Periodic Reporting for period 3 - Neuro-UTR (Mechanism and functional impact of ultra-long 3’ UTRs in the Drosophila nervous system)
Période du rapport: 2022-01-01 au 2023-06-30
The nervous system is composed of highly polarized cells of complex and dynamic architecture. The formation and maintenance of neurons and neural circuits require the coordinated expression of genes at each step of RNA metabolism: from transcription, processing, localized transport and translation, to degradation. To achieve this level of complexity, neurons employ mechanisms that increase RNA regulatory potential: alternative splicing, alternative polyadenylation, and non-coding RNA expression.
One particularly striking process is the recently discovered, drastic lengthening of the 3’ untranslated region (3’ UTR) of hundreds of genes, which occurs in neurons from flies to humans. The function of the resulting ultra-long 3’ UTRs is unknown. RNA deregulation plays a central role in neurological diseases; to understand underlying causes, it is essential to study regulatory processes and define the function of these novel 3’ UTRs.
Our study integrates the molecular mechanisms that govern biogenesis and function of ultra-long 3’ UTRs, from nucleus to synapse, in an animal model, the fruit fly Drosophila melanogaster. The results of this research will create a major impact on our understanding of neuronal gene regulation in health and disease.
One particularly striking process is the recently discovered, drastic lengthening of the 3’ untranslated region (3’ UTR) of hundreds of genes, which occurs in neurons from flies to humans. The function of the resulting ultra-long 3’ UTRs is unknown. RNA deregulation plays a central role in neurological diseases; to understand underlying causes, it is essential to study regulatory processes and define the function of these novel 3’ UTRs.
Our study integrates the molecular mechanisms that govern biogenesis and function of ultra-long 3’ UTRs, from nucleus to synapse, in an animal model, the fruit fly Drosophila melanogaster. The results of this research will create a major impact on our understanding of neuronal gene regulation in health and disease.
We unambiguously identified Drosophila neuron-specific 3’UTRs in vivo and found that ELAV mediates all processes of neuronal alternative polyadenylation. We succeeded in performing iCLIP in adult Drosophila brains to produce for the first time, a comprehensive identification of ELAV binding sites. We could show that ELAV specifically binds to proximal poly(A) sites of its functional targets in vivo. We made an unexpected discovery of how ELAV function can be safeguarded by a newly acquired role of its homologue FNE: when not directly repressed by ELAV, the transcript encoding FNE acquires a mini-exon, generating a new protein able to translocate to the nucleus and rescue ELAV-mediated alternative polyadenylation and alternative splicing.
We generated and optimized a protocol for global aUTR localisation in neurons of adult Drosophila brains, successfully implemented it and validated the purity of synaptosome fractions by electron microscopy. We developed and successfully applied several complementary RNA biochemistry approaches. A preliminary analysis of the data substantiates our work hypothesis that extended 3’UTRs undergo specific posttranscriptional regulation through specific proteins. These important findings on the global regulation of aUTRs in neurons significantly advance our understanding on post-transcriptional regulation of neuronal RNA signatures.
We generated and optimized a protocol for global aUTR localisation in neurons of adult Drosophila brains, successfully implemented it and validated the purity of synaptosome fractions by electron microscopy. We developed and successfully applied several complementary RNA biochemistry approaches. A preliminary analysis of the data substantiates our work hypothesis that extended 3’UTRs undergo specific posttranscriptional regulation through specific proteins. These important findings on the global regulation of aUTRs in neurons significantly advance our understanding on post-transcriptional regulation of neuronal RNA signatures.
We developed and optimized existing techniques to apply to whole tissues. These technologies have been described in tissue culture or other organisms but had never been successfully performed in endogenous tissue in flies. These techniques include iCLIP, UV-crosslinking RNA Affinity Purification (xRAP), UV-crosslinking RNA Immunopurification (xRIP), synaptosome purification, and Long-Read-Sequencing.
We developed a robust computational approach to quantify alternative poly(A) site usage from traditional mRNA-seq datasets, which are more prevalent than 3’-end sequencing datasets. Hence, our new tool can perform specialised analyses on publicly available datasets, which is a valuable resource for the RNA community.
These methods and their application in the scope of this project will reveal mechanisms of how alternative UTRs regulate gene expression complexity in neurons.
We generated and analysed flies lacking the aUTR of specific genes, and found one particularly interesting disease phenotype, which we are focusing on for functional and molecular characterization. We have made progress in the construction of genetic tools for global up- and down-regulation of aUTRs in vivo. Due to our unexpected finding of the involvement of FNE in aUTR regulation, we adjusted experimental setups and the design of in vivo mutagenesis, which will enable to create better models of aUTR deregulation, and better understand how neuronal UTRs regulate neuronal homeostasis in health and disease.
We developed a robust computational approach to quantify alternative poly(A) site usage from traditional mRNA-seq datasets, which are more prevalent than 3’-end sequencing datasets. Hence, our new tool can perform specialised analyses on publicly available datasets, which is a valuable resource for the RNA community.
These methods and their application in the scope of this project will reveal mechanisms of how alternative UTRs regulate gene expression complexity in neurons.
We generated and analysed flies lacking the aUTR of specific genes, and found one particularly interesting disease phenotype, which we are focusing on for functional and molecular characterization. We have made progress in the construction of genetic tools for global up- and down-regulation of aUTRs in vivo. Due to our unexpected finding of the involvement of FNE in aUTR regulation, we adjusted experimental setups and the design of in vivo mutagenesis, which will enable to create better models of aUTR deregulation, and better understand how neuronal UTRs regulate neuronal homeostasis in health and disease.