Sam68 sequestration and partial loss of function are associated with splicing alterations in FXTAS patients

Abstract

Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder caused by expansion of 55-200 CGG repeats in the 5'-UTR of the FMR1 gene. FXTAS is characterized by action tremor, gait ataxia and impaired executive cognitive functioning. It has been proposed that FXTAS is caused by titration of RNA-binding proteins by the expanded CGG repeats. Sam68 is an RNA-binding protein involved in alternative splicing regulation and its ablation in mouse leads to motor coordination defects. Here, we report that mRNAs containing expanded CGG repeats form large and dynamic intranuclear RNA aggregates that recruit several RNA-binding proteins sequentially, first Sam68, then hnRNP-G and MBNL1. Importantly, Sam68 is sequestered by expanded CGG repeats and thereby loses its splicing-regulatory function. Consequently, Sam68-responsive splicing is altered in FXTAS patients. Finally, we found that regulation of Sam68 tyrosine phosphorylation modulates its localization within CGG aggregates and that tautomycin prevents both Sam68 and CGG RNA aggregate formation. Overall, these data support an RNA gain-of-function mechanism for FXTAS neuropathology, and suggest possible target routes for treatment options.

Figure 1

Expanded CGG repeats form intranuclear RNA aggregates. (A) COS7 cells were transfected with a plasmid expressing either no (a), 20 (b), 40 (c), 60 (d) or 100 (e) CGG repeats, transferred to 0.1% serum to block cell divisions and analysed 24 h after transfection by RNA FISH using a (CCG)_8x-Cy3 DNA probe. (B) Primary cultures of hippocampal embryonic mouse neurons (a), differentiated PC12 (b), SKOV3 (c) and COS7 (d) cells were transfected with a plasmid expressing 60 CGG repeats and analysed 24 h after transfection by FISH. (C) COS7 cells were co-transfected with a plasmid expressing 60 CGG repeats and a plasmid expressing GFP-tagged lamin-A, and analysed by RNA FISH either 24 (a), 48 (b) or 72 (c) hours after transfection. In all the figures, the magnification is × 630. The scale bars represent 10 μm; nuclei were counterstained with DAPI and one representative experiment from at least three separate experiments is shown.

Figure 2

Sam68 colocalizes with CGG RNA aggregates. (A) COS7 cells were transfected with a plasmid expressing 60 CGG repeats and analysed by FISH/IF using an antibody against MBNL1 (a) 24 h after transfection. None of the antibodies against hnRNP-G that we tested supported FISH conditions. Consequently, endogenous hnRNP-G (b) was analysed by co-transfection of COS7 cells with a plasmid expressing 60 CGG repeats fused to three MS2 tags and a plasmid expressing the GFP-MS2 Coat Protein. The MS2 Coat Protein (MS2CP) possesses a very high and specific affinity for MS2 RNA tags. Endogenous hnRNP-G (b) was detected by IF and the CGG aggregates by localization of the GFP-MS2CP protein, which is bound to the MS2-(CGG)_60x RNA. In the absence of MS2-(CGG)_60x, GFP-MS2CP was diffuse in the nucleoplasm (data not shown). (B) Similar to panel A but analysed 72 h after transfection. (C) COS7 cells (a, b) or differentiated PC12 neuronal cells (c) were transfected with a plasmid expressing 60 CGG repeats and analysed by FISH/IF using an antibody against Sam68, 24 h (a and c) or 72 h after transfection (b). (D) The percentage of endogenous Sam68, MBNL1 and hnRNP-G colocalized within CGG RNA aggregates in transfected COS7 cells 24 or 72 h after transfection. In all the experiments, three independent transfections totalling a hundred cells were counted, and results are presented as mean±s.d.

Figure 3

Sam68 colocalization within nuclear RNA aggregates is specific to expanded CGG repeats. COS7 cells were transfected with a plasmid expressing either 60 CGG repeats (A), 160 AUUCU repeats (B), 960 CUG repeats (C) or 300 CCUG repeats (D). Endogenous Sam68 and CGG repeats were analysed 24 h after transfection by FISH/IF.

Figure 4

Sam68 is essential for recruitment of MBNL1 and hnRNP-G within CGG aggregates. (A) COS7 cells were co-transfected with a plasmid expressing 60 CGG repeats and a plasmid expressing either a control LacZ shRNA (a), or a Sam68 shRNA (b), and analysed 72 h after transfection by FISH/IF. Endogenous MBNL1 was detected by IF using an Alexa-488-labelled secondary antibody. Simultaneous detection of Sam68 by IF using a Cy5-labelled secondary antibody confirmed shRNA-mediated depletion of endogenous Sam68. (B) Endogenous hnRNP-G cannot be detected by FISH/IF. Thus, COS7 cells were co-transfected with a plasmid expressing 60 CGG repeats, a plasmid expressing GFP-hnRNP-G and either control (a) or Sam68 (b) shRNA and analysed 72 h after transfection by FISH. (C) shRNA-mediated depletion of endogenous Sam68 was confirmed by western blotting against Sam68. (D) The percentage of endogenous Sam68, MBNL1 and GFP-hnRNP-G colocalized within CGG RNA aggregates 72 h after transfection in COS7 cells transfected with a plasmid expressing either a control or a Sam68 shRNA.

Figure 5

Sam68 colocalizes with endogenous CGG aggregates. (A) Brain sections of mouse expressing either control eight CGG repeats (a) or expanded 98 CGG repeats (b) were analysed by FISH/IF. (B) Similar to panel A. Brain sections (hippocampal area) of age-matched control (a) or FXTAS (b) patients were analysed by FISH/IF. Magnification: × 630. Scale bar, 10 μm. (C) Brain (hippocampal area) sections of age-matched control (a) or FXTAS (b) patients were analysed by immunohistochemistry directed against Sam68. Magnification: × 350.

Figure 6

FRAP analysis uncovers an immobile fraction of GFP-Sam68 within CGG aggregates. (A) Photobleaching was performed 24 h after transfection of COS7 cells with GFP-Sam68 and a plasmid expressing either no CGG repeats (a) or 60 CGG repeats (b). The white circles denote the photobleached regions in the aggregates and the nucleoplasm. Representative images show a single z-section obtained before photobleaching (pre-bleach) and at the indicated time points after photobleaching. (B) Recovery curves of photobleached aggregates and nucleoplasm in cells expressing 0 or 60 CGG repeats are shown as relative fluorescence intensity versus time. In CGG-expressing cells, recovery reached a plateau at ∼60% around 300 s. Each data point is the average of 10 nuclei. The error bars indicate the s.e.m.'s.

Figure 7

Alternative splicing is altered in CGG-expressing cells. COS7 cells were co-transfected with a Bcl-x minigene (A), an SMN2 exon-7 minigene (B), or an ATP11B exon-28B minigene (C) and a plasmid expressing either no CGG repeat, or 60 CGG repeats, Sam68 or Sam68 shRNA. Bcl-xL/S, SMN2 and ATP11B splicing isoforms were identified 24 h after transfection by RNA extraction followed by RT–PCR.

Figure 8

Alternative splicing is altered in FXTAS patients. (A) qRT–PCR analysis of ATP11B exon-28B in brain RNA samples of three controls and four FXTAS patients. Results are expressed as the ratio between alternative ATP11B exon-28B and constitutive ATP11B exons 21–22. (B) qRT–PCR analysis of SMN2 exon-7 in brain RNA samples from three controls and four FXTAS patients. We were not able to discriminate between SMN1 and SMN2 constitutive exons; therefore, results are expressed as the ratio of the alternative SMN2 exon-7/PO mRNA.

Figure 9

Tyrosine phosphorylation reduces Sam68 colocalization within CGG aggregates. (A) COS7 cells were transfected with a plasmid expressing 60 CGG repeats and either co-transfected with a plasmid expressing a constitutively active YF447 mutant of the SIK/Brk kinase (a), or treated with 10 μM of tyrphostin/AG490 (b) or 20 μM dephostatin (c) and analysed by FISH/IF 24 h after transfection. (B) The percentage of endogenous Sam68 colocalized within CGG RNA aggregates in transfected COS7 cells 24 h after transfection. (C) Similar to panel A but phosphorylation of Sam68 was assayed by Sam68 immunoprecipitation followed by western blotting using a Sam68-Y440 phospho-specific antibody. (D) Similar to panel A but COS7 cells were also co-transfected with an ATP11B minigene. Inclusion of exon-28B was quantified 24 h after transfection by RNA extraction followed by RT–PCR.

Figure 10

Tautomycin abolishes CGG and Sam68 aggregate formation. (A) COS7 cells were co-transfected with a plasmid expressing 60 CGG repeats and either treated with solvent (a) or 1 μM of tautomycin (b), then analysed by FISH/IF 24 h after transfection. (B) The percentage of cells containing CGG RNA aggregates in transfected COS7 cells treated with solvent, 0.3 or 1 μM of tautomycin. (C) Similar to panel A but followed by RNA extraction 24 h after transfection and qRT–PCR quantification of the CGG repeats RNA and of the neomycin mRNA. CGG and neomycin cassettes are expressed from the same pcDNA3.1 plasmid, but under different promoters. (D) Similar to panel A or C but COS7 cells were also co-transfected with an ATP11B minigene. Inclusion of exon-28B was identified 24 h after transfection by RNA extraction followed by RT–PCR.