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'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.

FIGURE 1.

The strategy used to determine which part of a mRNA is necessary and sufficient for post-transcriptional regulation of gene expression in kinetoplastid protozoa, in this case the differential expression of the GPI-PLC gene between bloodstream and procyclic form trypanosomes. The procedure relies on the high rate of homologous recombination between exogenous DNA and the genome to insert cassettes at the initiation or stop codon of the gene. The selectable marker genes are shown in blue, the tubulin inter-ORF sequences in red, and the endogenous locus in black. Trans-splicing sites are indicated by filled circles; polyadenylation sites, by vertical lines.

FIGURE 2.

(a) Diagram showing the GPI-PLC alleles present in the cell lines used in this study. The mRNAs produced from each gene are shown above each allele and are labeled using the ORF identity (G, GPI-PLC; H, hyg^R; N, neo^R) and allele (wt and A to E). The selectable marker genes are shown in blue, the tubulin inter-ORF sequences in red, and the endogenous locus in black. Trans-splicing sites are indicated by filled circles; polyadenylation sites, by vertical lines. (b) Northern blot analysis of bloodstream and procyclic forms of each of the trypanosome cell lines. The GPI-PLC alleles present in each cell line are indicated above the blots. The identity of the mRNAs is shown to the right of each of the blots, and the probe used is indicated below each blot. EATRO 1125–derived cell lines were used in these experiments. B indicates bloodstream form RNA; P, procyclic form RNA. Ribosomal RNA is shown as a loading control.

FIGURE 3.

Northern blot analysis of neo^R and GPI-PLC mRNA expression in spontaneous revertants to G418 resistance. The probe used is indicated below each blot. Lister 427–derived cell lines were used in this experiment. B indicates bloodstream form RNA; P, procyclic form RNA. Ribosomal RNA is shown as a loading control. Two separate clones of the m2 revertants are shown (m2a and m2b).

FIGURE 4.

(a) Diagram showing the location of the HindIII sites in the GPI-PLC A allele and the location of the primers used for the inverse PCR reaction used to recover the allele from genomic DNA. (b) Gel showing the inverse PCR products from the GPI-PLC A allele in the five of the mutant cell lines analyzed.

FIGURE 5.

(a) Diagram showing the extent of the deletions in mutants 1 to 8 (m1 to m8). The lines above the genes show the location of the deletions that occurred between TA repeats. All (TA)_n (where n > 8) repeats present in the locus are shown (TA). Other deletions are shown below the line. The distance from the neo^R gene stop codon in kbp is shown. HindIII sites are indicated by H. (b) The extent of the duplication in mutant 7.