DDX3X and specific initiation factors modulate FMR1 repeat-associated non-AUG-initiated translation

Abstract

A CGG trinucleotide repeat expansion in the 5' UTR of FMR1 causes the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). This repeat supports a non-canonical mode of protein synthesis known as repeat-associated, non-AUG (RAN) translation. The mechanism underlying RAN translation at CGG repeats remains unclear. To identify modifiers of RAN translation and potential therapeutic targets, we performed a candidate-based screen of eukaryotic initiation factors and RNA helicases in cell-based assays and a Drosophila melanogaster model of FXTAS. We identified multiple modifiers of toxicity and RAN translation from an expanded CGG repeat in the context of the FMR1 5'UTR. These include the DEAD-box RNA helicase belle/DDX3X, the helicase accessory factors EIF4B/4H, and the start codon selectivity factors EIF1 and EIF5. Disrupting belle/DDX3X selectively inhibited FMR1 RAN translation in Drosophila in vivo and cultured human cells, and mitigated repeat-induced toxicity in Drosophila and primary rodent neurons. These findings implicate RNA secondary structure and start codon fidelity as critical elements mediating FMR1 RAN translation and identify potential targets for treating repeat-associated neurodegeneration.

Keywords: DDX3X; Fragile X-associated tremor/ataxia syndrome; RAN translation; RNA helicase; eIF.

Figure 1. A candidate‐based screen reveals modifiers of repeat‐associated toxicity in Drosophila

Candidate modifiers are categorized here based on their known functions in gene expression. Fly genes are listed in the left columns, while their human homologs are listed in the right columns. Disruption of genes highlighted in dark blue strongly suppressed (CGG)₉₀‐EGFP toxicity. Disruption of genes highlighted in light blue weakly suppressed (CGG)₉₀‐EGFP toxicity. Disruption of genes highlighted in light red enhanced (CGG)₉₀‐EGFP toxicity selectively. Disruption of genes highlighted in dark red enhanced the toxicity of both (CGG)₉₀‐EGFP and GMR‐GAL4 (these were toxic independent of the repeat.) All other genes are displayed in white. The methyl‐7‐guanosine (m⁷G) cap, eIF4F complex, 43S pre‐initiation complex (PIC), and ribosomes are indicated.

Figure 2. Knockdown of belle mitigates repeat‐associated toxicity by inhibiting RAN translation in Drosophila

1. A

   Representative photographs of fly eyes expressing (CGG)₉₀‐EGFP under a GMR‐GAL4 driver, with various belle disruptions.

2. B

   Quantitation of GMR‐GAL4 and (CGG)₉₀‐EGFP eye phenotypes with belle disruptions (Mann–Whitney U‐test with Bonferroni corrections for multiple comparisons; n = 35–77/genotype).

3. C, D

   Longevity assays of (CGG)₉₀‐EGFP; Tub5‐GS (log‐rank Mantel–Cox test with Bonferroni corrections for multiple comparisons; n = 110–219/genotype) and (CGG)₉₀‐EGFP; ElaV‐GS (n = 147–299/genotype) flies with belle knockdown.

4. E

   Western blots of the FMRpolyG‐EGFP RAN product in (CGG)₉₀‐EGFP; Tub5‐GS flies with and without belle knockdown by two independent shRNAs.

5. F

   Quantitation of FMRpolyG‐EGFP band density, normalized to β‐tubulin band density, from blots in (E) (Student's t‐test; n = 4–5/genotype).

6. G

   Abundance of (CGG)₉₀‐EGFP mRNA normalized to RPL32 mRNA, following belle knockdown, determined by qRT–PCR (n = 8/genotype).

7. H

   Western blot of AUG‐driven EGFP in EGFP; Tub5‐GS flies with and without belle knockdown.

8. I

   Quantitation of EGFP band density, normalized to β‐tubulin band density, from blot in (H) (n = 4/genotype).

Data information: For all panels, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥ 3 replicates).

Figure 3. Knockdown of DDX3X inhibits RAN translation in cultured human cells

1. A

   Dose–response curves showing the effects of two independent anti‐DDX3X siRNAs on the expression of AUG‐NL‐3xF (top) and (CGG)₁₀₀ +1 NL‐3xF (bottom) reporters. Plasmid‐based reporters were transfected into HeLa cells 24 h after knockdown, and reporter expression was quantified by luminescence. Nanoluciferase (NL) luminescence has been normalized to luminescence from firefly luciferase (FF), which was co‐transfected, in order to control for transfection variability. Asterisks refer to comparisons between anti‐DDX3X siRNAs and siRNAs against EGFP (siEGFP; two‐way ANOVA with Dunnett's multiple comparisons test; n = 12/condition).

2. B, C

   (CGG)_n +1 and (CGG)_n +2 NL‐3xF expression (normalized to FF) with and without DDX3X knockdown across a range of CGG repeat sizes. Black asterisks refer to comparisons between siDDX3X‐ and siEGFP‐treated cells; orange asterisks refer to comparisons between siDDX3X‐treated cells expressing AUG‐NL‐3xF and those expressing a different reporter (two‐way ANOVA with Tukey's multiple comparisons test; n = 17–30/condition).

3. D, E

   Western blots of FMRpolyG‐NL‐3xF and FMRpolyA‐NL‐3xF products with and without DDX3X knockdown across a range of repeat sizes.

Data information: For all panels, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 for the specified statistical test. All panels depict data as means ± SD (compiled from ≥ 3 replicates).

Figure 4. DDX3X facilitates expression of RAN products at the level of translation

1. The expression of in vitro‐transcribed AUG, +1 (CGG)₁₀₀, and +2 (CGG)₁₀₀ NL‐3xF RNAs following DDX3X knockdown in HeLa cells, expressed as NL luminescence normalized to FF luminescence. (Student's t‐test with Bonferroni corrections for multiple comparisons; n = 21/condition).

2. Abundance of reporter mRNAs following DDX3X knockdown and plasmid‐reporter transfection, determined by qRT–PCR (n = 7/condition). This panel depicts data as means ± SEM.

3. The expression of AUG‐NL‐3xF and +1 (CGG)₁₀₀ NL‐3xF in in vitro translation extracts, collected from HeLa cells treated with siRNAs against EGFP or DDX3X (two‐way ANOVA with Tukey's multiple comparisons test; n = 4/condition).

4. Enrichment of HSPA1A and +1 (CGG)₁₀₀ NL‐3xF mRNA following anti‐DDX3X RIP, relative to incubation with isotype control IgG. MALAT RNA, in contrast, is not enriched (Student's t‐test, n = 3). Data from the additional replicate are presented in Appendix Fig S8A.

5. The expression of +1 and +2 (CGG)₁₀₀ NL‐3xF plasmid reporters with and without an AUG inserted 5′ to the CGG repeat, with and without DDX3X knockdown. Black asterisks refer to comparisons between siDDX3X‐ and siEGFP‐treated cells; orange asterisks refer to comparisons between siDDX3X‐treated cells expressing either +1 or +2 (CGG)₁₀₀ NL‐3xF and those expressing the respective AUG‐driven variant (two‐way ANOVA with Tukey's multiple comparisons test; n = 11–12/condition).

6. The expression of NL‐3xF plasmids with initiator AUG codons mutated to near‐AUG codons, with and without DDX3X knockdown (two‐way ANOVA with Dunnett's multiple comparisons test; n = 18–24/condition). Black asterisks refer to comparisons between siEGFP‐treated and siDDX3X‐treated cells; orange asterisks refer to comparisons between siDDX3X‐treated cells expressing AUG‐NL‐3xF and those expressing a different reporter; white asterisks refer to comparisons between siDDX3X‐treated cells expressing +1 (CGG)₁₀₀ NL‐3xF and those expressing a different reporter.

7. The expression of in vitro‐transcribed near‐AUG reporter RNAs in in vitro translation extracts, collected from HeLa cells treated with siRNAs against EGFP or DDX3X. Experiments with independent, replicate lysates are presented in Appendix Fig S7C (n = 4/group).

Data information: For all panels, ns = non‐significant, ***P ≤ 0.001, ****P ≤ 0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥ 3 replicates).

Figure 5. EIF4B and EIF4H modulate RAN translation and general translation in Drosophila and cultured human cells

1. A

   Representative photographs of GMR‐GAL4; (CGG)₉₀‐EGFP fly eyes expressing manipulations of eIF4B and eIF4H.

2. B

   Quantitation of GMR‐GAL4, (CGG)₉₀‐EGFP eye phenotypes with eIF4B/H manipulations (Mann–Whitney U‐test with Bonferroni corrections for multiple comparisons; n = 26–55/genotype).

3. C, D

   The expression of plasmid‐based AUG‐NL and +1 (CGG)₁₀₀ NL‐3xF reporters (C), or co‐transfected AUG‐FF reporters (D), following knockdown of EIF4B or EIF4H. Black asterisks refer to comparisons between siEGFP‐ and siEIF4B/H‐treated cells; pink and blue asterisks refer to comparisons between siEIF4B‐ (pink) or siEIF4H‐ (blue) treated cells expressing AUG‐NL‐3xF and those expressing +1 (CGG)₁₀₀ (two‐way ANOVA with Tukey's multiple comparisons test, n = 9/condition).

4. E

   The expression of plasmid‐based AUG‐NL‐3xF and (CGG)₁₀₀ +1 NL‐3xF reporters with and without over‐expression of EIF4B, EIF4H, or both (two‐way ANOVA with Dunnett's multiple comparisons test; n = 20/condition). Asterisks refer to comparisons between cells over‐expressing either EGFP or EIF4B, EIF4H, or EIF4B and EIF4H and expressing the same reporter.

Data information: For all panels, ns = non‐significant, *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001 for the specified statistical test. All panels present data as means ± SD (compiled from ≥ 3 replicates).

Figure 6. EIF1 and EIF5 modulate RAN translation by determining AUG start codon specificity

1. The expression of plasmid‐based NL‐3xF reporters in HEK293 cells with and without over‐expression of EIF1 (two‐way ANOVA with Sidak's multiple comparisons test; n = 9–12/condition). Black asterisks refer to comparisons between empty vector‐transfected and EIF1‐transfected cells; green asterisks refer to comparisons between EIF1‐transfected cells expressing AUG‐NL‐3xF and those expressing a different reporter.

2. The expression of plasmid‐based NL‐3xF reporters in HEK293 cells with and without over‐expression of EIF5 (two‐way ANOVA with Sidak's multiple comparisons test; n = 9–12/condition). Black asterisks refer to comparisons between empty vector‐transfected and EIF5‐transfected cells; pink asterisks refer to comparisons between EIF5‐transfected cells expressing AUG‐NL‐3xF and those expressing a different reporter.

Data information: For all panels, ***P ≤ 0.001, ****P ≤ 0.0001 for the specified statistical test. Bars represent mean ± SEM (compiled from ≥ 3 replicates).

Figure 7. Knockdown of DDX3X mitigates (CGG)₁₀₀ toxicity in primary rodent neurons

1. A

   Sample micrographs collected by automated longitudinal fluorescence microscopy, demonstrating the automated determination of cell death.

2. B

   Anti‐DDX3X Western blot of B35 cells transfected with either of two independent anti‐DDX3X LNAs or a control LNA.

3. C

   The expression of EGFP in primary rat neurons transfected with (CGG)₁₀₀ (+1) EGFP and either anti‐DDX3X LNAs or a control (one‐way ANOVA with Tukey's multiple comparisons test; n = 2,408–5,689 cells/condition). All graphs depict pooled data, normalized first within the replicate.

4. D, E

   Transfection of anti‐DDX3X LNA #1 (D) or #2 (E) reduced the cumulative risk of death in (CGG)₁₀₀ (+1) EGFP‐expressing neurons (Cox proportional hazard analysis; n = 2,408–3,676 cells/condition).

Data information: For all panels, ****P ≤ 0.0001 for the specified statistical test (compiled from ≥ 3 replicates).