via electroporation, as reported previously); (4) Male/female adult pairs need not be separated prior to siRNA delivery by electroporation for effective gene suppression in both genders and (5) electroporation of siRNAs in medium is as efficient as in commercial electroporation buffer. Regarding the machinery of RNAi in schistosomes, a homologue of the C. elegans multi-membrane spanning, RNA importing protein SID-1 is identified in silico. The gene encoding this protein contains 21 exons and spans over 50 kb to potentially encode a 115,556 Mr protein (SmSID-1). These analyses, and a review of the literature, permit us to derive and present here a draft of potential RNAi pathways in schistosomes.

Characterization of the Src/Abl Hybrid Kinase SmTK6 of Schistosoma mansoni

“…Although standard protocols (dsRNA) (13,50) as well as novel approaches using different sets of specifically designed siRNAs 5 were applied by electroporation in adult schistosomes maintained in vitro, we obtained neither knockdown effects at the transcriptional level (determined by qPCR) nor phenotypic changes (as determined by confocal laser scanning microscopy (13)). SmTK6 may belong to the group of genes, which according to recent studies in this field were described as nonknockable genes (50).…”

Vaccinomics for the Major Blood Feeding Helminths of Humans

“…Parasitic helminths, by virtue of their often complex life cycles and large genomes, have rendered themselves refractory to many genetic manipulation tools that allow the exploration of gene function (Mann et al, , 2010. RNAi is now widely used to assess gene function in schistosomes, and appears to be particularly effective for genes expressed in tissues readily accessible to dsRNA, such as the tegument and gastrodermis (Krautz-Peterson et al, 2010;Stefanic et al, 2010). RNAi has been used to confirm gene function for a number of potential schistosome vaccine antigens and drug targets, and helps explain how some vaccines, which are based on these proteins, might exert their efficacy.…”

Using RNA Interference in Schistosoma mansoni

“…The empirical siRNA amount is 2.5 μg. Concentrations as low as 0.02-0.6 μg have been effective in schistosomula and adult worms, respectively (1,14). Concentrations as high as 15 μg have been used in adult parasites without apparently compromising target specificity (1, 14).…”

“…Among the more promising methodologies is RNA interference (RNAi, or gene silencing), a mechanism by which gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous mRNA transcripts. This technology currently represents the only approach for experimentally manipulating the expression of targeted endogenous schistosome genes, thereby providing insight into putative gene function (1,2). Given the abundance of schistosome transcriptome and genome sequences now available (3)(4)(5)(6)(7)(8)(9), RNAi has the potential to revolutionize investigation of the roles and importance of the genes of this globally important parasite.…”