Research Projects

Background: Neurons rely on the Integrated Stress Response (ISR) to withstand stress. However, chronic ISR is well known to induce neurodegeneration. ISR-driven eIF2α phosphorylation stops bulk translation yet still permits ATF4 and XRP1 synthesis, freeing mRNAs and RNA-binding proteins (RBPs) to condense into stress granules (SGs). SGs dissolve after acute stress, but under chronic stress, they stiffen and act as crucibles to seed irreversible protein aggregation, a hallmark of neurodegenerative diseases, as seen with TDP-43 in ALS/FTD. A newly appreciated SG type, the Chronic Stress Protective Response (CSPR) granule, emerges in Drosophila brain during chronic sensory quiescence. CSPR granules lack the canonical scaffold G3BP, sequester ATF4/XRP1, and can be dissolved by RNA-intercalating drugs, highlighting RNA as a structural lynchpin. Yet we still have no in vivo catalogue of the RNAs that nucleate or stabilise these newly appreciated SGs. Rationale: Our current knowledge of SG RNAs comes entirely from arsenite-stressed cell cultures, yielding conflicting profiles, from near-global capture to AU-rich subsets, each G3BP-baited and blind to G3BP-negative SGs. We will instead deploy Hyper-TRIBE in three blindness mutants, carT–/–, balaT–/–, norpAP24, whose neurons undergo robust chronic SGs. Fusing hyper-active ADARcd(E488Q) to Caprin and ATF4 will mark every bound RNA with A(Adenine)→I (Inosine) edits; deep sequencing will yield the first unbiased atlas of transcripts that stabilise chronic neuronal SGs. Hypothesis: We hypothesise that during sensory quiescence, Caprin drives the capture of a select set of neuronal mRNAs into SGs. Furthermore, ATF4, which carries a putative RNA-binding domain, may also play a part in this sequestration and provide structural support. Novelty: This study delivers the first in vivo, G3BP-independent transcriptome of chronic SGs. By combining Caprin and ATF4-Hyper-TRIBE with blind Drosophila, we sidestep arsenite artefacts and interrogate granules built by their native RBPs, exposing hidden RNA rules, grammars, and a new therapeutic foothold for neurodegeneration. Main experiments: Using CRISPR, will fuse Hyper-TRIBE to endogenous Caprin and ATF4. The tagged alleles and matching E488A and ADAR-only controls will be crossed into the three blindness mutants background. After confirming ISR-responsive expression and localisation to Caprin⁺/G3BP⁻ granules, adult heads will be collected, RNA extracted, and strand-specific libraries sequenced (greater or equal to 30 M paired-end reads per sample). A Hyper-TRIBER pipeline will call high-confidence A→I edits (greater or equal to 10× depth, FDR less then 0.05), and comparative analysis of Caprin- versus ATF4-marked sites will produce the definitive in vivo map of RNAs that hold chronic neuronal SGs together.