Destruction of noncoding RNAs

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SUMMARY Precise and dynamic control of gene expression is a balancing act between production and destruction. Although noncoding RNAs are critical regulators of gene expression with important roles in development and disease, our understanding of their destruction is still in its infancy. One of the reasons for this gap in knowledge is that some noncoding RNAs, like microRNAs and circular RNAs, are resistant to the known pathways that destroy protein- coding RNAs. Here I present my plan to identify how these two types of noncoding RNA are destroyed. MicroRNAs (miRNAs) are protected from degradation by their effector protein Argonaute. One way to degrade miRNAs is through target-directed miRNA degradation (TDMD), which occurs when a highly complementary viral or artificial RNA interacts with the miRNA. My postdoctoral work identified two of the first examples of endogenous targets that induce miRNA degradation, which also led to our discovery of an E3 ubiquitin ligase that mediates TDMD and the identification of 48 additional miRNAs (likely an underestimate) subject to TDMD. Our working model now is that the binding of highly complementary targets to the Argonaute–miRNA complex leads to recruitment of the E3 ligase, ubiquitination and proteosomal degradation of Argonaute, and release and degradation of the miRNA. With the discovery of TDMD effector proteins and the broad influence of TDMD on miRNA stability, a major gap is identifying the target RNAs that induce TDMD. A related problem is understanding why some targets are better than others. We will address these gaps by answering two questions: 1) Which endogenous RNAs induce miRNA degradation? 2) What are the pairing rules and cis-acting elements that promote TDMD? To do so, we will use transgenic mice, engineered cell lines, genome-wide approaches, and classic molecular biology techniques. Our second area of research is understanding how circular RNAs (circRNA) are degraded. The poster child of post-transcriptional circRNA regulation is Cdr1as, a conserved circRNA that is highly expressed in neurons and limits spontaneous synaptic vesicle release. Cdr1as contains a single near-perfect binding site that can be sliced by Ago2-loaded miR-671 and >70 seed sites for miR-7. Previously, I showed that Cdr1as can be regulated by the independent and cooperative actions of miR-671 and miR-7. How miR-7 induces destruction of Cdr1as is unclear as circRNAs should be resistant to miRNA-mediated deadenylation and decapping. Here, we will answer two questions: 1) How is Cdr1as degraded? 2) How are other unstable circRNAs degraded? The outcomes of our research will be the identification and improved understanding of dedicated pathways for degrading noncoding RNAs. These pathways have potential for broad impact as they are likely employed in diverse cell types and organisms and in response to a variety of stimuli.