Synergistic degradation of circRNAs by RNase K and lysosomes

Circular RNAs (circRNAs) are vital in many physiological and pathological events. Compared to the other processes of circRNA metabolism, circRNA degradation is less understood. Through screening and further characterization, RNAseK and lysosome are identified as circRNA degradation modules in metazoan, and RNase 1 as a lysosomal circRNA endoribonuclease in mammal. RNAseK and lysosome function synergistically with RNAseK degrading circRNAs outside and lysosome degrading those inside of the organelle. Mutations of RNAseK- or lysosome-sensitive sites in in vitro synthesized circRNA reporters lead to higher expression levels, and simultaneous mutations of both sensitive sites result in further increase. Under stress stimuli such as heat shock and ER stress, significantly decreased global circRNA levels are observed in C. elegans. RNAseK deficiency or lysosome inhibition diminishes the decreases in circRNA levels, and impedes the induction of stress response, suggesting that circRNA degradation by RNAseK and lysosome plays protective roles in C. elegans stress response. Overall design: To investigate circRNA degradation factors, we set out to establish a circReporter system with genome-wide CRIPSR-Cas9 knockout screen. Then, we performed circRNA and mRNA profiles in HEK293T cells, HeLa cells, 3T3-L1 cells, N2a cells and C. elegans upon RNase K knockdown or lysosomal inhibition by CQ or BafA1 treatment. We also performed circRNA and/or mRNA profiles in HEK293T cells by manipulating lysosomal RNases levels. We futher performed RNAseq for C. elegans upon stress stimuli.