Hyeshik Chang

Dicer recognizes the 5′ end of RNA for efficient and accurate processing

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 3′ end but also the 5′ 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.

Uridylation by TUT4 and TUT7 Marks mRNA for Degradation

Uridylation occurs pervasively on mRNAs, yet its mechanism and significance remain unknown. By applying TAIL-seq, we identify TUT4 and TUT7 (TUT4/7), also known as ZCCHC11 and ZCCHC6, respectively, as mRNA uridylation enzymes. Uridylation readily occurs on deadenylated mRNAs in cells. Consistently, purified TUT4/7 selectively recognize and uridylate RNAs with short A-tails (less than ∼ 25 nt) in vitro. PABPC1 antagonizes uridylation of polyadenylated mRNAs, contributing to the specificity for short A-tails. In cells depleted of TUT4/7, the vast majority of mRNAs lose the oligo-U-tails, and their half-lives are extended. Suppression of mRNA decay factors leads to the accumulation of oligo-uridylated mRNAs. In line with this, microRNA induces uridylation of its targets, and TUT4/7 are required for enhanced decay of microRNA targets. Our study explains the mechanism underlying selective uridylation of deadenylated mRNAs and demonstrates a fundamental role of oligo-U-tail as a molecular mark for global mRNA decay.

miRGator v3.0: a microRNA portal for deep sequencing, expression profiling and mRNA targeting

Biogenesis and molecular function are two key subjects in the field of microRNA (miRNA) research. Deep sequencing has become the principal technique in cataloging of miRNA repertoire and generating expression profiles in an unbiased manner. Here, we describe the miRGator v3.0 update (http://mirgator.kobic.re.kr) that compiled the deep sequencing miRNA data available in public and implemented several novel tools to facilitate exploration of massive data. The miR-seq browser supports users to examine short read alignment with the secondary structure and read count information available in concurrent windows. Features such as sequence editing, sorting, ordering, import and export of user data would be of great utility for studying iso-miRs, miRNA editing and modifications. miRNA–target relation is essential for understanding miRNA function. Coexpression analysis of miRNA and target mRNAs, based on miRNA-seq and RNA-seq data from the same sample, is visualized in the heat-map and network views where users can investigate the inverse correlation of gene expression and target relations, compiled from various databases of predicted and validated targets. By keeping datasets and analytic tools up-to-date, miRGator should continue to serve as an integrated resource for biogenesis and functional investigation of miRNAs.

Next-generation libraries for robust RNA interference-based genome-wide screens

Significance Genetic screening is a classic approach to identify genes acting in a biological process of interest. In mammalian cells, screens are commonly based on RNA interference (RNAi), in which a short interfering RNA (siRNA) or short-hairpin RNA (shRNA) triggers degradation of cellular messenger RNAs. RNAi approaches are prone to false-positive results because of siRNA/shRNA off-target effects and false-negative results because of siRNAs/shRNAs lacking activity. We previously established that these problems can be minimized with ultracomplex shRNA libraries. Here, we present next-generation shRNA libraries targeting the human and mouse genomes, for which we improved several features to increase shRNA activity. In a pilot screen, the new library yields complementary results to clustered regularly interspaced short palindromic repeats interference (CRISPRi), an orthogonal approach we developed recently. 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.

TUT7 controls the fate of precursor microRNAs by using three different uridylation mechanisms

Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis. Using biochemistry, single‐molecule, and deep sequencing techniques, we here investigate the mechanism by which human TUT7 (also known as ZCCHC6) recognizes and uridylates precursor miRNAs (pre‐miRNAs) in the absence of Lin28. We find that the overhang of a pre‐miRNA is the key structural element that is recognized by TUT7 and its paralogues, TUT4 (ZCCHC11) and TUT2 (GLD2/PAPD4). For group II pre‐miRNAs, which have a 1‐nt 3′ overhang, TUT7 restores the canonical end structure (2‐nt 3′ overhang) through mono‐uridylation, thereby promoting miRNA biogenesis. For pre‐miRNAs where the 3′ end is further recessed into the stem (as in 3′ trimmed pre‐miRNAs), TUT7 generates an oligo‐U tail that leads to degradation. In contrast to Lin28‐stimulated oligo‐uridylation, which is processive, a distributive mode is employed by TUT7 for both mono‐ and oligo‐uridylation in the absence of Lin28. The overhang length dictates the frequency (but not duration) of the TUT7‐RNA interaction, thus explaining how TUT7 differentiates pre‐miRNA species with different overhangs. Our study reveals dual roles and mechanisms of uridylation in repair and removal of defective pre‐miRNAs.

mTAIL-seq reveals dynamic poly(A) tail regulation in oocyte-to-embryo development

Eukaryotic mRNAs are subject to multiple types of tailing that critically influence mRNA stability and translatability. To investigate RNA tails at the genomic scale, we previously developed TAIL-seq, but its low sensitivity precluded its application to biological materials of minute quantity. In this study, we report a new version of TAIL-seq (mRNA TAIL-seq [mTAIL-seq]) with enhanced sequencing depth for mRNAs (by ∼1000-fold compared with the previous version). The improved method allows us to investigate the regulation of poly(A) tails in Drosophila oocytes and embryos. We found that maternal mRNAs are polyadenylated mainly during late oogenesis, prior to fertilization, and that further modulation occurs upon egg activation. Wispy, a noncanonical poly(A) polymerase, adenylates the vast majority of maternal mRNAs, with a few intriguing exceptions such as ribosomal protein transcripts. By comparing mTAIL-seq data with ribosome profiling data, we found a strong coupling between poly(A) tail length and translational efficiency during egg activation. Our data suggest that regulation of poly(A) tails in oocytes shapes the translatomic landscape of embryos, thereby directing the onset of animal development. By virtue of the high sensitivity, low cost, technical robustness, and broad accessibility, mTAIL-seq will be a potent tool to improve our understanding of mRNA tailing in diverse biological systems.

Temporal Landscape of MicroRNA-Mediated Host-Virus Crosstalk during Productive Human Cytomegalovirus Infection

Temporal profiles of miRNA activity during productive virus infection can provide fundamental insights into host-virus interactions. Most reported miRNA targetome analyses in the context of virus infection have been performed in latently infected cells and lack reliable models for quantifying the suppression efficacy at specific miRNA target sites. Here, we identified highly competent temporal miRNA targetomes during lytic HCMV infection by using AGO-CLIP-seq together with a bioinformatic method that quantifies miRNA functionality at a specific target site, called ACE-scoring. The repression efficiency at target sites correlates with the magnitude of the ACE-score, and temporal HCMV-encoded miRNA targetomes identified by ACE-scoring were significantly enriched in functional categories involved in pathways central for HCMV biology. Furthermore, comparative analysis between human and viral miRNA targetomes supports the existence of intimate cooperation and co-targeting between them. Our holistic survey provides a valuable resource for understanding host-virus interactions during lytic HCMV infection.

FitSearch: a robust way to interpret a yeast fitness profile in terms of drug's mode-of-action

BackgroundYeast deletion-mutant collections have been successfully used to infer the mode-of-action of drugs especially by profiling chemical-genetic and genetic-genetic interactions on a genome-wide scale. Although tens of thousands of those profiles are publicly available, a lack of an accurate method for mining such data has been a major bottleneck for more widespread use of these useful resources.ResultsFor general usage of those public resources, we designed FitRankDB as a general repository of fitness profiles, and developed a new search algorithm, FitSearch, for identifying the profiles that have a high similarity score with statistical significance for a given fitness profile. We demonstrated that our new repository and algorithm are highly beneficial to researchers who attempting to make hypotheses based on unknown modes-of-action of bioactive compounds, regardless of the types of experiments that have been performed using yeast deletion-mutant collection in various types of different measurement platforms, especially non-chip-based platforms.ConclusionsWe showed that our new database and algorithm are useful when attempting to construct a hypothesis regarding the unknown function of a bioactive compound through small-scale experiments with a yeast deletion collection in a platform independent manner. The FitRankDB and FitSearch enhance the ease of searching public yeast fitness profiles and obtaining insights into unknown mechanisms of action of drugs. FitSearch is freely available at http://fitsearch.kaist.ac.kr.

Construction of the first compendium of chemical-genetic profiles in the fission yeast Schizosaccharomyces pombe and comparative compendium approach.

Genome-wide chemical genetic profiles in Saccharomyces cerevisiae since the budding yeast deletion library construction have been successfully used to reveal unknown mode-of-actions of drugs. Here, we introduce comparative approach to infer drug target proteins more accurately using two compendiums of chemical-genetic profiles from the budding yeast S. cerevisiae and the fission yeast Schizosaccharomyces pombe. For the first time, we established DNA-chip based growth defect measurement of genome-wide deletion strains of S. pombe, and then applied 47 drugs to the pooled heterozygous deletion strains to generate chemical-genetic profiles in S. pombe. In our approach, putative drug targets were inferred from strains hypersensitive to given drugs by analyzing S. pombe and S. cerevisiae compendiums. Notably, many evidences in the literature revealed that the inferred target genes of fungicide and bactericide identified by such comparative approach are in fact the direct targets. Furthermore, by filtering out the genes with no essentiality, the multi-drug sensitivity genes, and the genes with less eukaryotic conservation, we created a set of drug target gene candidates that are expected to be directly affected by a given drug in human cells. Our study demonstrated that it is highly beneficial to construct the multiple compendiums of chemical genetic profiles using many different species. The fission yeast chemical-genetic compendium is available at http://pombe.kaist.ac.kr/compendium.

miRseqViewer: multi-panel visualization of sequence, structure and expression for analysis of microRNA sequencing data

SUMMARY Deep sequencing of small RNAs has become a routine process in recent years, but no dedicated viewer is as yet available to explore the sequence features simultaneously along with secondary structure and gene expression of microRNA (miRNA). We present a highly interactive application that visualizes the sequence alignment, secondary structure and normalized read counts in synchronous multipanel windows. This helps users to easily examine the relationships between the structure of precursor and the sequences and abundance of final products and thereby will facilitate the studies on miRNA biogenesis and regulation. The project manager handles multiple samples of multiple groups. The read alignment is imported in BAM file format. Implemented features comprise sorting, zooming, highlighting, editing, filtering, saving, exporting, etc. Currently, miRseqViewer supports 84 organisms whose annotation is available at miRBase. AVAILABILITY AND IMPLEMENTATION miRseqViewer, implemented in Java, is available at https://github.com/insoo078/mirseqviewer or at http://msv.kobic.re.kr. CONTACT sanghyuk@ewha.ac.kr.