Developmentally regulated instability of the GPI-PLC mRNA is dependent on a short-lived protein factor

Abstract

The expression of the vast majority of protein coding genes in trypanosomes is regulated exclusively at the post-transcriptional level. Developmentally regulated mRNAs that vary in levels of expression have provided an insight into one mechanism of regulation; a decrease in abundance is due to a shortened mRNA half-life. The decrease in half-life involves cis-acting elements in the 3' untranslated region of the mRNA. The trans-acting factors necessary for the increased rate of degradation remain uncharacterized. The GPI-PLC gene in Trypanosoma brucei encodes a phospholipase C expressed in mammalian bloodstream form, but not in the insect procyclic form. Here, it is reported that the differential expression of the GPI-PLC mRNA also results from a 10-fold difference in half-life. Second, the instability of the GPI-PLC mRNA in procyclic forms can be reversed by the inhibition of protein synthesis. Third, specifically blocking the translation of the GPI-PLC mRNA in procyclic forms by the inclusion of a hairpin in the 5' untranslated region does not result in stabilization of the mRNA. Thus, the effect of protein synthesis inhibitors in stabilizing the GPI-PLC mRNA operates in trans through a short-lived factor dependent on protein synthesis.

Figure 1

Estimation of GPI-PLC mRNA half-life following inhibition of mRNA maturation with sinefungin. Northern blots showing decay of GPI-PLC mRNA after sinefungin addition in (a) bloodstream forms and (b) procyclic forms and (c) transgenic procyclic forms expressing a GPI-PLC gene from a strong promoter. In each case, the same blot was probed with α- and β-tubulin to show the sinefungin-dependent appearance of dicistronic tubulin mRNAs and then for an rRNA to estimate comparative loading. The probe is indicated under each blot. The inset in panel (b) shows a five times longer exposure of region of the blots containing the GPI-PLC mRNA. (d) The GPI-PLC mRNA and rRNA were quantitated in (a) and (c) using a phosphorimager and the GPI-PLC mRNA determined as a concentration relative to the amount of rRNA. RNA size standards are shown in panels (a) and (b).

Figure 2

Inhibition of protein synthesis results in the appearance of GPI-PLC mRNA. Northern blot showing a time course after the addition of cycloheximide. The same blot was probed for EP procyclin and α- and β-tubulin to demonstrate that mRNA stabilization is not a general consequence of blocking translation and for an rRNA to estimate comparative loading. The probe is indicated under each blot. The GPI-PLC mRNA and rRNA were quantitated using a phosphorimager, and the GPI-PLC mRNA determined as a concentration relative to the amount of rRNA and is expressed in arbitrary units.

Figure 3

Estimation of the half-life of cycloheximide-induced GPI-PLC mRNA in procyclic forms. Cells were incubated with cycloheximide for 2 h, and then sinefungin added and samples were taken over a time course and analysed by northern blotting. After probing for GPI-PLC, the same blot was probed with α- and β-tubulin to show the sinefungin-dependent appearance of dicistronic tubulin mRNAs and then for an rRNA to estimate comparative loading. The probe is indicated under each blot. The predominant GPI-PLC mRNA indicated with an arrow and rRNA were quantitated using a phosphorimager and the GPI-PLC mRNA determined as a concentration relative to the amount of rRNA. RNA size standards are shown.

Figure 4

(a) Diagram showing the GPI-PLC genomic locus which is flanked by the HSP100 and β′-COP genes (49) and the GPI-PLC gene replacement strategy. The two major mRNAs are shown above the GPI-PLC gene with the mini-exon addition sites indicated by filled circles and the approximate location of the polyadenylation site by a vertical line. The ClaI HindIII restriction enzyme fragment cloned into the expression vector p1865 is indicated. The starting cells are GPI-PLC −/− and targeted insertion of a neo^R gene results in the return of a GPI-PLC gene to the endogenous locus. (b) Sequence detail around the initiation codon of the GPI-PLC showing the location and sequence of the hairpin introduced into the 5′UTR and the location of the premature stop codon.

Figure 5

Expression of GPI-PLC protein from GPI-PLC transgenes returned to the endogenous locus. The expression of GPI-PLC was tested in the parental GPI-PLC −/− cell line and two independent clones of cell lines containing transgenes encoding wild-type GPI-PLC or GPI-PLC with a hairpin in the 5′UTR or GPI-PLC with a stop codon replacing codon 15. A titration of 2 × 10⁶ (1 cell equivalent) to 2.5 × 10⁴ bloodstream form cells (1/80 cell equivalent) is shown as a measure of sensitivity. The antiserum used is indicated under each blot, anti-ISG65 was used as a loading control.

Figure 6

Comparison of GPI-PLC expression from transgenes. RNA from both bloodstream (B) and procyclic (P) forms of the same cell lines was analysed by northern blotting. The probe is indicated under each blot. One clone containing a wild-type GPI-PLC transgene was compared with two independent clones of each of two cell lines containing either GPI-PLC with a hairpin in the 5′UTR or GPI-PLC with a stop codon replacing codon 15. GPI-PLC +/+ and +/− bloodstream form RNA is shown for comparison, rRNA was used as a loading control and RNA size standards are shown.