Fig 1.
The uORFs of Drosophila ATF4 5′UTR mediate translation regulation.
(A) Structure of the 5′ UTR of the Drosophila ATF4-RA mRNA. The two uORFs, such as uORF1 and uORF2, were present in the 5′ UTR. The uORF2 overlaps with the ATF4 ORF, but in a different reading frame. (B) The luciferase reporter construct used in this experiment. The green arrow represents the wild type version of uORF1 and uORF2, and the X indicates a mutation in the initiation codon at each uORF. (C) Drosophila S2 cells were transfected with the indicated ATF4-Luc plasmid and a control Renilla luciferase plasmid. The transfected cells were treated with 1mM of DTT for the indicated time points, 0, 4, and 8 h. Relative light units (RLU) indicates a ratio of firefly luciferase activity normalized with Renilla luciferase activity, and each value was derived from three independent transfections. Error bars show ± s.e.m. *p<0.05, **p<0.005.
Fig 2.
In vivo ATF4 reporter, tub-ATF4.5′UTR>dsRed responds to protein misfolding.
(A) The structure of the ATF4 translational activation reporter, tub-ATF4.5′UTR>dsRed. The dsRed ORF replaces that of ATF4, thereby reporting ATF4′s translation. (B-E) Validation of the reporter response in vivo. Control eye discs did not show dsRed expression (B). Misexpression of Aβ activated the tub-ATF4.5′UTR>dsRed reporter, as evidenced by the expression of dsRed (C). The ATF4 reporter pattern in response to Rh-1G69D misexpression in the eye disc (D). Endogenous ATF4 expression detected using anti-ATF4 antibody labeling (E).
Fig 3.
The expression of ATF4.5′UTR>dsRed upon ER stress.
2nd instar larvae expressing ATF4 reporter were starved for 4 h and then fed with control (A, E, I, M, Q) or 10 μg/mL tunicamycin (B, F, J, N, R) or 1 μM thapsigargin (C, G, K, O, S) or 5 mM DTT (D, H, L, P, T) in Schneider′s Drosophila medium for 5 h. Next, whole mount labeling of tissues were performed with the anti-dsRed antibody. DsRed expressions in salivary gland (E-H), gut (I-L) and fat body (M-P) increased in response to ER stress-inducing agents. In brain (A-D) and mapighian tubule (Q-T), feeding with ER stress-inducing agents did not noticeably alter dsRed expression. The scale bars in (A, E, M) represent 100 μm.
Fig 4.
tub-ATF4.5′UTR>dsRed expression is activated in response to nutritional deprivation.
(A, B) tub-ATF4.5′UTR>dsRed reporter in the larval brain. dsRed was observed in the brain (white arrowhead). However, the expression was not significantly changed in larvae reared in protein deficient food. dsRed expression did not co-localize with anti-repo labeling, which marks glial cells (green). (C, D) dsRed expression in larval tissues expressing tub-ATF4.5′UTR>dsRed reporter. The in vivo ATF4 reporter expression was enhanced in response to nutritional restriction for 18 h in 2nd instar larvae. Specifically, gut and fat body showed high levels of dsRed. (E-J) show higher magnification images of the inset in (C, D). The scale bar in (A) and (E) represents 100 μm for (A, B and E-J) and that in C, D represents 500 μm.
Fig 5.
In vivo ATF4 reporter is activated in the photoreceptor cells.
(A) At 37% pupal development (37h APF), retina were stained with anti-dsRed (red), anti-armadillo (arrowhead, marker for zonula adherens, green), and anti-actin-phalloidin (blue), respectively. DsRed is expressed at this stage. (B) At 48% of pupal development (48h APF), ATF4 reporter was still active, but, rhodopsin was not detected at this stage. (C) At 96% pupal development retina (96h APF), ATF4 reporter activity was detected at the photoreceptor cells. (D) The expression of dsRed in the adult fly retina, but not in the photoreceptor. Red indicates ATF4 reporter and green is rhodopsin staining. The scale bar in (A′′′) represents 5 μm for (A-C) and that in D represents 50 μm.
Fig 6.
tub-ATF4.5′UTR>dsRed expression in the male reproductive organ.
Dissected male reproductive tissue is stained with anti-dsRed antibody. DsRed signal is shown in the testis, accessory gland, and the ejaculatory duct. The scale bar in (A) represents 100 μm.