Induction of cap-independent BiP (hsp-3) and Bcl-2 (ced-9) translation in response to eIF4G (IFG-1) depletion in C. elegans

Abstract

During apoptosis, activated caspases cleave the translation initiation factor eIF4G. This cleavage disrupts cap-dependent mRNA translation initiation within the cell. However, a specific subset of mRNAs can still be recruited for protein synthesis in a cap-independent manner by the residual initiation machinery. Many of these mRNAs, including cell death related mRNAs, contain internal ribosome entry sites (IRESes) that promote their enhanced translation during apoptosis. Still other mRNAs have little dependence on the cap recognition mechanism. The expression of the encoded proteins, both anti- and pro-apoptotic, allows for an initial period of attempted cell survival, then commitment to cell death when damage is extensive. In this study we address the translational regulation of the stress and apoptosis-related mRNAs in C. elegans: BiP (hsp-3) (hsp-4), Hif-1 (hif-1), p53 (cep-1), Bcl-2 (ced-9) and Apaf-1 (ced-4). Altered translational efficiency of these messages was observed upon depletion of cap-dependent translation and induction of apoptosis within the C. elegans gonad. Our findings suggest a physiological link between the cap-independent mechanism and the enhanced translation of hsp-3 and ced-9. This increase in the efficiency of translation may be integral to the stress response during the induction of physiological apoptosis.

Keywords: Apaf-1; Bcl-2; BiP; cap-independent translation; eIF4G; germ cell apoptosis.

Figure 1. A splicing defect increases the proportion of non-cap-associated (p130) to cap-associated (p170) IFG-1 isoforms. (A) Diagram depicting the Mos transposon insert in the ifg-1 gene. The Mos transposon is inserted at 3 bp downstream of the exon-intron 5 junction. The strain was outcrossed 5 times to ensure the absence of additional mutations.(B) Northern blot hybridization depicts ifg-1 message populations. Northern blotting of wild type mRNA displays mRNAs of 2500 nt (a) and 4500 nt (b) indicative of p130 and p170 variants. To control for RNA loading, the blot was stripped and reprobed for gpd-3 RNA. Full-length p170 mRNA (4500 nt) was reduced by 42% and an additional mRNA variant (m*) at approximately 6000 nt was detected in the ifg-1::mos strain that accounted for 17% of all ifg-1 mRNAs. The ratio of p130:p170 mRNA increased 2-fold as p130 mRNA and the total ifg-1 mRNA amounts changed little relative to gpd-3 mRNA. (C) Diagrams representing the four partially spliced variants of ifg-1 mRNA due to the Mos transposon insertion. These splice variants were characterized by RT-PCR and sequencing. All three Mos-containing mRNAs encode nonsense codons in all three reading frames. (D) Western Blot for IFG-1 using the central antibody that detects both the p170 and p130 isoforms. Wild type worms express equal amounts of each IFG-1 isoform (p130:p170 ratio is 1.2), whereas the ifg-1::mos strain p130 abundance is 3.1-fold higher than p170. IFG-1 p170 consistently shows a much greater reduction, likely due to its instability. In the ifg-1::mos strain the total amount of IFG-1 expression is also reduced 30% in comparison to the wild type strain. Western blotting for actin was used as a loading control.

Figure 2.ifg-1::mos transgenic lines exhibits a marked increase in apoptotic corpses in the germ line and a decrease in fertility. (A) A graph depicting CED-1::GFP decorated corpses in the wild type, ifg-1::mos, and ced-9ts strains over time. There is an initial increase in apoptosis in all of the strains as they mature from the L3 larval stage, in which sperm fills the gonad and there is little to no apoptosis. During the L4 larval stage oocytes begin to progress through the gonad and the level of constitutive apoptosis increases. The level of apoptosis within the adult wild type gonad remains constant over time. However, the levels of apoptosis in the ifg-1::mos and ced-9ts lines continue to increase during the adult stage. *ifg-1::mos significantly different from wild type strain P < 0.05, ** P < 0.001, # ced-9ts significantly differs from the wild type strain P < 0.01, ## P < 0.001, § ifg-1::mos significantly differs from ced-9ts strain P < 0.001. (B and C) Fluorescence and DIC image of wild type, ifg-1::mos (D and E) and ced-9ts (F and G) strains expressing the CED-1::GFP apoptotic marker after 24 h at 25 °C. DIC images confirm normal growth and differentiation of oocytes within the gonads of mutant strains. Fertility and extent of larval/adult development were assayed in the wild type (H) and ifg-1::mos (I) strains at 25 °C. Brood size was quantified and the extent of embryonic, larval and adult development was assessed by visual morphology at 24-h intervals for 96 h following egg laying.

Figure 3.hsp-3 mRNA is translated more efficiently after the reduction of cap-associated p170 protein abundance. Polysome profiles depicting the continuous monitoring of absorbance at 254nm during sucrose gradient fractionation of worm lysates from wild type (A), ifg-1::mos (B) and ced-9ts (C) strains. These gradients represent 1 of 4 biological replicates of wild type and ced-9ts strains and 1 of 2 biological replicates of the ifg-1::mos strain. The profiles of biological replicates closely resemble one another and confirm the observed changes in translational efficiency. RNA distributions quantified by qPCR in each gradient fraction indicating changes in translational efficiency of gpd-3 in the ifg-1::mos (D) and ced-9ts (E) strains in comparison to the wild type strains. The percent mRNA was quantified by calculating the amount of a specific target mRNA in each fraction then dividing by the total amount of that mRNA across the entire gradient and multiplying by 100. qPCR quantified RNA distributions indicating changes in the translational efficiency of hsp-3 in the ifg-1::mos (F) and ced-9ts (G) strains in comparison to the wild type strain. Changes in translational efficiency of hsp-4 in the ifg-1::mos (H) and ced-9ts (I) strains are indicated by qPCR quantification of gradient fractions. All qPCR results from the ced-9ts strain were confirmed in four biological replicates and ifg-1::mos strain in biological duplicates.

Figure 4. 5′ UTR extended hsp-3 mRNA is not detected. (A) Diagram indicating the coverage of the hsp-3 mRNA probe. The arrow indicates the RPA probe extending upstream from exon 1 and covering all potential mRNA variants. (B) RNase Protection Assays were conducted by hybridizing the 5′ probe to total mRNA in wild type, ced-9ts, ifg-1::mos and wild type heat shocked strains. The arrows indicate the two detected hsp-3 mRNA 5′ variants. Control RNase Protection Assay detecting act-1 mRNA is shown to demonstrate RNA quality.

Figure 5. Other stress and apoptotic mRNAs, ced-9 (Bcl-2), hif-1 (HIF-1), cep-1 (p53) and ced-4 (Apaf-1) have differing abilities to be translated after depletion of cap-associated IFG-1 p170. RNA distributions were quantified by qPCR in wild type, ifg-1::mos, and ced-9ts strains from polysome profiles depicted in Figure 3. These gradients represent 1 of 4 biological replicates of wild type and ced-9ts strains and 1 of 2 biological replicates of the ifg-1::mos strain. Changes efficiency of ced-9 mRNA translation was determined in ifg-1::mos (A) and ced-9ts (B) strains in comparison to the wild type strain. RNA distributions quantified by qPCR indicating changes in translational efficiency of hif-1 in the ifg-1::mos (C) and ced-9ts (D) strains in comparison to the wild type strains. qPCR quantified RNA distributions indicating changes in the translational efficiency of cep-1 in the ifg-1::mos (E) and ced-9ts (F) strains in comparison to the wild type strain. Changes efficiency of total ced-4 mRNA translation were determined in ifg-1::mos (G) and ced-9ts (H) strains in comparison to the wild type strain. All qPCR data was confirmed in biological duplicates.

Figure 6.ced-4 mRNA structure and mode of translation differs from that of Apaf-1. (A) Diagram comparing C. elegans ced-4 and mammalian Apaf-1 mRNAs. ced-4 is the second gene in an operon with a canonical trans-splice splice site adjacent to its translation start site leaving no encoded 5′ UTR. However, the intergenic region upstream of this splice site has sequence typical of an IRES, such as polypyrimidine tracts and multiple AUG start sites. The arrows indicates the RPA probes extending upstream from exon 1 into the intergenic region. The probes were used to attempt to detect extended ced-4 message variants. (B) RNase Protection Assays were conducted by hybridizing the 5′ probe to total mRNA in wild type, ced-9ts, ifg-1::mos and wild type heat shocked strains. The arrows indicate the SL2 trans-spliced message variant. A band consistent with extended ced-4 mRNA that could not be confirmed with longer ced-4 mRNA probe or other characterization methods (*). (C) RNase Protection Assay using a probe spanning the entire intergenic region. Arrows indicate the detected ced-4 5′ end points. The 3′ UTR of the upstream gene was also protected as indicated by the specified arrows. Control RNase Protection Assay detecting act-1 mRNA is shown to demonstrate RNA quality. (D) Verification of ced-4 mRNA endpoints by circularized RT-PCR. Total mRNA was isolated from C. elegans wild type worms. These mRNAs were decapped using TAP to allow T4 ligation of the 5′ end and the poly(A) tail of mRNAs. cDNA across the junction was then synthesized. An additional nested PCR was performed and the resulting cDNA amplifications were subcloned and sequenced. Agarose gel electrophoresis of nested PCR across the ligated junction is shown. Extended mRNA variants differed due to poly(A) tail size and alternative 3′ end points.

Figure 7. Model for the induction of cap-independent translation of stress-related mRNAs during apoptosis. A. Model comparing the translational efficiencies of C. elegans and mammalian stress related mRNAs. Wide width arrows indicate the increased translational efficiency of C. elegans hsp-3 and ced-9 mRNAs and human BiP, Bcl-2, Apaf-1, Hif-1 and p53. The decreased translational efficiency of other C. elegans mRNAs, ced-4, hif-1 and cep-1, is shown by an “x.” * indicates the genetic manipulation of the ifg-1::mos strain. B. The induction of apoptosis in humans leads to the activation of caspases and cleavage of the cap-dependent translation initiation factor eIF4GI and constitutively cap-independent translation initiation factor p97. This cleavage leads to the induction of cap-independent protein synthesis. As a result, the translational efficiency is increased for pro- and anti-apoptotic messages such as pro-apoptotic Apaf-1 and recovery mRNAs Bcl-2 and BiP. The translation of Bcl-2 and BiP promotes periods of cellular recovery from stress. Subsequent translation of Apaf-1 leads to cellular suicide. In the C. elegans gonad, apoptosis activated caspase, CED-3 leads to C. elegans eIF4GI (IFG-1 p170) and p97 (IFG-1 p130) cleavage. This cleavage leads to an increase in p130-driven translation of stress-related mRNAs hsp-3 and ced-9. * indicates that genetic manipulation in the ifg-1::mos and ced-9ts strains leads to the induction of cap-independent protein synthesis. # indicates a proapoptotic function for CED-9.