The lysosome is an essential element of cellular metabolism, functioning as both a recycling center and signaling hub. Autophagy targets macromolecules to the inside of the lysosome to be broken-down. Specifically, selective autophagy uses autophagic adapters to target specific macromolecules to the autophagosome1. The mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway converges at the lysosomal membrane to integrate nutrient, growth factor, and stress signals to ultimately control cell growth and metabolism2. The signaling and recycling functions of the lysosome work in concert; mTORC1 senses the nutrient state of the cell and induces autophagy when nutrients are low. To date, research has emphasized the role of proteins at the lysosome; autophagy targets misfolded proteins and protein aggregates to lysosomes and protein complexes such as mTORC1 mediates lysosomal signaling. It is now recognized that there is a role for autophagy in degrading both protein-RNA complexes, such as ribosomes and stress granules, and RNA, such as RNA viruses3. For this reasons we are interested in the identifying and characterizing messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) that are targeted to the lysosome by selective autophagy. Our lab has developed a technique, termed LysoIP, for the rapid isolation of intact lysosomes using an affinity-tag expressed on the lysosomal membrane. LysoIP has been used to establish metabolite concentrations in the lysosome4 as well as a lysosomal proteome (unpublished). Based on the hypothesis that specific mRNAs or lncRNAs are targeted for lysosomal degradation by selective autophagy, we used LysoIP to purify RNA at lysosomes. Our preliminary data reveals a population of mRNAs and lncRNAs that increase in abundance at the lysosome when cells are starved of nutrients. The top RNAs in this population include mRNAs for spliceosome and cytoskeleton regulators, a lncRNA characterized as a transcriptional regulator and oncogene, and mRNAs and lncRNAs of unknown function. While we appreciate that some of these RNAs may localize to the lysosomal membrane, we hypothesize that most of this population includes RNAs targeted for selective autophagy as a cell starvation response. To understand how and why selective autophagy targets this RNA population to the lysosome we propose the following aims: 1. Identify the mechanism by which the starvation-induced RNA population localizes to the lysosome. 2. Determine the signaling mechanism necessary to localize starvation-induced RNA to lysosomes. 3. Investigate the function of localizing starvation-induced spliceosome regulator mRNA to the lysosome.
This project will elucidate how and why starvation induced selective autophagy targets specific mRNAs and lncRNAs to the lysosome. Defects in autophagy are linked to neurodegenerative diseases, while increasing basal levels of autophagy may also have positive impacts on lifespan. Results from this work will inform our understanding of how the lysosome interacts with mRNA and lncRNA to control cell state, and the potential effect that has on alternative splicing.
