Repeat-associated non-AUG translation from antisense CCG repeats in fragile X tremor/ataxia syndrome

Abstract

Objective: Repeat-associated non-AUG (RAN) translation drives production of toxic proteins from pathogenic repeat sequences in multiple untreatable neurodegenerative disorders. Fragile X-associated tremor/ataxia syndrome (FXTAS) is one such condition, resulting from a CGG trinucleotide repeat expansion in the 5' leader sequence of the FMR1 gene. RAN proteins from the CGG repeat accumulate in ubiquitinated inclusions in FXTAS patient brains and elicit toxicity. In addition to the CGG repeat, an antisense mRNA containing a CCG repeat is also transcribed from the FMR1 locus. We evaluated whether this antisense CCG repeat supports RAN translation and contributes to pathology in FXTAS patients.

Methods: We generated a series of CCG RAN translation-specific reporters and utilized them to measure RAN translation from CCG repeats in multiple reading frames in transfected cells. We also developed antibodies against predicted CCG RAN proteins and used immunohistochemistry and immunofluorescence on FXTAS patient tissues to measure their accumulation and distribution.

Results: RAN translation from CCG repeats is supported in all 3 potential reading frames, generating polyproline, polyarginine, and polyalanine proteins, respectively. Their production occurs whether or not the natural AUG start upstream of the repeat in the proline reading frame is present. All 3 frames show greater translation at larger repeat sizes. Antibodies targeted to the antisense FMR polyproline and polyalanine proteins selectively stain nuclear and cytoplasmic aggregates in FXTAS patients and colocalize with ubiquitinated neuronal inclusions.

Interpretation: RAN translation from antisense CCG repeats generates novel proteins that accumulate in ubiquitinated inclusions in FXTAS patients. Ann Neurol 2016;80:871-881.

Figure 1. ASFMR1 transcript and putative RAN translation products

A) The FMR1 locus is bidirectionally transcribed with a start site (TSS) between exons 2 and 3 in the antisense orientation. ASFMR1a mRNA includes an AUG start codon upstream of the CCG repeat, creating a polyproline containing open reading frame (ASFMRP). RAN translation of ASFMR1a mRNA could result in proline (+0 reading frame, ASFMRpolyP), arginine (+1, ASFMRpolyR), and alanine (+2, ASFMRpolyA) repeat proteins. Underlined regions are epitopes used for antibody generation. B) ASFMR1 RAN specific reporters contain the ASFMR1 mRNA sequence including the repeat upstream of a C-terminally 3xFLAG tagged nanoLuciferase lacking a start codon (GGG-NL). Expression constructs were generated for each reading frame by addition of nucleotide frameshifts (+/−FS) at different CCG repeat lengths and with or without the AUG start codon for ASFMRP (+/−AUG).

Figure 2. RAN translation from CCG repeats in the proline reading frame of ASFMR1

A) Anti-FLAG western blot of whole cell lysates from COS-7 cells transfected with the indicated reporters for ASFMRP and ASFMRpolyP. Molecular weight of ASFMR1 derived proteins increased with expanded repeats (ATG P_x). Removal of the AUG start codon for ASFMRP (noATG P_x, left) eliminated these proteins in the absence of a repeat, but did not prevent their generation at larger repeat sizes. GAPDH served as a loading control and AUG-NL and GGG-NL served as positive and negative controls, respectively. B) NanoLuciferase activity from indicated constructs. For all figures, the Y axis is mean ± standard deviation expressed as fold change above GGG-NL (n>3). *=p<0.05 by Fisher’s LSD with Bonferroni correction for individual comparisons to GGG-NL and by ANOVA for repeat length dependent differences among RAN reporters. C) Localization of ASFMRP and ASFMRpolyP in transfected COS-7 cells stained for FLAG (green). There was no change in distribution with increasing repeat size and FLAG positive inclusions were not observed. 4′,6-Diamidino-2-phenylindole (DAPI, blue) was used to counterstain nuclei.

Figure 3. RAN translation from CGC repeats in the arginine reading frame of ASFMR1

A) western blot against FLAG in cells expressing the indicated ASMFRpolyR reporters. GAPDH was used as a loading control. “noATG” indicates the ATG codon in the proline reading frame was removed. B) NanoLuciferase activity derived from the indicated constructs 24hrs post transfection. *=p<0.05 by Fisher’s LSD with Bonferroni correction for individual comparisons to GGG-NL and by ANOVA for repeat length dependent differences among RAN reporters. C) FLAG staining of ASFMRpolyR constructs transfected into COS-7 cells. ASFMRpolyR staining (green) shifted to the nucleus in the presence of the expanded CCG repeats and co-localized (arrows) with the nucleolar marker nucleolin (red). DAPI (blue) was used to counter stain nuclei.

Figure 4. RAN translation from GCC repeats in the alanine reading frame of ASFMR1

A) western blot against FLAG on lysates from COS-7 cells transfected with indicated ASFMRpolyA reporters. Red asterisks indicate bands generated from initiation 3′ to the repeat site but in the human sequence at a non-canonical start codon. “noATG” indicates the ATG codon in the proline reading frame was absent. B) NanoLuciferase activity from ASFMRpolyA constructs compared to GGG-NL. *=p<0.05 by Fisher’s LSD with Bonferroni correction for individual comparisons to GGG-NL and by ANOVA for repeat length dependent differences among RAN reporters. C) Localization of ASFMRpolyA (green) was primarily nuclear (arrows) compared to AUG-NL which was cytoplasmic. DAPI (blue) was used to counterstain nuclei.

Figure 5. ASFMRpolyP and ASFMRpolyA antibody validation

A and C) Western blots of constructs probed with FLAG or antibody generated against ASFMRpolyP (α–ASpolyP, panel A), or against ASFMRpolyA (α–ASpolyA, panel C). GAPDH was used as a loading control. B and D) α–ASpolyP (red, panel B) recognized ASFMRPolyP but not AUG-NL by co-immunofluorescence. Similarly, α–ASpolyA (red, panel D) specifically recognized ASFMRpolyA but not AUG-NL.

Figure 6. ASFMRpolyP accumulates in FXTAS brain tissue and intranuclear inclusions

A) Control and FXTAS tissue from the indicated brain regions stained with α–ASpolyP. Hematoxylin (blue) was used as a counterstain to identify nuclei. In addition to strong perinuclear staining, nuclear aggregates (arrows) were observed in FXTAS cases that were not seen in control tissue. B) Higher magnification of intranuclear neuronal inclusions from the indicated brain regions from FXTAS cases probed with α–ASpolyP. C) Pre-immune sera for α–ASpolyP did not show staining in control or FXTAS patient cortex. D) Immunofluorescence of FXTAS hippocampus showed colocalization of ASFMRpolyP (green) with ubiquitin (red). DAPI (blue) was used to identify nuclei. Scale bar is 20μm. HIPP: hippocampus; CTX: frontal cortex; CB: cerebellum; MB: midbrain.

Figure 7. ASFMRpolyA RAN proteins aggregate in FXTAS brain tissue

A) Control and FXTAS tissue from the indicated brain regions stained with α–ASpolyA. Hematoxylin (blue) was used to identify nuclei. In addition to strong perinuclear staining, nuclear aggregates (arrows) were observed in FXTAS cases that were not seen in control tissue. B) Higher magnification of intranuclear neuronal inclusions from the indicated brain regions from FXTAS cases probed with α–ASpolyA. C) Pre-immune sera for α–ASpolyA did not show specific staining in control or FXTAS patient cortex. D) Co-immunofluorescence on FXTAS hippocampus showed colocalization of ASFMRpolyA (green) with ubiquitin (red). DAPI (blue) was used to identify nuclei. Scale bar is 20μm. HIPP: hippocampus; CTX: frontal cortex; CB: cerebellum; MB: midbrain.