A short bifunctional element operates to positively or negatively regulateESAG9expression in different developmental forms ofTrypanosoma brucei

Abstract

SummaryIn their mammalian host trypanosomes generate 'stumpy' forms from proliferative 'slender' forms as an adaptation for transmission to their tsetse fly vector. This transition is characterised by the repression of many genes while quiescent stumpy forms accumulate during each wave of parasitaemia. However, a subset of genes are upregulated either as an adaptation for transmission or to sustain infection chronicity. Among this group are ESAG9 proteins, whose genes were originally identified as a component of some telomeric variant surface glycoprotein gene expression sites, although many members of this diverse family are also transcribed elsewhere in the genome. ESAG9 genes are among the most highly regulated genes in transmissible stumpy forms, encoding a group of secreted proteins of cryptic function. To understand their developmental silencing in slender forms and activation in stumpy forms, the post-transcriptional control signals for a well conserved ESAG9 gene have been mapped. This identified a precise RNA sequence element of 34 nucleotides that contributes to gene expression silencing in slender forms but also acts positively, activating gene expression in stumpy forms. We predict that this bifunctional RNA sequence element is targeted by competing negative and positive regulatory factors in distinct developmental forms of the parasite. Analysis of the 39UTR regulatory regions flanking the highly diverse ESAG9 family reveals that the linear regulatory sequence is not highly conserved, suggesting that RNA structure is important for interactions with regulatory proteins.

Genome-wide RNAi selection identifies a regulator of transmission stage-enriched gene families and cell-type differentiation in Trypanosoma brucei

“…When the resulting cell line was assayed with a titration of G418, cells transfected with the NeoR gene coupled to the ESAG9 flanking sequence were killed at ≥10μg/ml G418, whereas cells transfected with the same construct but where the NeoR gene was flanked by the constitutively-expressed aldolase gene 3’UTR were viable at >750μg/ml G418 (Fig 1B). This confirmed our previous analyses demonstrating that the ESAG9 downstream sequences repress gene expression in proliferative slender forms [22]. …”

“…For ESAG9 , a previous mapping of signals in the 3’UTR that contribute to stumpy-enriched expression identified a short domain that contributed to expression in stumpy forms, but with more subtle effects in monomorphs [22]. We have confirmed regulatory control via this domain in our reporter experiments, since deletion of the element resulted in increased NeoR expression and reduced sensitivity to G418 (S2 Fig).…”

“…Genome-wide screens require use of the s427 monomorphic bloodstream form cell line, competent for efficient transfection with a tetracycline-inducible RNAi library [23–26]. These cells, like slender forms in natural (pleomorphic) bloodstream infections, express little ESAG9 mRNA due to posttranscriptional repression [18, 22]. To carry out the screen, we initially transfected the s427 monomorphic parental cell line with a construct comprising the neomycin resistance ( NeoR ) gene flanked downstream by a 1057nt fragment containing the previously characterised 3’UTR control signals and intergenic sequence of the ESAG9 gene Tb927.5.4620 [22] (‘ ESAG9-EQ long 3’UTR’ in Fig 1A).…”

“…These cells, like slender forms in natural (pleomorphic) bloodstream infections, express little ESAG9 mRNA due to posttranscriptional repression [18, 22]. To carry out the screen, we initially transfected the s427 monomorphic parental cell line with a construct comprising the neomycin resistance ( NeoR ) gene flanked downstream by a 1057nt fragment containing the previously characterised 3’UTR control signals and intergenic sequence of the ESAG9 gene Tb927.5.4620 [22] (‘ ESAG9-EQ long 3’UTR’ in Fig 1A). When the resulting cell line was assayed with a titration of G418, cells transfected with the NeoR gene coupled to the ESAG9 flanking sequence were killed at ≥10μg/ml G418, whereas cells transfected with the same construct but where the NeoR gene was flanked by the constitutively-expressed aldolase gene 3’UTR were viable at >750μg/ml G418 (Fig 1B).…”

“…The stumpy-enriched expression of the ESAG9 gene family is regulated through their 3’UTR [22]. Exploiting this, here we report a genome-wide RNAi selectional regimen designed to isolate repressors of stumpy-enriched gene expression operative in proliferative bloodstream slender forms.…”