Discovery of Novel Isoforms of Huntingtin Reveals a New Hominid-Specific Exon

<div><p>Huntington’s disease (HD) is a devastating neurological disorder that is caused by an expansion of the poly-Q tract in exon 1 of the Huntingtin gene (HTT). HTT is an evolutionarily conserved and ubiquitously expressed protein that has been linked to a variety of functions including transcriptional regulation, mitochondrial function, and vesicle transport. This large protein has numerous caspase and calpain cleavage sites and can be decorated with several post-translational modifications such as phosphorylations, acetylations, sumoylations, and palmitoylations. However, the exact function of HTT and the role played by its modifications in the cell are still not well understood. Scrutiny of HTT function has been focused on a single, full length mRNA. In this study, we report the discovery of 5 novel <i>HTT</i> mRNA splice isoforms that are expressed in normal and <i>HTT</i>-expanded human embryonic stem cell (hESC) lines as well as in cortical neurons differentiated from hESCs. Interestingly, none of the novel isoforms generates a truncated protein. Instead, 4 of the 5 new isoforms specifically eliminate domains and modifications to generate smaller HTT proteins. The fifth novel isoform incorporates a previously unreported additional exon, dubbed 41b, which is hominid-specific and introduces a potential phosphorylation site in the protein. The discovery of this hominid-specific isoform may shed light on human-specific pathogenic mechanisms of HTT, which could not be investigated with current mouse models of the disease.</p></div

Identification of a novel HTT isoform incorporating a previously unreported hominid-specific exon.

<p>(A) RNAseq analysis showing the clear incorporation of a non-reported exon. (B) Alignment of the genomic sequences of exon 41b of mouse and primates, demonstrating the very recent acquisition of this exon along human evolution. Only hominidae family members (in red) have both splice donor and acceptor and maintain the frame. (C) RT-PCR with primers specific for HTT-41b isoform unmistakably detects expression of HTT-41b in hES cells, without amplifying the canonical HTT isoform. (D) Quantification of HTT-41b isoform expression through qRT-PCR at different time points upon neural differentiation of hES cells. Results are shown as mean+SEM of 3–6 independent replicates.</p

Time course of <i>HTT</i> isoform expression in hESCs differentiating to telencephalic neural fate.

<p>(A-D) RUES2 hESCs were differentiated to neural fate by blocking both branches of TGFβ signaling (default mechanism) as described in Materials and Methods. Values are normalized by GAPDH and displayed as fold change to day 0 values. Only the HTT-Δ10 isoform consistently decreases as the cells differentiate, while all three other isoforms maintain their expression levels unchanged. Error bars represent the standard error of the mean of 3 to 6 independent replicates. * p<0.05 vs d0.</p

HTT protein consequences of the 4 novel shorter isoforms.

<p>(A, C-E) Alignment of sequencing data obtained from amplicons shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127687#pone.0127687.g002" target="_blank">Fig 2</a> to canonical HTT, confirming the isoform sequences obtained from the RNAseq analysis. Red protein sequences represent predicted lost amino acids. Amino acids highlighted with an asterisk represent Ser434 in (A) and Asp552 in (C), sites of phosphorylation and caspase cleavage, respectively. (B) Alignment of human, mouse, frog and zebrafish HTT mRNA sequences show that Exon10 is specifically missing in the gene model of frog Htt, while it is present in mammalians (human, mouse) and fish (zebrafish).</p

PCR and qPCR validation of novel HTT isoforms.

<p>(A) RT-PCR results with primers specific for each individual splice isoform, detecting expression of HTT-Δ10, Δ12, Δ13 and Δ46 in hESCs cells. A plasmid containing the canonical full-length HTT was used to control for non-specific amplification of canonical HTT mRNA. (B) Quantification of HTT isoform expression in hES cells through qPCR in GENEA019 and GENEA020 hESCs. Data represents mean + SEM of 3 replicates.</p

RNA-seq analysis reveals novel isoforms of <i>HTT</i>.

<p>Using Tuxedo software, 10 putative <i>HTT</i> splice isoforms were detected in RNA-seq data. (A) RUES2 RNA-seq reads were aligned to the hg19 genome, demonstrating good coverage of HTT mRNA used for the isoform analysis. (B) Diagram depicting the 5 HTT isoforms validated in this study. (C) Frequency of all detected isoforms across the three RNA-seq samples. aSA: alternative splice acceptor, aSD: alternative splice donor.</p