A novel strategy to identify the location of necessary and sufficient cis-acting regulatory mRNA elements in trypanosomes

Abstract

Expression of nearly all protein coding genes in trypanosomes is regulated post-transcriptionally, predominantly at the level of mRNA half-life. The identification of cis-acting elements involved in mRNA stability has been hindered by a lack of ability to screen for loss-of-regulation mutants. The method described in this article allows the region containing the necessary and sufficient elements within a mRNA to be identified and uses antibiotic resistance genes as both selectable markers and reporters. In the case of unstable mRNAs, the strategy can be extended by performing a screen for spontaneous loss-of-function mutants in regulatory parts of a mRNA. The method was validated by using the GPI-PLC mRNA, which is unstable in procyclic form trypanosomes and showed that the 3 0 UTR of the GPI-PLC mRNA contains all elements required for developmentally regulated instability. Loss-of-instability mutants all contained deletions within the 2300-nucleotide-long 3 0 UTR, and their analysis showed that a deletion including the last 800 nt of the gene stabilized the mRNA. The method is nonpresumptive, allows far more rapid screening for cis-elements than existing procedures, and has the advantage of identifying functional mutants. It is applicable to all eukaryotes using polycistronic transcription.

Codon choice directs constitutive mRNA levels in trypanosomes

“…Many mRNAs down regulated in PCFs (when compared to BSFs) were expressed at levels lower than predicted by geCAI scores. This might be expected if the intrinsic geCAI expression level of these mRNAs is modulated by cis -elements in the UTRs to destabilise the mRNA ( Webb et al, 2005a ; Clayton, 2013 ). Conversely, many mRNAs upregulated in PCFs relative to BSFs were expressed at levels greater that would be predicted by the geCAI score.…”

“…Cis -elements have been identified in the 3’UTRs that differentially affect stability and/or rate of translation of mRNAs in specific developmental stages; the best characterised being those in procyclin mRNAs ( Furger et al, 1997 ; Hehl et al, 1994 ), cytochrome oxidase subunits ( Mayho et al, 2006 ), and a short stem loop, necessary and sufficient for response to external purine concentration ( Fernández-Moya et al, 2014 ). Manipulation of these elements rarely quantitatively recapitulates the observed variations in mRNA levels in vivo whereas transfer of an entire 3’UTR can ( Webb et al, 2005a ), this may reflect a combinatorial mechanism necessary for complete developmental regulation.…”

Functionally related transcripts have common RNA motifs for specific RNA-binding proteins in trypanosomes

“…Mature transcripts contain regulatory motifs located in the 5'- and 3'-untranslated regions (UTRs) that modulate transcript abundance by specific interaction with RNA-binding proteins (RBPs). These cis -elements are involved in the control of mRNA transport, stability, and translation efficiency [ 18 , 19 ]. Several RBPs form, together with mRNAs, a network of messenger-ribonucleoprotein (mRNP) complexes directing post-transcriptional regulation in response to diverse stimuli [ 20 ].…”

Coordinate regulation of a family of promastigote-enriched mRNAs by the 3′UTR PRE element in Leishmania mexicana

“…These two technologies together not only provide insights about known regulatory elements, but also may hasten the discovery of novel elements. It may be possible to identify additional regulatory elements in the 3′ UTRs using a bioinformatic search of regulated mRNAs, and then test these using traditional or emerging biochemical experimental techniques [74].…”