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Non-Coding RNA - Small Non-Coding RNA 09:00 - 10:00 Saturday, 30Th May, 2020 Talk Session Chair Jeremy Wilusz

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Non-Coding RNA - Small Non-Coding RNA 09:00 - 10:00 Saturday, 30Th May, 2020 Talk Session Chair Jeremy Wilusz Plenary Session 5a: Non-coding RNA - Small Non-coding RNA 09:00 - 10:00 Saturday, 30th May, 2020 Talk Session Chair Jeremy Wilusz 366 Cryo-EM structures of Human Drosha and DGCR8 bound to an RNA substrate reveal how Microprocessor recognizes primary microRNAs Alexander Partin1, Kaiming Zhang2, Byung-Cheon Jeong1, Emily Herrell1, Shanshan Li2, Wah Chiu3, Yunsun Nam1 1University of Texas Southwestern Medical Center, Dallas, TX, USA. 2Stanford University, Menlo Park, CA, USA. 3Stanford University, Menlo Park, TX, USA Abstract Metazoan microRNAs require specific maturation steps initiated by the Microprocessor complex, comprised of Drosha and DGCR8. A lack of structural information for the assembled Microprocessor/RNA complex has hindered our understanding of how Drosha/DGCR8 recognizes primary microRNA transcripts (pri-miRNAs). Here we present a cryo-electron microscopy structure of human Microprocessor with a pri-miRNA docked in the active site, poised for cleavage. The basal junction is recognized by a four-way intramolecular junction in Drosha, triggered by structural elements we have named the Belt and Wedge. The Belt and the Wedge clamp the single-stranded RNA arms while simultaneously contacting the double-stranded RNA stem, thereby recognizing a dsRNA-ssRNA junction. The Belt forms a two-helix extension that folds over the front of the basal junction and contributes crucially to both efficiency and accuracy of processing. Thus, our model shows that Drosha does not contain a PAZ-like domain, as had been previously suggested. Two dsRBDs form a molecular ruler to measure the length of the stem between the two dsRNA-ssRNA junctions. A second structure, in a partially-docked state, reveals how DGCR8 may drive the initial steps in RNA binding. Collectively, we derive a molecular model to explain how Microprocessor recognizes a pri-miRNA and accurately identifies the cleavage site. Presenting author email [email protected] Topic category Small Non-coding RNAs in Eukaryotes 12 Mechanisms of phosphorylation-mediated regulation of the RNAi effector protein Argonaute 2 Brianna Bibel1,2, Elad Elkayam2,3, Leemor Joshua-Tor2,3 1Watson School of Biological Sciences, Cold Spring Harbor, NY, USA. 2W.M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA. 3Howard Hughes Medical Institute, Cold Spring Harbor, NY, USA Abstract Argonaute (Ago) proteins play a central role in gene regulation through a process called RNA interference (RNAi). Ago binds to small RNAs (sRNAs) such as small interfering RNA (siRNA) and microRNA (miRNA) to form the functional core of the RNA Induced Silencing Complex (RISC), which targets mRNAs with complementary sequences, ultimately leading to down-regulation of the corresponding genes. It was previously shown that phosphorylation of a cluster of residues (824-834) in the “eukaryotic insertion” (EI) of Ago by CK1α can alleviate this repression in a variety of cell types. However, the mechanisms of such regulation were unknown. Using in-vitro phosphorylation assays, we show that binding of miRNA-loaded Ago2 to target RNA primes the EI for phosphorylation. Furthermore, we show that this phosphorylation reduces the affinity of the Ago-miRNA complex to its target mRNA. While the EI of Ago2 contains 5 potential phosphorylation sites, we propose that specific residues in the EI are more susceptible to phosphorylation and dictate the pattern and sequence of the phosphorylation events. We also examined the structural requirements for the target to induce phosphorylation. These findings shed light on the manner in which phosphorylation of the EI acts to regulate the RNAi pathway central to gene regulation. The high conservation of potential phosphosites in the EI (both among Ago homologs and throughout eukaryotes) suggests that such a regulatory strategy may be a shared mechanism with broader impact. Presenting author email [email protected] Topic category Small Non-coding RNAs in Eukaryotes 364 The Widespread Scope and Molecular Basis of Target-Directed MicroRNA Degradation Charlie Shi1,2, Elena Kingston1,2, Benjamin Kleaveland1,2, Michael Stubna1,2, David Bartel1,2 1Whitehead Institute for Biomedical Research, Cambridge, MA, USA. 2Massachusetts Institute of Technology, Cambridge, MA, USA Abstract Most microRNAs (miRNAs) are quite stable, with half-lives of a day or more. However, more rapid degradation can be observed in the presence of a target with extensive complementarity to the miRNA 3′ region. This phenomenon, called target-directed miRNA degradation (TDMD), was initially observed when examining the effects of either miRNA antisense reagents or certain viral transcripts. For example, the HSUR1 transcript of Herpesvirus saimiri triggers the degradation of miR-27a, a host miRNA with antiviral effects. In addition, four examples of TDMD triggered by cellular transcripts have recently been reported in mammals and fish. Many of the same transcripts that direct miRNA degradation also direct 3′ tailing and trimming of the miRNA, which has led to speculation that target-directed tailing and trimming (TDTT) might be required for TDMD. However, the molecular mechanism of TDMD has been unclear. We carried out a CRISPRi screen designed to identify proteins required for endogenous target-directed degradation of miR-7. This screen and subsequent analyses identified a set of proteins that work together to mediate target-directed miR-7 degradation as well as HSUR1-directed degradation of miR-27a. Small-RNA sequencing of mouse and human cells that lack one of these proteins required for TDMD indicated that >20 miRNAs are targeted for degradation by endogenous transcripts in mammals—a large increase over the four previously reported examples. Analogous loss-of-function experiments expanded the implied scope of endogenous TDMD to flies and nematodes, with >10 miRNAs affected in each case. Results of other experiments speak to the mechanism of TDMD and the relationship between TDTT and TDMD. Presenting author email [email protected] Topic category Small Non-coding RNAs in Eukaryotes.
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