Neuron-specific alternative polyadenylation in nervous system development and function
Final Report Abstract
The 3’ termini of metazoan mRNAs determine transcript stability, translation efficiency and subcellular localization. A subset of genes exhibit synchronous lengthening of their 3’ UTR during the course of Drosophila embryogenesis. These extended 3’ UTR sequences are selectively expressed in the nervous system. The pan-neuronal RNA- binding protein ELAV coordinates 3’ extension by binding to proximal poly(A) signals and inhibiting processing at that site. Studies in zebrafish, mouse and human suggest that 3’ UTR extension is a conserved feature of neurogenesis. For this project, I proposed two aims: first, to determine the function of the 3’ UTR extension of specific genes in the Drosophila nervous system, and second, to gain insight into the mechanism of ELAV- mediated 3’ UTR extension. Preliminary data indicated that flies lacking the extended portion of the ago1 3’ UTR suffer from neurodegeneration. Further testing was consistent with the hypothesis that 3’ UTR extensions cause neurological phenotypes, but also showed a significant variability in the behavioral results. I decided to substantially improve the experimental controls, which involved generating additional, independent mutant alleles. This significantly postponed the proposed phenotypic, biochemical, and whole-genome experiments. In total, I generated mutants for five ELAV targets. In addition, In situ hybridization on adult brains indicated that 3’ UTR extensions localize to synaptic regions. MS2-bearing flies were generated using CRISPR/Cas9-assisted homologous recombination in order to perform live imaging in the future. The second aim was based on preliminary evidence suggesting that promoter regions play a role in ELAV-mediated 3’ UTR extension. I generated transgenic flies carrying reporter constructs with a number of promoter/extension combinations, and showed that extension-associated promoter sequences are required for ELAV-mediated 3’ UTR extension. Computational and genetic analyses provide strong evidence that promoterproximal RNA Polymerase II pausing and the promoter-associated GAGA element help foster efficient ELAV-mediated 3’ UTR extension. ELAV ChIP-Seq assays identified ELAV in the promoter regions of extended genes, suggesting that the interaction between ELAV and promoter elements acts to “poise” 3’ UTR extension.
Publications
- Alternative polyadenylation coupled to transcription initiation: Insights from ELAV-mediated 3' UTR extension. RNA Biology 2015, 12: 918-921
Hilgers V
(See online at https://doi.org/10.1080/15476286.2015.1060393) - ELAV links paused Pol II to alternative polyadenylation in the Drosophila nervous system. Molecular Cell 2015, 57: 341-348
Oktaba K, Zhang W, Lotz TS, Jun DJ, Lemke SB, Ng SP, Esposito E, Levine M, Hilgers V
(See online at https://doi.org/10.1016/j.molcel.2014.11.024)
