Figure 4. Delineation of mRNA cis-elements that regulate ttv expression.

(A) Western blots of staged embryonic extracts from flies transgenic for the indicated constructs were probed to visualize expression of Ttv or GFP. LC denotes loading control (i) Truncation of the 5’ UTR to eliminate upstream AUG sequences and removal of the 3’ UTR permits translation of maternally loaded Ttv-myc transcript in 0–3 hour embryos (upper band) while the endogenous protein (lower band) is not expressed at this time. (ii–v) matTub>Gal4 was used drive expression of UAS constructs in which GFP coding sequence was flanked by the indicated regions of ttv. (ii) GFP expression was detected in 0–3 hour extracts in the absence of ttv regulatory sequences as well as when only 5’ (iv) or 3’ (v) UTRs were present. (iii) In contrast, GFP expression was blocked in early embryos and initiated at ~3 hours similar to endogenous Ttv when the ORF was flanked by both 5’ and 3’ UTR sequences. (B–F) Translational regulation visualized in live animals. GFP fluorescence is absent from a female transgenic for a construct in which GFP is flanked by ttv 5’ and 3’ UTRs. (C) In contrast, ovarian expression results in bright fluorescence in a female transgenic for matTub>Gal4 and a GFP construct that lacks ttv UTRs. (D–E) Merged images showing DAPI stained nuclei and GFP fluorescence in egg chambers. (D) Egg chambers from an animal carrying matTub>Gal4 and the reporter construct with ttv 5’ and 3’ UTRs showing the absence of GFP expression and only yolk autofluorescence. (E) In contrast a reporter that lacks ttv UTRs generates high levels of GFP expression in nurse cells (nc) and oocytes (oo). Note that expression cannot be detected in follicle cells (fc). (F) Time-lapse series of a 5’ttv-UAS-GFP-3’ttv embryo shows that significant GFP fluorescence develops only after three hours post-egg laying.