Aberrantly spliced HTT, a new player in Huntington's disease pathogenesis

Abstract

Huntington's disease (HD) is an adult-onset neurodegenerative disorder caused by a mutated CAG repeat in the huntingtin gene that is translated into an expanded polyglutamine tract. The clinical manifestation of HD is a progressive physical, cognitive, and psychiatric deterioration that is eventually fatal. The mutant huntingtin protein is processed into several smaller fragments, which have been implicated as critical factors in HD pathogenesis. The search for proteases responsible for their production has led to the identification of several cleavage sites on the huntingtin protein. However, the origin of the small N-terminal fragments that are found in HD postmortem brains has remained elusive. Recent mapping of huntingtin fragments in a mouse model demonstrated that the smallest N-terminal fragment is an exon 1 protein. This discovery spurred our hypothesis that mis-splicing as opposed to proteolysis could be generating the smallest huntingtin fragment. We demonstrated that mis-splicing of mutant huntingtin intron 1 does indeed occur and results in a short polyadenylated mRNA, which is translated into an exon 1 protein. The exon 1 protein fragment is highly pathogenic. Transgenic mouse models containing just human huntingtin exon 1 develop a rapid onset of HD-like symptoms. Our finding that a small, mis-spliced HTT transcript and corresponding exon 1 protein are produced in the context of an expanded CAG repeat has unraveled a new molecular mechanism in HD pathogenesis. Here we present detailed models of how mis-splicing could be facilitated, what challenges remain in this model, and implications for therapeutic studies.

Keywords: HTT exon 1; Huntington’s disease; SRSF6; huntingtin fragment; mis-splicing.

Figure 1. Architecture of the 5′-splice site for HTT intron 1. (A) The exon 1-intron 1 junction is conserved between human and mouse and predicted to be a strong splice site. There is an in-frame stop codon within the first four bases of intron 1. 5′-splice site consensus sequences for high GC isochores showing nucleotide conservation at the respective positions (plotted with data from ref. 7). Cytosines in the -1 and -2 positions, as for HTT exon 1-intron 1 splice site, are rare. (B) The 3′ end of HTT exon 1 is underrepresented in next generation sequencing. Read coverage is shown for the HTT 5′UTR through the beginning of intron 1. Coverage is very shallow across the 3′ end of exon 1. Histograms of read coverage were created across the HTT gene. Reads were tabulated for 150 base stretches. Coverage follows a normal distribution with the 150 base stretch at the end of exon 1/beginning of intron 1 being consistently one of the lowest regions.

Figure 2. Diagram of the huntingtin transcript producing an exon 1 protein. The expanded repeat is represented by hash marks in exon 1, the stop codon as a red polygon, and polyA signal with a star. We observed exon 1-intron 1 transcripts for mouse and human mutant huntingtin that corresponded with cleavage at polyA signals in intron 1. Because of the immediate stop codon, the short transcript is translated into an exon 1 protein.

Figure 3. Involvement of SRSF6 in HTT mis-splicing. (A) SRSF6 is a classical SR protein with two RNA recognition motifs (RRM) and a serine and arginine rich (SR) domain. The SRSF6 binding motif resembles a CAG repeat. (B) We propose that SRSF6 binds to the expanded CAG repeat (hash marks). Two possible scenarios could arise from this: SRSF6 binding could interfere with U1 snRNP protection of the cryptic polyA signals in intron 1 by depleting the local pool of U1 snRNP by direct interaction; or SRSF6 binding interferes with the assembly of a stable and productive spliceosome at the 5′-splice site.

Figure 4. Human RNA-Seq data from Allen Brain Atlas demonstrates low representation of exon 1 coverage. RNA-Seq data from Allen is available as a table of exonic RPKMs. Plotted are HTT RPKMs for three individuals in four brain regions: cerebellar cortex (CBC), primary motor cortex (M1C), primary somatosensory cortex (S1C), and striatum (STR). RPKMs were averaged for all HTT exons (black) compared with RPKMs for exon 1 alone (red).