A New Strategy of RNA Interference That Targets Heterologous Sequences Reveals CITFA1 as an Essential Component of Class I Transcription Factor A in Trypanosoma brucei

Abstract

Conditional gene silencing by RNA interference in Trypanosoma brucei can be inconclusive if knockdowns are inefficient or have off-target effects. To enable efficient, specific silencing of single-copy genes in mammalian-infective, bloodstream form trypanosomes, we developed a system that targets the heterologous and functional Trypanosoma cruzi U2AF35 3= untranslated region (UTR) (Tc3) or, alternatively, the sequence of the PTP tag, which can be fused to any mRNA of interest. Two cell lines were created, single-marker Tc3 (smTc3) and smPTP, which conditionally express Tc3 and PTP double-stranded RNA (dsRNA), respectively. The system depends on manipulating both alleles of the gene of interest so that cells exclusively express the target mRNA as a fusion to one of these heterologous sequences. We generated allele integration vectors in which the C-terminal part of a gene's coding sequence can be fused to either heterologous sequence in a single cloning step. We first tested this system with CITFA7, which encodes a well-characterized subunit of the class I transcription factor A (CITFA), an essential factor for transcription initiation by RNA polymerase I. Targeting either Tc3 or PTP fused to the CITFA7 mRNA resulted in gene knockdowns that were as efficient and specific as targeting the endogenous CITFA7 mRNA. Moreover, application of this system to CITFA1, which could not be silenced by established methods, demonstrated that the gene encodes an essential CITFA subunit that mediates binding of the transcription factor complex to RNA polymerase I promoters.

eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei

“…Discordant results for individual genes and the Alsford study (Alsford et al 2011) are not unknown (Badjatia et al 2013). The inability to obtain a knockout TbE5 line may be technical rather than linked to the function of the gene, and alternative approaches (Kim et al 2013;Merritt and Stuart 2013;Park et al 2014) are in progress.…”

Dynein Light Chain LC8 Is Required for RNA Polymerase I-Mediated Transcription in Trypanosoma brucei, Facilitating Assembly and Promoter Binding of Class I Transcription Factor A

“…Immune serum against LC8 was generated by immunization of Sprague-Dawley rats with rGST-LC8, according to a standard protocol (47). rGST-LC8-specific antibodies were purified from serum by blot-immobilized antigen, as previously detailed (46). In contrast to immune serum, the purified antibody did not detect a nonspecific band present in E. coli that comigrated with thrombin-digested LC8 (data not shown).…”

“…smC2-PTP cells allow the conditional silencing of CITFA2 through targeting of the PTP tag coding sequence and were generated in two steps. Starting with a previously published smPTP cell line (46), which conditionally expresses doublestranded RNA (dsRNA) targeting the PTP tag sequence, we first used site-directed integration of SphI-linearized pPURO-PTP-CITFA2 into one CITFA2 allele to fuse the PTP tag sequence to the 5= end of the CITFA2 coding region. In a second step, the remaining CITFA2 allele was replaced by a PCR product in which 100 bp of CITFA2 gene flanks surrounded the hygromycin phosphotransferase coding sequence.…”

“…To analyze the effect of LC8 silencing on transcription by RNA Pol I, total RNA was prepared by the hot-phenol method, as described previously (46). For the analysis of rRNA, total RNA was separated in Reliant precast 1.25% SeaKem Gold agarose RNA gels (Lonza), and rRNA was detected by ethidium bromide staining.…”

“…Chromatin was sonicated until fragments averaged 200 to 400 bp in length. The precipitated DNA was analyzed by quantitative PCR (qPCR) using consensus primers for the slightly varying copies of the RRNA and BES promoters and a primer pair for the ␤/␣-tubulin intergenic region, which were specified previously (33,46). The percent immunoprecipitated DNA (percent IP) was calculated relative to the input material and corrected by subtracting the percent IP of the negative-control ChIP.…”