Fig 1: Schematic depicting mechanisms influencing incomplete splicing of HTT in HD. In the normal situation (left panel), U1 snRNP protects cryptic polyA sites from being utilized and defines the 5′ splice site at the exon 1/intron 1 junction. Transcription by PolII is fast and thus HTT is properly spliced and the full-length mRNA is produced. In HD (right panel), there is an increased amount of SRSF6 binding to the elongated CAG repeat, which could sequester U1 snRNP resulting in interference with 5′ spliceosome formation and cryptic polyA site de-protection. In addition, transcription throughout the 5′ end of the gene is slower, kinetically allowing a higher probability of cryptic polyA site usage. Together, these mechanisms lead to the generation of HTTexon1
Fig 2: SRSF6 levels modulate incomplete splicing of Htt. a–e Overexpression of SRSF6 increases the amount of incomplete splicing. Mouse Srsf6 (a) and human SRSF6 (b), respectively, were over-expressed to the same extent in the CAG7 and CAG100 cell lines. c Western blot data confirmed the over-expression of mouse and human SRSF6 for both CAG repeat lengths. - = mock; m = mouse SRSF6; h = human SRSF6. Uncropped blots can be found in Supplementary Fig. 8A. d Spliced exon 1–exon 2 transcripts were not significantly changed due to the overexpression of mouse or human SRSF6. e Intron 1 containing transcripts were increased to statistically significantly levels when human SRSF6 was over-expressed. Individual data points and the mean ± s.e.m. are shown. n = 8 independent experiments/CAG-length; two-way ANOVA with Tukey post hoc. Effect of SRSF6 overexpression for a given CAG-length: ###p < 0.001. Effects due to CAG-length for a given treatment: ***p < 0.001. f–i Knock-down of SRSF6 by siRNA treatment (s12740, ThermoFisher) decreased the amount of incomplete splicing. SRSF6 levels were decreased by siRNA treatment on transcript (f) and protein (g) levels for both CAG repeat lengths. scr = scramble; si = siRNA treatment. Uncropped blots can be found in Supplementary Fig. 8B. h Spliced exon 1–exon 2 transcripts were not changed due to the knock-down of SRSF6. i There was a statistically significant reduction in the amount of incompletely spliced minigene in the CAG100 line. Individual data points and the mean ± s.e.m. are shown. n = 3–6 independent experiments/CAG-length; two-way ANOVA with Tukey post-hoc. Treatment for a given CAG-length: *p < 0.05, **p < 0.01, ***p < 0.001. Treatment x CAG-length ###p < 0.001
Fig 3: Characterisation of the Srsf6 knockout mouse line. (a) Schematic of mouse Srsf6 with scissors denoting approximate Cas9 cleavage sites used to generate the knockout allele. Sanger sequencing was used to confirm the deletion breakpoint for the Δ990 bp line. UTR untranslated region. (b) qPCR analysis showed Srsf6 mRNA levels to be 50% of WT whereas, Srsf4 or Srsf5 levels were unchanged in cortex from 2 month old Srsf6+/− Δ990 mice. n = 7 WT and 4 Srsf6+/− mice. (c) Western blot analysis showed a 50% reduction in SRSF6 in cerebellum and cortex from 2 month old Srsf6+/− mice compared to WT littermates. See Supplementary Fig. S7 for uncropped blots and total protein loading controls. n = 5/genotype. Statistical analyses were by unpaired Student’s t-tests. Test statistics can be found in Supplementary Table S4. WT wild type.
Fig 4: QuantiGene analysis of Htt transcripts in brain regions from the progeny of the zQ175 and Srsf6+/− mouse cross. (a) Schematic of the location of the QuantiGene plex probe sets on the mouse Htt transcript. (b) Srsf6+/− female mice were bred to zQ175 knockin male mice to generate progeny with four genotypes: WT, Srsf6 heterozygous knockout (Srsf6+/−), zQ175 knockin and double mutants (zQ175::Srsf6+/−). (c) Httexon1 was detected in the cortex, striatum, hippocampus and cerebellum of 2 month old zQ175 mice but was not altered by heterozygosity for Srsf6 knockout. (d) Full-length Htt was measured in the cortex, striatum, hippocampus and cerebellum using the Htt exon 50–53 assay. Cortical full-length Htt was significantly lower in zQ175 mice compared to WT and this was not changed by heterozygosity for Srsf6 knockout. n = 6/genotype. Statistical analysis was by one-way ANOVA with Bonferroni correction for multiple pairwise comparisons, ***p < 0.001, p < 0.2 values are indicated. Test statistics can be found in Supplementary Table S5. WT wild type.
Fig 5: Measurement of Httexon1 and Htt transcripts in WT, zQ175 and zQ175::Srsf6+/− MEFs after transfection with an siRNA targeting Srsf6 (siSRSF6). QuantiGene analysis was used to measure Httexon1 and Htt mRNA levels 72 h after siSRSF6 or siNC transfection. Neither Httexon1 nor full-length Htt levels were changed in MEFs treated with siSRSF6 compared to siNC. n = 3 biological replicates/genotype. Statistical analysis was by two-way ANOVA, ***p < 0.001. Test statistics can be found in Supplementary Table S10.
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