A novel strategy to identify the location of necessary and sufficient cis-acting regulatory mRNA elements in trypanosomes.
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'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'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.
Webb
Helena
H
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
Burns
Roisin
R
Kimblin
Nicola
N
Ellis
Louise
L
Carrington
Mark
M
D020413:3' Untranslated Regions; D000483:Alleles; D000818:Animals; D001483:Base Sequence; D017353:Gene Deletion; D018507:Gene Expression Regulation, Developmental; D017125:Genes, Protozoan; D005819:Genetic Markers; D006207:Half-Life; D016297:Mutagenesis, Site-Directed; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D016053:RNA, Protozoan / Q000378:metabolism*; D011995:Recombination, Genetic; D038621:Regulatory Sequences, Ribonucleic Acid; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA; D020040:Trans-Splicing; D014158:Transcription, Genetic; D019076:Transgenes; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000254:growth & development
D020413:3' Untranslated Regions; D005819:Genetic Markers; D012333:RNA, Messenger; D016053:RNA, Protozoan; D038621:Regulatory Sequences, Ribonucleic Acid