Fragile X mental retardation 1 gene enhances the translation of large autism-related proteins

Abstract

Mutations in the fragile X mental retardation 1 gene (FMR1) cause the most common inherited human autism spectrum disorder. FMR1 influences messenger RNA (mRNA) translation, but identifying functional targets has been difficult. We analyzed quiescent Drosophila oocytes, which, like neural synapses, depend heavily on translating stored mRNA. Ribosome profiling revealed that FMR1 enhances rather than represses the translation of mRNAs that overlap previously identified FMR1 targets, and acts preferentially on large proteins. Human homologs of at least 20 targets are associated with dominant intellectual disability, and 30 others with recessive neurodevelopmental dysfunction. Stored oocytes lacking FMR1 usually generate embryos with severe neural defects, unlike stored wild-type oocytes, which suggests that translation of multiple large proteins by stored mRNAs is defective in fragile X syndrome and possibly other autism spectrum disorders.

Figure 1.. Fmr1 is specifically required during the storage of mature, quiescent stage 14 oocytes in the ovary.

(A) Schematic of a Drosophila ovariole with immature pre-checkpoint follicles and two stored mature stage 14 follicles. (B) Plot shows arrested mature follicle stability (red) following feeding protocol as described (Supplemental). (C) Fmr1 knockdown (line #1 and line #2), but not knockdowns of controls or other indicated genes, specifically reduces 10-day stored (red) but not 1-day stored (black) oocytes from developing into hatching larvae. (D) Fmr1 germline RNAi during storage progressively reduces the fraction of mature oocytes competent after 1, 4, 7 or 10 days of storage to support development. Re-feeding females to promote maturation of fresh stage 8 follicles restores full developmental potential.

Figure 2.. Stored FMR1-depleted oocytes frequently generate embryos with neural defects.

(A) Control oocytes stored in vivo for 1 day or 10 days support normal embryonic nervous system development (22C10 antibody). Fmr1 RNAi oocytes stored in vivo for 1 day support normal development but after storage for 10 days produce highly abnormal nervous system development. (B) Normal ventral nerve cord (BP102 antibody) from embryo developed from 10 day control oocyte (C), abnormal ventral nerve cords including broken or fused connectives (arrows) from three embryos developed from 10 day Fmr1 RNAi oocytes (D) Normal nerve cord from a control oocyte stored 14 days. (E) Summary of nervous system development in embryos from Control (GFP) and Fmr1 RNAi oocytes. Bar = 20μm.

Figure 3.. FMR1 stimulates the translation during storage of transcripts from multiple intellectual disability and autism genes.

(A) Translational profile (log₁₀TPM; transcripts per million) and mRNA abundance profile (mRNA-seq, log₁₀TPM) are highly similar between control vs. Fmr1-RNAi oocytes (stored 1–2d). (B) Top genes translationally reduced by Fmr1 RNAi from 11 ribosome footprinting experiments do not show significant changes in mRNA levels. (C) Significance vs. fold change plot reveals 421 candidate targets translationally stimulated by FMR1 (p<0.01; t-test). Protein size class indicated by color. (D) Cumulative plot of translation (Fmr1 RNAi/control) as a function of protein size (left), or translational efficiency (TE, right) defined as ribosome footprinting TPM(Fmr1 RNAi)/mRNA-seq TPM. (E) Translation of large mRNAs in Fmr1 RNAi vs. controls is reduced independent of TE. (F) Normalized read depth is plotted for two FMR1 targets (Poe and Huwe1) and two non-targets (Orb and Top2). In Fmr1 RNAi oocytes, target gene footprints are reduced at all positions along the mRNA.

Figure 4.. Poe is required for oocyte storage and neural development.

(A) Poe mutation accelerates oocyte decline during storage. (B) Poe mutant oocytes frequently fail to support normal neural development after prolonged storage. (C) POE antibody staining (see Methods) during follicle development, showing germline granules that arise in maturing follicles. Bar = 3μm. (D) Many POE granules are seen in wild type stage 10 follicles, but not in Poe RNAi, Poe⁰¹⁶⁵⁹, Fmr1 RNAi, or null Fmr1^3/Δ50 follicles. Fmr1 RNAi combined with POE overexpression recovers POE granules. Bar = 20μm.