Conditional U1 Gene Silencing in Toxoplasma gondii

Abstract

The functional characterisation of essential genes in apicomplexan parasites, such as Toxoplasma gondii or Plasmodium falciparum, relies on conditional mutagenesis systems. Here we present a novel strategy based on U1 snRNP-mediated gene silencing. U1 snRNP is critical in pre-mRNA splicing by defining the exon-intron boundaries. When a U1 recognition site is placed into the 3'-terminal exon or adjacent to the termination codon, pre-mRNA is cleaved at the 3'-end and degraded, leading to an efficient knockdown of the gene of interest (GOI). Here we describe a simple method that combines endogenous tagging with DiCre-mediated positioning of U1 recognition sites adjacent to the termination codon of the GOI which leads to a conditional knockdown of the GOI upon rapamycin-induction. Specific knockdown mutants of the reporter gene GFP and several endogenous genes of T. gondii including the clathrin heavy chain gene 1 (chc1), the vacuolar protein sorting gene 26 (vps26), and the dynamin-related protein C gene (drpC) were silenced using this approach and demonstrate the potential of this technology. We also discuss advantages and disadvantages of this method in comparison to other technologies in more detail.

Novel Components of the Toxoplasma Inner Membrane Complex Revealed by BioID

“…We endogenously tagged both the target gene and the upstream gene with an epitope tag followed downstream by an loxP site, thereby flanking the target gene with loxP sites. We performed this genetic engineering in DiCre- ku80 :: KillerRed flox YFP parasites that express inducible Cre recombinase and also activate yellow fluorescent protein (YFP) expression upon induction with rapamycin ( 30 ). In both target strains, we observed the loss of the 3× HA-tagged target protein at the IMC as well as concomitant expression of YFP upon induction of Cre activity ( Fig.…”

“… Gene disruption of selected ISP3 BioID hits using a combinatorial epitope-tagging/Cre- lox strategy. (A) IFA showing parasites with IMC18 endogenously tagged in the DiCre- ku80::KillerRed flox YFP background ( 30 ), grown with or without rapamycin induces Cre recombinase, resulting in excision of the tagged gene. Cre-mediated recombination is monitored by a chromosomal copy of loxP-KillerRed - loxP - YFP under the control of the p5RT70 promoter, which drives YFP expression after the floxed KillerRed gene is excised.…”

Distinct contribution of Toxoplasma gondii rhomboid proteases 4 and 5 to micronemal protein protease 1 activity during invasion

“…ROM5 has previously been shown to cleave a variety of MICs including P. falciparum AMA1 in cell-based assays (Brossier et al, 2005;Dowse et al, 2005;Baker et al, 2006); therefore, we investigated its contribution to MICs cleavage through both knockdown and knockout approaches. Knockdown of ROM5 was achieved using the U1-snRNP-mediated gene silencing methodology recently adapted to T. gondii (Pieperhoff et al, 2014). This method involved appending U1 snRNP recognition sequences just downstream of the terminal exon to prevent alternative polyadenylation, thus leading to highly efficient mRNA decay and gene silencing (Gunderson et al, 1998;Fortes et al, 2003).…”

“…This method involved appending U1 snRNP recognition sequences just downstream of the terminal exon to prevent alternative polyadenylation, thus leading to highly efficient mRNA decay and gene silencing (Gunderson et al, 1998;Fortes et al, 2003). This methodology combined with Cremediated positioning of U1 sequences downstream of the terminal exon of a gene-of-interest (GOI) has been successfully used in T. gondii to knockdown various genes (Pieperhoff et al, 2014).…”

“…To knockdown ROM5 (ToxoDB accession no: TGME49_294690), we modified the plasmid pLIC-HA-FLAGflox(3′UTR-SAG1-HXGPRT)-4xU1 (a gift from Dr. Markus Meissner) used for C-terminal knock-in and U1-mediated silencing (Pieperhoff et al, 2014). Genomic DNA corresponding to the ROM5 C-terminus was amplified by listed primers and cloned into the plasmid pLIC-3ty-flox(3′UTR-SAG1-HXGPRT)-4xU1 between KpnI and NsiI sites.…”