Abstract
We have previously shown that the incomplete splicing of exon 1 to exon 2 of the HTT gene results in the production of a small polyadenylated transcript (Httexon1) that encodes the highly pathogenic exon 1 HTT protein. There is evidence to suggest that the splicing factor SRSF6 is involved in the mechanism that underlies this aberrant splicing event. Therefore, we set out to test this hypothesis, by manipulating SRSF6 levels in Huntington’s disease models in which an expanded CAG repeat had been knocked in to the endogenous Htt gene. We began by generating mice that were knocked out for Srsf6, and demonstrated that reduction of SRSF6 to 50% of wild type levels had no effect on incomplete splicing in zQ175 knockin mice. We found that nullizygosity for Srsf6 was embryonic lethal, and therefore, to decrease SRSF6 levels further, we established mouse embryonic fibroblasts (MEFs) from wild type, zQ175, and zQ175::Srsf6+/− mice and transfected them with an Srsf6 siRNA. The incomplete splicing of Htt was recapitulated in the MEFs and we demonstrated that ablation of SRSF6 did not modulate the levels of the Httexon1 transcript. We conclude that SRSF6 is not required for the incomplete splicing of HTT in Huntington’s disease.
Highlights
We have previously shown that the incomplete splicing of exon 1 to exon 2 of the HTT gene results in the production of a small polyadenylated transcript (Httexon1) that encodes the highly pathogenic exon 1 HTT protein
We show that zQ175 mouse embryonic fibroblasts (MEFs) express detectable and quantifiable levels of Httexon[1] and that, as we observed in brain, Httexon[1] and full-length Htt mRNA levels are unaffected by heterozygosity for Srsf[6] knockout
Our previous data suggested that the splicing factor serine/arginine-rich splicing factor 6 (SRSF6) might play a critical role in the mechanism that, in the context of expanded CAG repeats, underlies the incomplete splicing of the HTT gene resulting in the production of the highly pathogenic exon 1 HTT p rotein[9,10]
Summary
We have previously shown that the incomplete splicing of exon 1 to exon 2 of the HTT gene results in the production of a small polyadenylated transcript (Httexon1) that encodes the highly pathogenic exon 1 HTT protein. This is a attractive target as it is upstream of the pathogenic HTT protein, and understanding how the HTT mRNA is processed in the context of Huntington’s disease could inform future therapeutic strategies Both human HTT and mouse Htt are known to be transcribed into five mature protein-coding mRNAs: three endogenous 67 exon full-length transcripts that differ by the length of their 3′ UTRs and encode a 350 kDa protein[7,8] and, in the context of an expanded CAG repeat, two transcripts that contain exon 1 and intron 1 sequences (HTTexon1), and are translated to produce the exon 1 HTT p rotein[9]. They all function as constitutive and alternative splicing factors but have been shown to operate in a number of co-transcriptional and co-translational processes
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