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Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity.

Brain somatic mutations confer genomic diversity in the human brain and cause neurodevelopmental disorders. Recently, brain somatic activating mutations in MTOR have been identified as a major etiology of intractable epilepsy in patients with cortical malformations. However, the molecular genetic mechanism of how brain somatic mutations in MTOR cause intractable epilepsy has remained elusive. In this study, translational profiling of intractable epilepsy mouse models with brain somatic mutations and genome-edited cells revealed a novel translational dysregulation mechanism and mTOR activation-sensitive targets mediated by human MTOR mutations that lead to intractable epilepsy with cortical malformation. These mTOR targets were found to be regulated by novel mTOR- responsive 5'-UTR motifs, distinct from known mTOR inhibition-sensitive targets regulated by 5' terminal oligopyrimidine motifs. Novel mTOR target genes were validated in patient brain tissues, and the mTOR downstream effector eIF4E was identified as a new therapeutic target in intractable epilepsy via pharmacological or genetic inhibition. We show that metformin, an FDA-approved eIF4E inhibitor, suppresses intractable epilepsy. Altogether, the present study describes translational dysregulation resulting from brain somatic mutations in MTOR, as well as the pathogenesis and potential therapeutic targets of intractable epilepsy., Introduction Brain somatic mutations are genetic variations arising in somatic cells of the brain during development and aging, and confer genomic diversity in the human brain (1, 2). However, their [...]

A hallmark of RNA silencing is a class of approximately 22-nucleotide RNAs that are processed from double-stranded RNA precursors by Dicer. Accurate processing by Dicer is crucial for the functionality of microRNAs (miRNAs). The current model posits that Dicer selects cleavage sites by measuring a set distance from the 3' overhang of the double-stranded RNA terminus. Here we report that human Dicer anchors not only the 39 end but also the 59 end, with the cleavage site determined mainly by the distance (~22 nucleotides) from the 5' end (5' counting rule). This cleavage requires a 5'-terminal phosphate group. Further, we identify a novel basic motif (5' pocket) in human Dicer that recognizes the 5'-phosphorylated end. The 5' counting rule and the 5' anchoring residues are conserved in Drosophila Dicer-1, but not in Giardia Dicer. Mutations in the 5' pocket reduce processing efficiency and alter cleavage sites in vitro. Consistently, miRNA biogenesis is perturbed in vivo when Dicer-null embryonic stem cells are replenished with the 5'-pocket mutant. Thus, 5'-end recognition by Dicer is important for precise and effective biogenesis of miRNAs. Insights from this study should also afford practical benefits to the design of small hairpin RNAs., RNase III proteins have central roles in RNA silencing by processing double-stranded (ds)RNA precursors into small RNA duplexes (1). Drosha cleaves a primary precursor of miRNA (pri-miRNA) to release a [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2010.11.018 Byline: Young-Kook Kim (1), Inha Heo (1), V. Narry Kim (1) Abstract: Small regulatory RNAs and their associated proteins are subject to diverse modifications that can impinge on their abundance and function. Some of the modifications are under the influence of cellular signaling, thus contributing to the dynamic regulation of RNA silencing. Author Affiliation: (1) School of Biological Sciences and Center for National Creative Research, Seoul National University, Seoul 151-742, Korea

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2009.11.020 Byline: Seogang Hyun (1), Jung Hyun Lee (1), Hua Jin (1), JinWu Nam (1), Bumjin Namkoong (1), Gina Lee (2), Jongkyeong Chung (2), V. Narry Kim (1) Keywords: SIGNALING; RNA; HUMDISEASE Abstract: How body size is determined is a long-standing question in biology, yet its regulatory mechanisms remain largely unknown. Here, we find that a conserved microRNA miR-8 and its target, USH, regulate body size in Drosophila. miR-8 null flies are smaller in size and defective in insulin signaling in fat body that is the fly counterpart of liver and adipose tissue. Fat body-specific expression and clonal analyses reveal that miR-8 activates PI3K, thereby promoting fat cell growth cell-autonomously and enhancing organismal growth non-cell-autonomously. Comparative analyses identify USH and its human homolog, FOG2, as the targets of fly miR-8 and human miR-200, respectively. USH/FOG2 inhibits PI3K activity, suppressing cell growth in both flies and humans. FOG2 directly binds to p85[alpha], the regulatory subunit of PI3K, and interferes with the formation of a PI3K complex. Our study identifies two novel regulators of insulin signaling, miR-8/miR-200 and USH/FOG2, and suggests their roles in adolescent growth, aging, and cancer. Author Affiliation: (1) School of Biological Sciences and National Creative Research Center, Seoul National University, Seoul, 151-742, Korea (2) Department of Biological Sciences and National Creative Research Center, Korea Advanced Institute of Science and Technology, Daejon, 305-701, Korea Article History: Received 4 June 2009; Revised 21 September 2009; Accepted 10 November 2009 Article Note: (miscellaneous) Published: December 10, 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2009.09.013 Byline: Inha Heo (1), V. Narry Kim (1) Abstract: Every regulator should be regulated, and this holds true for small RNAs and their associated proteins. Knowledge has begun to emerge of the various mechanisms that impose specificity on the expression and function of RNA silencing factors. Recent papers, including one in this issue of Cell (), now reveal the posttranslational modifications that take part in the regulation of the core RNA silencing factors, Ago, Piwi, and TRBP. Author Affiliation: (1) School of Biological Sciences and National Creative Research Initiatives Center, Seoul National University, Seoul 151-742, Korea

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2009.08.002 Byline: Inha Heo (1), Chirlmin Joo (1), Young-Kook Kim (1), Minju Ha (1), Mi-Jeong Yoon (1), Jun Cho (1), Kyu-Hyeon Yeom (1), Jinju Han (1), V. Narry Kim (1) Keywords: RNA; PROTEINS; STEMCELL Abstract: As key regulators in cellular functions, microRNAs (miRNAs) themselves need to be tightly controlled. Lin28, a pluripotency factor, was reported to downregulate let-7 miRNA by inducing uridylation of let-7 precursor (pre-let-7). But the enzyme responsible for the uridylation remained unknown. Here we identify a noncanonical poly (A) polymerase, TUTase4 (TUT4), as the uridylyl transferase for pre-let-7. Lin28 recruits TUT4 to pre-let-7 by recognizing a tetra-nucleotide sequence motif (GGAG) in the terminal loop. TUT4 in turn adds an oligouridine tail to the pre-let-7, which blocks Dicer processing. Other miRNAs with the same sequence motif (miR-107, -143, and -200c) are regulated through the same mechanism. Knockdown of TUT4 and Lin28 reduces the level of stem cell markers, suggesting that they are required for stem cell maintenance. This study uncovers the role of TUT4 and Lin28 as specific suppressors of miRNA biogenesis, which has implications for stem cell research and cancer biology. Author Affiliation: (1) Creative Research Center and School of Biological Sciences, Seoul National University, Seoul, Korea 151-742 Article History: Received 6 April 2009; Revised 16 June 2009; Accepted 3 August 2009 Article Note: (miscellaneous) Published: August 20, 2009