Targeted 3'-end RNA sequencing uncovers cryptic polyadenylation in Huntington's disease linked to somatic instability and CAG repeat purity

Velasco-Bilbao, A.; Manterola, M.; Herrero-Reiriz, A.; Carazo-Hidalgo, M.; Misiukiewicz, A.; Arnold-Garcia, O.; Perez-Navarro, E.; Hallegger, M.; Ule, J.; Rabano, A.; Lopez de Munain, A.; Olejniczak, M.; Brito, V.; Blazquez, L.

doi:10.64898/2026.01.03.697463

bioRxiv · 10.64898/2026.01.03.697463

Targeted 3'-end RNA sequencing uncovers cryptic polyadenylation in Huntington's disease linked to somatic instability and CAG repeat purity

Velasco-Bilbao, A.·Manterola, M.·Herrero-Reiriz, A.·Carazo-Hidalgo, M.·Misiukiewicz, A.·Arnold-Garcia, O.·Perez-Navarro, E.·Hallegger, M.·Ule, J.·Rabano, A.·Lopez de Munain, A.·Olejniczak, M.·Brito, V.·Blazquez, L.

View original source ↗Export metadata ↓

Abstract

Huntingtons disease (HD) is a progressive neurodegenerative disorder caused by expanded CAG repeats in the first exon of the HTT gene, which encodes for huntingtin (HTT) protein. Full-penetrance is established at 40 repeats, but beyond, somatic repeat instability in the brain and CAG repeat purity modulate disease onset and severity. Previous studies have described that expanded repeats induce the incomplete splicing of HTT intron 1 to express the most pathogenic HTT isoform, known as HTT1a. Yet, the lack of a robust and sensitive method to evaluate HTT RNA-misprocessing has limited our understanding of HTT1a expression in HD pathophysiology. Here we describe a targeted RNA sequencing approach, known as 3-end targeted RNA sequencing or 3TRS, to simultaneously quantify multiple HTT transcripts generated by canonical and cryptic polyadenylation in several HD models. We show that activation of HTT cryptic polyadenylation is highly selective and requires long and uninterrupted CAG repeat expansions. In HD knock-in mice and human postmortem brain, cryptic HTT expression strongly correlates with brain-specific somatic repeat instability, supporting a model where ultralong and unstable CAG repeats drive toxicity by activating HTT RNA-misprocessing. Overall, 3TRS provides a robust framework to investigate HTT1a biogenesis and expression and to evaluate HTT-lowering therapeutic strategies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/697463v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@168d8e1org.highwire.dtl.DTLVardef@76ba3forg.highwire.dtl.DTLVardef@bdaf93org.highwire.dtl.DTLVardef@151e581_HPS_FORMAT_FIGEXP M_FIG C_FIG

Explore related subjects

molecular biology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Velasco-Bilbao, A., Manterola, M., Herrero-Reiriz, A., Carazo-Hidalgo, M., Misiukiewicz, A., Arnold-Garcia, O., Perez-Navarro, E., Hallegger, M., Ule, J., Rabano, A., Lopez de Munain, A., Olejniczak, M., Brito, V., Blazquez, L.. 2026-01-03. Targeted 3'-end RNA sequencing uncovers cryptic polyadenylation in Huntington's disease linked to somatic instability and CAG repeat purity. https://doi.org/10.64898/2026.01.03.697463

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

An ssRNA Phage Single-gene Lysis Protein Targeting MraY Reveals Convergent Evolution Across the RNA-DNA Phage Boundary

Small lytic phages of the ssRNA Leviviricetes and ssDNA Microviricetes achieve host lysis by expression of a single protein, designated as an Sgl protein. Four ssRNA Sgls have been shown to inhibit steps in peptidoglycan biosynthesis: three target MurJ, the Lipid II flippase, and one targets MurA, the committed step of the pathway. A fifth Sgl, the E protein of the canonical ssDNA microvirus {varphi}X174, inhibits MraY, which catalyzes the synthesis of the first lipid-linked intermediate. This inhibition requires SlyD, a cytoplasmic FKBP-type peptidyl-prolyl cis-trans isomerase. The Sgl of the Caulobacter ssRNA phage {varphi}Cb5 (Cebevirus halophobicum) has not been characterized. Here, we present physiological evidence that Sgl{varphi}Cb5 targets MraY. Moreover, we show that, despite the absence of detectable sequence similarity, Sgl{varphi}Cb5 shares several features with the {varphi}X174 E protein, including the distribution of hydrophobic and charged residues, a proposed N-out, C-in membrane topology, and retention of lytic activity in an Sgl{varphi}Cb5 -LacZ fusion. Furthermore, expression of the Caulobacter cytoplasmic FKBP-type peptidyl-prolyl cis-trans isomerase CC2799 enhances Sgl{varphi}Cb5 -mediated lysis in E. coli. These parallels between Sgl function in a DNA phage and an RNA phage either represent remarkably consonant convergent evolution or some evolutionary cross-over event for which there is currently no precedent.

molecular biology↗

bioRxiv2026

HERC1-mediated orphan subunit quality control maintains proteasome homeostasis and is disrupted in HERC1-associated neurodevelopmental disease

Biallelic hypomorphic variants in the E3 ubiquitin ligase HERC1 cause a neurological disorder clinically characterized by macrocephaly, dysmorphic facies, and psychomotor delay. Identifying the substrates of HERC1 is essential for understanding its biological functions and the mechanisms underlying disease-associated variants. To address this, we performed a quantitative proteomic analysis using biotinylated ubiquitin to identify HERC1 targets and examine their regulation in cells expressing HERC1 with or without ubiquitin ligase activity. This approach revealed a broad set of HERC1-dependent ubiquitylation substrates, including proteins from major multiprotein complexes such as the proteasome, the translation initiation machinery, and pathways involved in cell metabolism. Focusing on the proteasome, the most prominently represented complex, we identified several regulatory particle subunits, including PSMC6, PSME1, and PSME2. Specifically, within the 19S regulatory particle, we demonstrated that ubiquitylation of the PSMC6 subunit involves the formation of K48-linked polyubiquitin chains and depends on HERC1 activity. Other 19S subunits, such as PSMC2, PSMC4, and PSMC5, undergo a similar modification. Mechanistically, we show that HERC1 selectively recognizes unassembled subunits, promotes their ubiquitylation, and targets them for proteasomal degradation. Notably, in HERC1-deficient cells, disruption of this mechanism impaired proteasomal activity. At the disease level, quantitative proteomic analysis of the pathogenic HERC1 variant c.1448G>A (p.Gly483Glu) likewise identified altered ubiquitylation of proteasome subunits and components of the translation initiation machinery. Biochemical analyses further revealed impaired ubiquitylation of PSMC6 by the mutant HERC1 protein, associated with a loss of interaction between HERC1 and PSMC6. Together, these findings establish a link between HERC1-associated neurological disorders and defective ubiquitylation pathways. They elucidate the molecular mechanisms by which HERC1 regulates proteostasis and demonstrate how disruption of these processes contributes to disease pathogenesis, thereby highlighting potential avenues for therapeutic intervention.

molecular biology↗

bioRxiv2026

PKR inhibition prevents maladaptive responses to endoplasmic reticulum (ER) stress in early-onset dystonia DYT-PRKRA.

DYT-PRKRA is a rare, childhood-onset dystonia caused by mutations in the PRKRA gene, which encodes the protein PACT. PACT activates protein kinase R (PKR) in response to endoplasmic reticulum (ER) stress. In DYT-PRKRA patient cells, the PACT-PKR interaction is enhanced, increasing sensitivity to ER stress-induced apoptosis. The canonical protein kinase activated by ER stress is PKR-like endoplasmic reticulum kinase (PERK), which phosphorylates the subunit of eukaryotic translation initiation factor 2 (eIF2) to initiate the integrated stress response (ISR) and restore cellular homeostasis via the transcription factor ATF4. However, under persistent stress, ATF4 induces CHOP, shifting the adaptive response toward apoptosis. PKR, also activated by ER stress, phosphorylates eIF2, but its contribution to cellular homeostasis remains understudied. This work shows, for the first time, that PKR activation is essential for CHOP induction during ISR, directly linking enhanced apoptosis in DYT-PRKRA cells to prolonged PKR activation and heightened CHOP induction. Strikingly, a small-molecule PKR inhibitor effectively blocks CHOP expression and significantly decreases apoptosis in DYT-PRKRA cells, suggesting PKR inhibition as an effective therapeutic strategy for DYT-PRKRA.

molecular biology↗