The Experts below are selected from a list of 8958 Experts worldwide ranked by ideXlab platform
V. Narry Kim - One of the best experts on this subject based on the ideXlab platform.
-
fCLIP-seq for transcriptomic footprinting of dsRNA-binding proteins: Lessons from Drosha.
Methods (San Diego Calif.), 2018Co-Authors: Baekgyu Kim, V. Narry KimAbstract:Abstract CLIP-seq (crosslinking immunoprecipitation and sequencing) is widely used to map the binding sites of a protein of interest on the transcriptome, and generally employs UV to induce the covalent bonds between protein and RNA, which allows stringent washing. However, dsRNA is inefficiently crosslinked by UV, making it difficult to study the interactions between dsRNA binding proteins and their substrates. A dsRNA endoribonuclease Drosha initiates the maturation of microRNA (miRNA) by cleaving primary miRNA (pri-miRNA). Despite the importance of Drosha in miRNA maturation and sequence determination, accurate mapping of Drosha cleavage sites has not been feasible due to rapid processing, modification, and degradation of the cleaved products in cells. Here, we present a high-throughput sequencing method that allows the mapping of in vivo Drosha cleavage sites at single nucleotide resolution, termed formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq). The fCLIP-seq protocol has been improved significantly over the standard CLIP-seq methods by (1) using formaldehyde for efficient and reversible crosslinking, (2) employing polyethylene glycol and adaptors with randomized sequences to enhance ligation efficiency and minimize bias, and (3) performing ligation after elution, which exposes the RNA termini for efficient ligation. fCLIP-seq successfully captures the nascent products of Drosha, which allows precise mapping of the Drosha processing sites. Moreover, from the analysis of the distinctive cleavage pattern, we discover previously unknown substrates of Drosha. fCLIP-seq is a useful tool to obtain transcriptome-wide information on Drosha activity and can be applied further to investigate other dsRNA-protein interactions.
-
Genome-wide Mapping of Drosha Cleavage Sites on Primary MicroRNAs and Noncanonical Substrates
Molecular cell, 2017Co-Authors: Baekgyu Kim, Kyowon Jeong, V. Narry KimAbstract:MicroRNA (miRNA) maturation is initiated by Drosha, a double-stranded RNA (dsRNA)-specific RNase III enzyme. By cleaving primary miRNAs (pri-miRNAs) at specific positions, Drosha serves as a main determinant of miRNA sequences and a highly selective gatekeeper for the canonical miRNA pathway. However, the sites of Drosha-mediated processing have not been annotated, and it remains unclear to what extent Drosha functions outside the miRNA pathway. Here, we establish a protocol termed "formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq)," which allows identification of Drosha cleavage sites at single-nucleotide resolution. fCLIP identifies numerous processing sites, suggesting widespread end modifications during miRNA maturation. fCLIP also finds many pri-miRNAs that undergo alternative processing, yielding multiple miRNA isoforms. Moreover, we discovered dozens of Drosha substrates on non-miRNA loci, which may serve as cis-elements for Drosha-mediated gene regulation. We anticipate that fCLIP-seq could be a general tool for investigating interactions between dsRNA-binding proteins and structured RNAs.
-
Re-evaluation of the roles of Drosha, Exportin 5, and DICER in microRNA biogenesis
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Young Kook Kim, Boseon Kim, V. Narry KimAbstract:Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by Drosha, nuclear export of precursor miRNA (pre-miRNA) by Exportin 5 (XPO5), and cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted Drosha, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in Drosha-deleted cells, whereas we detected a few Drosha-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to Drosha knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3′ end, yielding miRNAs from the 5′ strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of Drosha and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research.
-
Structure of Human Drosha
Cell, 2015Co-Authors: S. Chul Kwon, V. Narry Kim, Tuan Anh Nguyen, Yeon-gil Choi, Sungchul Hohng, Jae-sung WooAbstract:MicroRNA maturation is initiated by RNase III Drosha that cleaves the stem loop of primary microRNA. Drosha functions together with its cofactor DGCR8 in a heterotrimeric complex known as Microprocessor. Here, we report the X-ray structure of Drosha in complex with the C-terminal helix of DGCR8. We find that Drosha contains two DGCR8-binding sites, one on each RNase III domain (RIIID), which mediate the assembly of Microprocessor. The overall structure of Drosha is surprisingly similar to that of Dicer despite no sequence homology apart from the C-terminal part, suggesting that Drosha may have evolved from a Dicer homolog. Drosha exhibits unique features, including non-canonical zinc-finger motifs, a long insertion in the first RIIID, and the kinked link between Connector helix and RIIID, which explains the 11-bp-measuring "ruler" activity of Drosha. Our study implicates the evolutionary origin of Drosha and elucidates the molecular basis of Microprocessor assembly and primary microRNA processing.
-
Posttranscriptional Crossregulation between Drosha and DGCR8
Cell, 2009Co-Authors: Jinju Han, Young Kook Kim, S. Chul Kwon, Jakob Skou Pedersen, Cassandra D. Belair, Kyu-hyeon Yeom, Woo-young Yang, David Haussler, Robert Blelloch, V. Narry KimAbstract:The Drosha-DGCR8 complex, also known as Microprocessor, is essential for microRNA (miRNA) maturation. Drosha functions as the catalytic subunit, while DGCR8 (also known as Pasha) recognizes the RNA substrate. Although the action mechanism of this complex has been intensively studied, it remains unclear how Drosha and DGCR8 are regulated and if these proteins have any additional role(s) apart from miRNA processing. Here, we report that Drosha and DGCR8 regulate each other posttranscriptionally. The Drosha-DGCR8 complex cleaves the hairpin structures embedded in the DGCR8 mRNA and thereby destabilizes the mRNA. We further find that DGCR8 stabilizes the Drosha protein via protein-protein interaction. This crossregulation between Drosha and DGCR8 may contribute to the homeostatic control of miRNA biogenesis. Furthermore, microarray analyses suggest that a number of mRNAs may be downregulated in a Microprocessor-dependent, miRNA-independent manner. Our study reveals a previously unsuspected function of Microprocessor in mRNA stability control.
Janggi Choi - One of the best experts on this subject based on the ideXlab platform.
-
lower and upper stem single stranded rna junctions together determine the Drosha cleavage site
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Yonggan Wu, Janggi Choi, Haoquan WuAbstract:Microprocessor [Drosha–DGCR8 (DiGeorge syndrome critical region gene 8) complex] processing of primary microRNA (pri-miRNA) is the critical first step in miRNA biogenesis, but how the Drosha cleavage site is determined has been unclear. Previous models proposed that the Drosha–DGCR8 complex measures either ∼22 nt from the upper stem–single-stranded RNA (ssRNA, terminal loop) junction or ∼11 nt from the lower stem–ssRNA junction to determine the cleavage site. Here, using miRNA-offset RNAs to determine the Drosha cleavage site, we show that the Microprocessor measures the distances from both the lower and upper stem–ssRNA junctions to determine the cleavage site in human cells, and optimal distances from both structures are critical to the precision of Drosha processing. If the distances are not optimal, Drosha tends to cleave at multiple sites, which can, in turn, generate multiple 5′ isomiRs. Thus, our results also reveal a mechanism of 5′ isomiR generation.
-
Lower and upper stem–single-stranded RNA junctions together determine the Drosha cleavage site
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Janggi ChoiAbstract:Microprocessor [Drosha–DGCR8 (DiGeorge syndrome critical region gene 8) complex] processing of primary microRNA (pri-miRNA) is the critical first step in miRNA biogenesis, but how the Drosha cleavage site is determined has been unclear. Previous models proposed that the Drosha–DGCR8 complex measures either ∼22 nt from the upper stem–single-stranded RNA (ssRNA, terminal loop) junction or ∼11 nt from the lower stem–ssRNA junction to determine the cleavage site. Here, using miRNA-offset RNAs to determine the Drosha cleavage site, we show that the Microprocessor measures the distances from both the lower and upper stem–ssRNA junctions to determine the cleavage site in human cells, and optimal distances from both structures are critical to the precision of Drosha processing. If the distances are not optimal, Drosha tends to cleave at multiple sites, which can, in turn, generate multiple 5′ isomiRs. Thus, our results also reveal a mechanism of 5′ isomiR generation.
Bharat Ramratnam - One of the best experts on this subject based on the ideXlab platform.
-
Acetylation of Drosha on the N-Terminus Inhibits Its Degradation by Ubiquitination
PloS one, 2013Co-Authors: Xiaoli Tang, Sicheng Wen, Dong Zheng, Lynne Tucker, Lulu Cao, Dennis Pantazatos, Steven F. Moss, Bharat RamratnamAbstract:The RNase III enzyme Drosha initiates microRNA (miRNA) biogenesis in the nucleus by cleaving primary miRNA transcripts into shorter precursor molecules that are subsequently exported into the cytoplasm for further processing. While numerous disease states appear to be associated with aberrant expression of Drosha, the molecular mechanisms that regulate its protein levels are largely unknown. Here, we report that ubiquitination and acetylation regulate Drosha protein levels oppositely. Deacetylase inhibitors trichostatin A (TSA) and nicotinamide (NIA) increase Drosha protein level as measured by western blot but have no effects on its mRNA level in HEK293T cells. TSA increases miRNA-143 production in a miRNA sensor assay and in a qPCR analysis in HEK293T cells. Treatment of AGS and HEK293T cells with proteasome inhibitors MG132 or Omuralide increases Drosha protein levels. Furthermore, the N-terminal, but not the C-terminal Drosha can be acetylated by multiple acetyl transferases including p300, CBP and GCN5. Acetylation of Drosha competes with its ubquitination, inhibiting the degradation induced by the ubiquitin-proteasome pathway, thereby increasing Drosha protein levels. Infection of the gastric mucosa AGS cells by H. pylori, the gastric cancer associated carcinogen, leads to the ubiquitination and reduction of Drosha protein levels. H. pylori infection of AGS cells has no significant effects on Drosha mRNA levels. Our findings establish a central mechanism of protein homeostasis as playing a critical role in miRNA biogenesis.
-
Phosphorylation of the RNase III enzyme Drosha at Serine300 or Serine302 is required for its nuclear localization
Nucleic acids research, 2010Co-Authors: Xiaoli Tang, Lynne Tucker, Yingjie Zhang, Bharat RamratnamAbstract:The RNaseIII enzyme Drosha plays a pivotal role in microRNA (miRNA) biogenesis by cleaving primary miRNA transcripts to generate precursor miRNA in the nucleus. The RNA binding and enzymatic domains of Drosha have been characterized and are on its C-terminus. Its N-terminus harbors a nuclear localization signal. Using a series of truncated Drosha constructs, we narrowed down the segment responsible for nuclear translocation to a domain between aa 270 and aa 390. We further identified two phosphorylation sites at Serine300 (S300) and Serine302 (S302) by mass spectrometric analysis. Double mutations of S→A at S300 and S302 completely disrupted nuclear localization. Single mutation of S→A at S300 or S302, however, had no effect on nuclear localization indicating that phosphorylation at either site is sufficient to locate Drosha to the nucleus. Furthermore, mimicking phosphorylation status by mutating S→E at S300 and/or S→D at S302 restored nuclear localization. Our findings add a further layer of complexity to the molecular anatomy of Drosha as it relates to miRNA biogenesis.
Yan Zeng - One of the best experts on this subject based on the ideXlab platform.
-
Cloning, expression, and characterization of the zebrafish Dicer and Drosha enzymes.
Biochemical and biophysical research communications, 2019Co-Authors: Qiuhuan Tian, Xiaoxiao Zhang, Yan ZengAbstract:The biogenesis of animal microRNAs (miRNAs) involves transcription followed by a series of processing steps, with Drosha and Dicer being two key enzymes that cleave primary miRNA (pri-miRNA) and precursor miRNA (pre-miRNA) transcripts, respectively. While human and fly Dicer and human Drosha are well studied, their homologs in other organisms have not been biochemically characterized, leaving open the question of whether their miRNA substrate specificities and regulatory functions are conserved. Zebrafish is a widely used model organism, but its miRNA processing enzymes have never been reconstituted and analyzed. In this study we cloned and constructed expression plasmids encoding zebrafish Dicer, Drosha, and their accessory proteins TARBP2 and DGCR8. After transfection of human cell cultures, we isolated the recombinant protein complexes. We found that zebrafish Dicer bound TARBP2, but Dicer alone exhibited significant pre-miRNA processing activity. On the other hand, zebrafish Drosha associated with DGCR8, and both were required to cleave pri-miRNAs. The Drosha/DGCR8 holoenzyme preferred pri-miRNAs with a large terminal loop, an extended duplex region, and flanking single stranded RNAs. These results lay the foundation for future studies of the regulatory roles and conserved mechanisms of Drosha and Dicer.
-
The insertion in the double-stranded RNA binding domain of human Drosha is important for its function.
Biochimica et biophysica acta. Gene regulatory mechanisms, 2017Co-Authors: Xiaoxiao Zhang, Jian Lin, Haochu Huang, Bin Yin, Yan ZengAbstract:microRNAs (miRNAs) are first transcribed as long, primary transcripts, which are then processed by multiple enzymes and proteins to generate the single-stranded, approximately 22-nucleotide (nt)-long mature miRNAs. A critical step in animal miRNA biogenesis is the cleavage of primary miRNA transcripts (pri-miRNAs) to produce precursor miRNAs (pre-miRNAs) by the enzyme Drosha. How Drosha recognizes its substrates remains incompletely understood. In this study we constructed a series of human Drosha mutants and examined their enzymatic activities and interaction with RNAs. We found that the N-terminal region is required for the nuclear localization and cellular function of Drosha. And in contrast to previous reports, we showed that the double-stranded RNA binding domain (RBD) of Drosha exhibited a weak but noticeable affinity for RNA. Compared to the RBDs of other RNA-binding proteins, the RBD of Drosha has a short insert, whose mutations reduced RNA binding and pri-miRNA cleavage. Overexpression of Drosha RBD mutants in a reporter assay corroborated their deficiencies in Drosha activity in cell cultures. In addition, we found that point mutations in the RNaseIIIb domain of Drosha implicated in Wilms tumors differentially affected cleavage of the 5′ and 3′ strands of pri-miRNAs in vitro. In conclusion, our results provided important insights into the mechanism of pri-miRNA processing by human Drosha.
-
efficient processing of primary microrna hairpins by Drosha requires flanking nonstructured rna sequences
Journal of Biological Chemistry, 2005Co-Authors: Yan Zeng, Bryan R CullenAbstract:Abstract Drosha is a member of the ribonuclease (RNase) III family that selectively processes RNAs with prominent double-stranded features. Drosha plays a key role in the generation of precursor microRNAs from primary microRNA (pri-miRNA) transcripts in animal cells, yet how Drosha recognizes its RNA substrates remains incompletely understood. Previous studies have indicated that, within the context of a larger pri-miRNA, an ∼80-nucleotide-long RNA hairpin structure is necessary for processing by Drosha. Here, by performing in vitro Drosha processing reactions with RNA substrates of various sizes and structures, we show that Drosha function also requires single-stranded RNA extensions located outside the pri-miRNA hairpin. The sequence of these RNA extensions was largely unimportant, but a strong secondary structure within the extension or a blunt-ended pri-miRNA hairpin blocked Drosha cleavage. The requirement for single-stranded extensions on the pri-miRNA hairpin substrate for Drosha processing is currently unique among the RNase III enzymes.
-
Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha.
The EMBO journal, 2004Co-Authors: Yan Zeng, Bryan R CullenAbstract:A critical step during human microRNA maturation is the processing of the primary microRNA transcript by the nuclear RNaseIII enzyme Drosha to generate the ∼60-nucleotide precursor microRNA hairpin. How Drosha recognizes primary RNA substrates and selects its cleavage sites has remained a mystery, especially given that the known targets for Drosha processing show no discernable sequence homology. Here, we show that human Drosha selectively cleaves RNA hairpins bearing a large (⩾10 nucleotides) terminal loop. From the junction of the loop and the adjacent stem, Drosha then cleaves approximately two helical RNA turns into the stem to produce the precursor microRNA. Beyond the precursor microRNA cleavage sites, approximately one helix turn of stem extension is also essential for efficient processing. While the sites of Drosha cleavage are determined largely by the distance from the terminal loop, variations in stem structure and sequence around the cleavage site can fine-tune the actual cleavage sites chosen.
Baekgyu Kim - One of the best experts on this subject based on the ideXlab platform.
-
fCLIP-seq for transcriptomic footprinting of dsRNA-binding proteins: Lessons from Drosha
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2019Co-Authors: Baekgyu Kim, Narry V. KimAbstract:CLIP-seq (crosslinking immunoprecipitation and sequencing) is widely used to map the binding sites of a protein of interest on the transcriptome, and generally employs UV to induce the covalent bonds between protein and RNA, which allows stringent washing. However, dsRNA is inefficiently crosslinked by UV, making it difficult to study the interactions between dsRNA binding proteins and their substrates. A dsRNA endoribonuclease Drosha initiates the maturation of microRNA (miRNA) by cleaving primary miRNA (pri-miRNA). Despite the importance of Drosha in miRNA maturation and sequence determination, accurate mapping of Drosha cleavage sites has not been feasible due to rapid processing, modification, and degradation of the cleaved products in cells. Here, we present a high-throughput sequencing method that allows the mapping of in vivo Drosha cleavage sites at single nucleotide resolution, termed formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq). The fCLIP-seq protocol has been improved significantly over the standard CLIP-seq methods by (1) using formaldehyde for efficient and reversible crosslinking, (2) employing polyethylene glycol and adaptors with randomized sequences to enhance ligation efficiency and minimize bias, and (3) performing ligation after elution, which exposes the RNA termini for efficient ligation. fCLIP-seq successfully captures the nascent products of Drosha, which allows precise mapping of the Drosha processing sites. Moreover, from the analysis of the distinctive cleavage pattern, we discover previously unknown substrates of Drosha. fCLIP-seq is a useful tool to obtain transcriptome-wide information on Drosha activity and can be applied further to investigate other dsRNA-protein interactions. © 2018 Elsevier In
-
fCLIP-seq for transcriptomic footprinting of dsRNA-binding proteins: Lessons from Drosha.
Methods (San Diego Calif.), 2018Co-Authors: Baekgyu Kim, V. Narry KimAbstract:Abstract CLIP-seq (crosslinking immunoprecipitation and sequencing) is widely used to map the binding sites of a protein of interest on the transcriptome, and generally employs UV to induce the covalent bonds between protein and RNA, which allows stringent washing. However, dsRNA is inefficiently crosslinked by UV, making it difficult to study the interactions between dsRNA binding proteins and their substrates. A dsRNA endoribonuclease Drosha initiates the maturation of microRNA (miRNA) by cleaving primary miRNA (pri-miRNA). Despite the importance of Drosha in miRNA maturation and sequence determination, accurate mapping of Drosha cleavage sites has not been feasible due to rapid processing, modification, and degradation of the cleaved products in cells. Here, we present a high-throughput sequencing method that allows the mapping of in vivo Drosha cleavage sites at single nucleotide resolution, termed formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq). The fCLIP-seq protocol has been improved significantly over the standard CLIP-seq methods by (1) using formaldehyde for efficient and reversible crosslinking, (2) employing polyethylene glycol and adaptors with randomized sequences to enhance ligation efficiency and minimize bias, and (3) performing ligation after elution, which exposes the RNA termini for efficient ligation. fCLIP-seq successfully captures the nascent products of Drosha, which allows precise mapping of the Drosha processing sites. Moreover, from the analysis of the distinctive cleavage pattern, we discover previously unknown substrates of Drosha. fCLIP-seq is a useful tool to obtain transcriptome-wide information on Drosha activity and can be applied further to investigate other dsRNA-protein interactions.
-
Genome-wide Mapping of Drosha Cleavage Sites on Primary MicroRNAs and Noncanonical Substrates
CELL PRESS, 2018Co-Authors: Baekgyu Kim, Kyowon Jeong, Narry V. KimAbstract:MicroRNA (miRNA) maturation is initiated by Drosha, a double-stranded RNA (dsRNA)-specific RNase III enzyme. By cleaving primary miRNAs (pri-miRNAs) at specific positions, Drosha serves as a main determinant of miRNA sequences and a highly selective gatekeeper for the canonical miRNA pathway. However, the sites of Drosha-mediated processing have not been annotated, and it remains unclear to what extent Drosha functions outside the miRNA pathway. Here, we establish a protocol termed “formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq),” which allows identification of Drosha cleavage sites at single-nucleotide resolution. fCLIP identifies numerous processing sites, suggesting widespread end modifications during miRNA maturation. fCLIP also finds many pri-miRNAs that undergo alternative processing, yielding multiple miRNA isoforms. Moreover, we discovered dozens of Drosha substrates on non-miRNA loci, which may serve as cis-elements for Drosha-mediated gene regulation. We anticipate that fCLIP-seq could be a general tool for investigating interactions between dsRNA-binding proteins and structured RNAs. © 2017 Elsevier Inc9
-
Genome-wide Mapping of Drosha Cleavage Sites on Primary MicroRNAs and Noncanonical Substrates
Molecular cell, 2017Co-Authors: Baekgyu Kim, Kyowon Jeong, V. Narry KimAbstract:MicroRNA (miRNA) maturation is initiated by Drosha, a double-stranded RNA (dsRNA)-specific RNase III enzyme. By cleaving primary miRNAs (pri-miRNAs) at specific positions, Drosha serves as a main determinant of miRNA sequences and a highly selective gatekeeper for the canonical miRNA pathway. However, the sites of Drosha-mediated processing have not been annotated, and it remains unclear to what extent Drosha functions outside the miRNA pathway. Here, we establish a protocol termed "formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq)," which allows identification of Drosha cleavage sites at single-nucleotide resolution. fCLIP identifies numerous processing sites, suggesting widespread end modifications during miRNA maturation. fCLIP also finds many pri-miRNAs that undergo alternative processing, yielding multiple miRNA isoforms. Moreover, we discovered dozens of Drosha substrates on non-miRNA loci, which may serve as cis-elements for Drosha-mediated gene regulation. We anticipate that fCLIP-seq could be a general tool for investigating interactions between dsRNA-binding proteins and structured RNAs.
