Poly(A)-independent regulation of maternal hunchback translation in the Drosophila embryo

Abstract

Development of the Drosophila abdomen requires repression of maternal hunchback (hb) mRNA translation in the posterior of the embryo. This regulation involves at least four components: nanos response elements within the hb 3' untranslated region and the activities of Pumilio (PUM), Nanos (NOS), and Brain tumor. To study this regulation, we have developed an RNA injection assay that faithfully recapitulates the regulation of the endogenous hb message. Previous studies have suggested that NOS and PUM can regulate translation by directing poly(A) removal. We have found that RNAs that lack a poly(A) tail and cannot be polyadenylated and RNAs that contain translational activating sequences in place of the poly(A) tail are still repressed in the posterior. These data demonstrate that the poly(A) tail is not required for regulation and suggest that NOS and PUM can regulate hb translation by two mechanisms: removal of the poly(A) tail and a poly(A)-independent pathway that directly affects translation.

Figure 1

Translational regulation of hb does not require a poly(A) tail. (A) Radiolabeled, m⁷GpppG-capped RNAs (500 pg/μl) corresponding to the hb 3′ UTR were injected into the anterior of WT embryos. Uninjected RNA controls (U) show transcript size without (−) and with (+) a poly(A) tail. A poly(A) tail is added to a hb 3′ UTR that lacks a poly(A) tail (lane 1). hb 3′ UTRs that lack a poly(A) tail and are end-blocked with cordycepin (lane 2) do not have a poly(A) tail added. Mutation of the polyadenylation signal (AAUAAA to AAUACA) also blocks addition of a poly(A) tail to these transcripts (lane 3). The injections shown were performed in the same experiment and analyzed on the same gel; intervening lanes have been removed. (B). HFH_WT RNAs (50 pg/μl) without (A0) a poly(A) tail were injected and incubated 60 min. n is the number of independent injections analyzed. † indicates that the data set is significantly different (P < 0.0001) than 1. Comparison of A25 with A0 (AAUACA) RNAs indicates that these are significantly different data sets (P < 0.0001). Reporters that lack a poly(A) tail are efficiently translated and differentially regulated in the anterior versus the posterior. mRNAs lacking a poly(A) tail that are also end-blocked with cordycepin are repressed in the posterior of the embryo. HFH_WT RNAs bearing a mutant polyadenylation signal (AAUAAA to AAUCAA) were injected at a concentration of 200 pg/μl and incubated for 60 min.

Figure 2

NRE-dependent translational repression in the presence of the HSL. (A) HFH RNAs (50 pg/μl) with WT or mutant (GU) NREs containing the HSL in place of the poly(A) tail were injected into WT or nos mutant embryos and incubated 60 min. n is the number of independent injections analyzed. † indicates that this data set is significantly different (P < 0.0001) from 1, the remaining data sets are not. Parallel injections were performed with HFH_WT RNAs (50 pg/μl) containing a poly(A) tail to confirm that poly(A)-containing mRNAs are regulated in a similar manner. Comparison of the aggregate data for HFH_WT A25 versus HFH_WT HSL indicates that populations are significantly different (P < 0.04). (B) Radiolabeled, m⁷GpppG-capped RNAs (500 pg/μl) corresponding to the hb 3′ UTR were injected into the anterior (A) or posterior (P) of embryos. (Left) Polyadenylated hb 3′ UTR. Uninjected controls (U) showing transcript size without (−) and with (+) a poly(A) tail. The poly(A) tail remains intact when injected into the anterior, but is rapidly removed when injected into the posterior. (Right) hb 3′ UTR with the HSL. Uninjected controls (U) showing transcript size without (−) and with (+) the HSL. The HSL remains intact in both anterior- and posterior-injected samples. (C) Reporter RNAs were injected and incubated for 60 min, the embryos were then divided and either assayed for F-Luc and R-Luc activities or harvested for total RNA. HFH_WT A25- or HSL-containing mRNAs were coinjected with HCH_GU A25 and R-Luc A25 reporters at a concentration of 50 ng/μl. Equal amounts of total RNA were used for reverse transcriptase–PCR with primers against the chloramphenicol acetyltransferase or F-Luc coding sequences. The numbers shown are for the experiment pictured. Analysis of all of the data demonstrate that no significant differences are found in the stability of poly(A)-containing mRNAs (A/P 1.4 ± 0.3; n = 5) or HSL-containing mRNAs (A/P 1.1 ± 0.2; n = 5) injected into the anterior versus the posterior of the embryo.

Figure 3

Poly(A)- and HSL-containing mRNAs are repressed differently. (A) HFH RNAs (50 pg/μl) containing either a poly(A) tail (Left) or the HSL (Right) were incubated 30, 60, or 90 min after injection into WT embryos. The A/P ratio for poly(A)-containing mRNAs continued to increase with longer incubation, consistent with the message being actively translated in the anterior, but silenced or inactivated in the posterior. Comparison of the 60- and 90-min time points for HFH_WT A25 demonstrates that they are significantly different (P < 0.005). In contrast, the HSL-containing mRNAs reach a maximal A/P difference at 60 min even though luciferase expression levels continue to increase. (B) Reporter RNAs were injected and incubated for the designated time and harvested for total RNA. Activity readings were gathered from embryos injected with the same batches of RNA, but they were not processed in parallel. HFH_WT A25- or HSL-containing mRNAs were coinjected with HCH_GU A25 and R-Luc A25 reporters at a concentration of 50 ng/μl. Embryo equivalent amounts of total RNA were used for reverse transcriptase–PCR with primers against the chloramphenicol acetyltransferase or F-Luc coding sequences. The quantitation shown is for the experiment pictured. Analysis of aggregate data demonstrate no significant differences in the stability of poly(A)-containing mRNAs (60′:1.4 ± 0.3; 90′: 0.9 ± 0.2) or HSL-containing mRNAs (60′: 1.1 ± 0.2; 90′: 1.1 ± 0.1) injected into the anterior versus the posterior of the embryo throughout the length of the study.

Figure 4

NRE-directed poly(A) removal is not cap-dependent. Radiolabeled RNAs (500 pg/μl) corresponding to the hb 3′ UTR were capped with either ApppG or m⁷GpppG and injected into the anterior (A) or posterior (P) of embryos. Uninjected controls (U) show transcript size without (−) and with (+) a poly(A) tail. The poly(A) tail remains intact when injected into the anterior, but is rapidly removed when injected into the posterior regardless of the structure of the cap.