Authors: Fei Peng, Jie Xu, Bai Cui...

RNase III DROSHA is upregulated in multiple cancers and contributes to tumor progression by hitherto unclear mechanisms. Here, we demonstrate that DROSHA interacts with β-Catenin to transactivate STC1 in an RNA cleavage-independent manner, contributing to breast cancer stem-like cell (BCSC) properties. DROSHA mRNA stability is enhanced by N-methyladenosine (mA) modification which is activated by AURKA in BCSCs. AURKA stabilizes METTL14 by inhibiting its ubiquitylation and degradation to promote DROSHA mRNA methylation. Moreover, binding of AURKA to DROSHA transcript further strengthens the binding of the mA reader IGF2BP2 to stabilize mA-modified DROSHA. In addition, wild-type DROSHA, but not an mA methylation-deficient mutant, enhances BCSC stemness maintenance, while inhibition of DROSHA mA modification attenuates BCSC traits. Our study unveils the AURKA-induced oncogenic mA modification as a key regulator of DROSHA in breast cancer and identifies a novel DROSHA transcriptional function in promoting the BCSC phenotype.

Topics: Adenosine; Adult; Aged; Animals; Aurora Kinase A; Breast Neoplasms; Female; Glycoproteins; HEK293 Cells; Humans; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplastic Stem Cells; Oncogene Proteins; RNA Stability; Ribonuclease III; Signal Transduction; Transcriptional Activation; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

PubMed: 32859993
DOI: 10.1038/s41422-020-00397-2

Authors: Pavan Kumar Kakumani, Yunkoo Ko, Sushmitha Ramakrishna...

MicroRNAs are sequentially processed by RNase III enzymes Drosha and Dicer. miR-451 is a highly conserved miRNA in vertebrates which bypasses Dicer processing and instead relies on AGO2 for its maturation. miR-451 is highly expressed in erythrocytes and regulates the differentiation of erythroblasts into mature red blood cells. However, the mechanistic details underlying miR-451 biogenesis in erythrocytes remains obscure. Here, we report that the RNA binding protein CSDE1 which is required for the development of erythroblasts into erythrocytes, controls the expression of miR-451 in erythroleukemia cells. CSDE1 binds miR-451 and regulates AGO2 processing of pre-miR-451 through its N-terminal domains. CSDE1 further interacts with PARN and promotes the trimming of intermediate miR-451 to the mature length. Together, our results demonstrate that CSDE1 promotes biogenesis of miR-451 in erythroid progenitors.

Topics: Animals; MicroRNAs; Ribonuclease III; RNA-Binding Proteins; Humans

PubMed: 37493604
DOI: 10.1093/nar/gkad619

Authors: Ankur Garg, Renfu Shang, Todor Cvetanovic...

MicroRNA (miRNA) biogenesis is initiated upon cleavage of a primary miRNA (pri-miRNA) hairpin by the Microprocessor (MP), composed of the Drosha RNase III enzyme and its partner DGCR8. Multiple pri-miRNA sequence motifs affect MP recognition, fidelity, and efficiency. Here, we performed cryoelectron microscopy (cryo-EM) and biochemical studies of several let-7 family pri-miRNAs in complex with human MP. We show that MP has the structural plasticity to accommodate a range of pri-miRNAs. These structures revealed key features of the 5' UG sequence motif, more comprehensively represented as the "flipped U with paired N" (fUN) motif. Our analysis explains how cleavage of class-II pri-let-7 members harboring a bulged nucleotide generates a non-canonical precursor with a 1-nt 3' overhang. Finally, the MP-SRSF3-pri-let-7f1 structure reveals how SRSF3 contributes to MP fidelity by interacting with the CNNC motif and Drosha's Piwi/Argonaute/Zwille (PAZ)-like domain. Overall, this study sheds light on the mechanisms for flexible recognition, accurate cleavage, and regulated processing of different pri-miRNAs by MP.

Topics: MicroRNAs; Humans; Ribonuclease III; RNA-Binding Proteins; Cryoelectron Microscopy; RNA Processing, Post-Transcriptional; Nucleic Acid Conformation; HEK293 Cells; Protein Binding

PubMed: 39368465
DOI: 10.1016/j.molcel.2024.09.008

Review

Authors: Allen W Nicholson

Double-stranded(ds) RNA has diverse roles in gene expression and regulation, host defense, and genome surveillance in bacterial and eukaryotic cells. A central aspect of dsRNA function is its selective recognition and cleavage by members of the ribonuclease III (RNase III) family of divalent-metal-ion-dependent phosphodiesterases. The processing of dsRNA by RNase III family members is an essential step in the maturation and decay of coding and noncoding RNAs, including miRNAs and siRNAs. RNase III, as first purified from Escherichia coli, has served as a biochemically well-characterized prototype, and other bacterial orthologs provided the first structural information. RNase III family members share a unique fold (RNase III domain) that can dimerize to form a structure that binds dsRNA and cleaves phosphodiesters on each strand, providing the characteristic 2 nt, 3'-overhang product ends. Ongoing studies are uncovering the functions of additional domains, including, inter alia, the dsRNA-binding and PAZ domains that cooperate with the RNase III domain to select target sites, regulate activity, confer processivity, and support the recognition of structurally diverse substrates. RNase III enzymes function in multicomponent assemblies that are regulated by diverse inputs, and at least one RNase III-related polypeptide can function as a noncatalytic, dsRNA-binding protein. This review summarizes the current knowledge of the mechanisms of catalysis and target site selection of RNase III family members, and also addresses less well understood aspects of these enzymes and their interactions with dsRNA.

Topics: Animals; Humans; Models, Molecular; Protein Conformation; RNA, Double-Stranded; Ribonuclease III; Substrate Specificity

PubMed: 24124076
DOI: 10.1002/wrna.1195

Authors: Caroline Thivierge, Maxime Bellefeuille, Sarah-Slim Diwan...

MicroRNAs (miRNAs) play crucial roles in physiological functions and disease, but the regulation of their nuclear biogenesis remains poorly understood. Here, BioID on Drosha, the catalytic subunit of the microprocessor complex, reveals its proximity to splicing factor proline- and glutamine (Q)-rich (SFPQ), a multifunctional RNA-binding protein (RBP) involved in forming paraspeckle nuclear condensates. SFPQ depletion impacts both primary and mature miRNA expression, while other paraspeckle proteins (PSPs) or the paraspeckle scaffolding RNA NEAT1 do not, indicating a paraspeckle-independent role. Comprehensive transcriptomic analyses show that SFPQ loss broadly affects RNAs and miRNA host gene (HG) expression, influencing both their transcription and the stability of their products. Notably, SFPQ protects the oncogenic miR-17∼92 polycistron from degradation by the nuclear exosome targeting (NEXT)-exosome complex and is tightly linked with its overexpression across a broad variety of cancers. Our findings reveal a dual role for SFPQ in regulating miRNA HG transcription and stability, as well as its significance in cancers.

Topics: Humans; MicroRNAs; PTB-Associated Splicing Factor; Cell Nucleus; Transcription, Genetic; Ribonuclease III; RNA-Binding Proteins; HeLa Cells

PubMed: 39250314
DOI: 10.1016/j.celrep.2024.114695

Authors: Jie Gao, Chen-Fei Liu, Ping-Ping Liu...

Invertebrates mainly rely on sequence-specific RNA interference (RNAi) to resist viral infections. Increasing studies show that double-stranded RNA (dsRNA) can induce sequence-independent protection and that Dicer-2, the key RNAi player that cleaves long dsRNA into small interfering RNA (siRNA), is necessary for this protection. However, how this protection occurs remains unknown. Herein, we report that it is caused by adenosine triphosphate (ATP)-hydrolysis accompanying the dsRNA-cleavage. Dicer-2 helicase domain is ATP-dependent; therefore, the cleavage consumes ATP. ATP depletion activates adenosine monophosphate-activated protein kinase (Ampk) and induces nuclear localization of Fork head box O (FoxO), a key transcriptional factor for dsRNA-induced genes. siRNAs that do not require processing cannot activate the transcriptional response. This study reveals a unique nonspecific antiviral mechanism other than the specific RNAi in shrimp. This mechanism is functionally similar to, but mechanistically different from, the dsRNA-activated antiviral response in vertebrates and suggests an interesting evolution of innate antiviral immunity.

Topics: Animals; RNA, Double-Stranded; Ribonuclease III; AMP-Activated Protein Kinases; Adenosine Triphosphate; RNA Interference; RNA, Small Interfering; Immunity, Innate; Transcription, Genetic

PubMed: 39047046
DOI: 10.1073/pnas.2409233121

Review

Authors: Fumiaki Sato, Soken Tsuchiya, Stephen J Meltzer...

MicroRNAs (miRNAs) comprise species of short noncoding RNA that regulate gene expression post-transcriptionally. Recent studies have demonstrated that epigenetic mechanisms, including DNA methylation and histone modification, not only regulate the expression of protein-encoding genes, but also miRNAs, such as let-7a, miR-9, miR-34a, miR-124, miR-137, miR-148 and miR-203. Conversely, another subset of miRNAs controls the expression of important epigenetic regulators, including DNA methyltransferases, histone deacetylases and polycomb group genes. This complicated network of feedback between miRNAs and epigenetic pathways appears to form an epigenetics-miRNA regulatory circuit, and to organize the whole gene expression profile. When this regulatory circuit is disrupted, normal physiological functions are interfered with, contributing to various disease processes. The present minireview details recent discoveries involving the epigenetics-miRNA regulatory circuit, suggesting possible biological insights into gene-regulatory mechanisms that may underlie a variety of diseases.

Topics: Animals; DNA (Cytosine-5-)-Methyltransferases; DNA Methyltransferase 3A; Epigenesis, Genetic; Gene Expression Regulation; Genomic Imprinting; Histone Deacetylases; Humans; MicroRNAs; Polycomb-Group Proteins; Proteins; RNA Polymerase II; RNA-Binding Proteins; Repressor Proteins; Ribonuclease III

PubMed: 21395977
DOI: 10.1111/j.1742-4658.2011.08089.x

Authors: Yingzi Cui, Xuehui Lyu, Li Ding...

MicroRNAs (miRNAs) have essential functions during embryonic development, and their dysregulation causes cancer. Altered global miRNA abundance is found in different tissues and tumours, which implies that precise control of miRNA dosage is important, but the underlying mechanism(s) of this control remain unknown. The protein complex Microprocessor, which comprises one DROSHA and two DGCR8 proteins, is essential for miRNA biogenesis. Here we identify a developmentally regulated miRNA dosage control mechanism that involves alternative transcription initiation (ATI) of DGCR8. ATI occurs downstream of a stem-loop in DGCR8 mRNA to bypass an autoregulatory feedback loop during mouse embryonic stem (mES) cell differentiation. Deletion of the stem-loop causes imbalanced DGCR8:DROSHA protein stoichiometry that drives irreversible Microprocessor aggregation, reduced primary miRNA processing, decreased mature miRNA abundance, and widespread de-repression of lipid metabolic mRNA targets. Although global miRNA dosage control is not essential for mES cells to exit from pluripotency, its dysregulation alters lipid metabolic pathways and interferes with embryonic development by disrupting germ layer specification in vitro and in vivo. This miRNA dosage control mechanism is conserved in humans. Our results identify a promoter switch that balances Microprocessor autoregulation and aggregation to precisely control global miRNA dosage and govern stem cell fate decisions during early embryonic development.

Topics: Animals; Gene Dosage; Gene Expression Regulation, Developmental; Germ Layers; Hep G2 Cells; Humans; K562 Cells; Lipid Metabolism; Mice; MicroRNAs; Promoter Regions, Genetic; RNA-Binding Proteins; Ribonuclease III; Transcription Initiation, Genetic

PubMed: 33953397
DOI: 10.1038/s41586-021-03524-0

Review

Authors: Donald L Court, Jianhua Gan, Yu-He Liang...

RNase III is a global regulator of gene expression in Escherichia coli that is instrumental in the maturation of ribosomal and other structural RNAs. We examine here how RNase III itself is regulated in response to growth and other environmental changes encountered by the cell and how, by binding or processing double-stranded RNA (dsRNA) intermediates, RNase III controls the expression of genes. Recent insight into the mechanism of dsRNA binding and processing, gained from structural studies of RNase III, is reviewed. Structural studies also reveal new cleavage sites in the enzyme that can generate longer 3' overhangs.

Topics: 3' Untranslated Regions; 5' Untranslated Regions; Amino Acid Motifs; Bacteriophage lambda; Catalysis; Clustered Regularly Interspaced Short Palindromic Repeats; Escherichia coli; Escherichia coli Proteins; Eukaryotic Cells; Gene Expression Regulation, Bacterial; Nucleic Acid Conformation; Operon; Prokaryotic Cells; Protein Processing, Post-Translational; RNA; RNA Processing, Post-Transcriptional; RNA, Bacterial; RNA, Double-Stranded; RNA, Ribosomal; RNA, Small Untranslated; Ribonuclease III; Structure-Activity Relationship; Substrate Specificity; Virus Diseases

PubMed: 24274754
DOI: 10.1146/annurev-genet-110711-155618

Review

Authors: Kinga Ciechanowska, Maria Pokornowska, Anna Kurzyńska-Kokorniak...

Ribonuclease Dicer belongs to the family of RNase III endoribonucleases, the enzymes that specifically hydrolyze phosphodiester bonds found in double-stranded regions of RNAs. Dicer enzymes are mostly known for their essential role in the biogenesis of small regulatory RNAs. A typical Dicer-type RNase consists of a helicase domain, a domain of unknown function (DUF283), a PAZ (Piwi-Argonaute-Zwille) domain, two RNase III domains, and a double-stranded RNA binding domain; however, the domain composition of Dicers varies among species. Dicer and its homologues developed only in eukaryotes; nevertheless, the two enzymatic domains of Dicer, helicase and RNase III, display high sequence similarity to their prokaryotic orthologs. Evolutionary studies indicate that a combination of the helicase and RNase III domains in a single protein is a eukaryotic signature and is supposed to be one of the critical events that triggered the consolidation of the eukaryotic RNA interference. In this review, we provide the genetic insight into the domain organization and structure of Dicer proteins found in vertebrate and invertebrate animals, plants and fungi. We also discuss, in the context of the individual domains, domain deletion variants and partner proteins, a variety of Dicers' functions not only related to small RNA biogenesis pathways.

Topics: Animals; Evolution, Molecular; Fungi; Gene Deletion; Humans; Models, Molecular; Plants; Protein Conformation; Protein Domains; Ribonuclease III

PubMed: 33435485
DOI: 10.3390/ijms22020616