Differential localization of messenger ribonucleoprotein particles (mRNPs) to the translation machinery or to mRNP aggregates such as P-bodies and stress granules is a key step to determine the translational fate of an mRNA. Since RNA misregulation underlies several neurodegenerative diseases, I seek to understand the effect of granulophagy, the turnover of mRNP aggregates by autophagy, on mRNA decay. I will investigate if granulophagy induces the decay of mRNAs that associate with mRNP aggregates as it may represent a novel mRNA decay pathway. Using RNA-seq, I have identified mRNAs whose levels are stabilized when autophagy is inhibited in yeast. Interestingly, a subset of mRNAs is enriched in the stabilized mRNAs suggesting that autophagy may preferentially degrade specific mRNAs. I will establish if these mRNAs are targeted for granulophagy by using fluorescent in situ hybridization to test if they associate with mRNP aggregates and localize to the vacuole. Additionally, I will validate that these mRNAs are degraded by autophagy by examining their decay rates in wild type cells and cells that are defective for autophagy. These experiments will corroborate that a novel mRNA decay pathway mediated by autophagy exists in yeast. Our preliminary data also suggests that mRNAs can be preferentially targeted for autophagic decay. To explore this possibility, I will test if cis elements encoded in the mRNAs, RNA binding proteins (RBPs) or segments of the mRNA mediate targeting. First, I will use bioinformatics to test if cis elements are enriched in the targeted mRNAs. Second, I will take a candidate approach and test if known RBPs can mediate specificity. Lastly, I will systematically dissect a mRNA to find domains required for its targeting. These assays will define the requirements for specificity in autophagic mRNA decay. Lastly, I will investigate if autophagic mRNA degradation is a conserved process that occurs in higher eukaryotes such as humans by identifying if mRNAs are targeted to autophagy in HEK293T cells. Upon characterization of human mRNAs subjected to autophagic decay, I anticipate testing if RNA aggregates found in neurodegenerative diseases result from defects in granulophagy or autophagic decay of mRNAs. In summary, I seek to uncover a novel mRNA decay mechanism in yeast and humans, thus, serving as a basis to uncover dysfunctions related to neurodegeneration.
This project is relevant to public health as it has the potential to uncover cellular dysfunctions that can contribute to neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Cells obtained from patients afflicted with these diseases contain RNA-protein aggregates, but unlike normal RNA-protein aggregates they strongly accumulate and do not turnover (Lowe, 1989). By studying the impact of autophagy-mediated RNA-protein aggregate turnover on mRNA decay, this study has strong potential to unlock how pathogenic aggregates can contribute to neurodegenerative disease.
|Garcia, Jennifer F; Parker, Roy (2015) MS2 coat proteins bound to yeast mRNAs block 5' to 3' degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system. RNA 21:1393-5|