RESUMO
The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) posed a major threat to global health. Although the World Health Organization ended the public health emergency status, antiviral drugs are needed to address new variants of SARS-CoV-2 and future pandemics. To identify novel broad-spectrum coronavirus drugs, we developed a high-content imaging platform compatible with high-throughput screening. The platform is broadly applicable as it can be adapted to include various cell types, viruses, antibodies, and dyes. We demonstrated that the antiviral activity of compounds against SARS-CoV-2 variants (Omicron BA.5 and Omicron XBB.1.5), SARS-CoV, and human coronavirus 229E could easily be assessed. The inclusion of cellular dyes and immunostaining in combination with in-depth image analysis enabled us to identify compounds that induced undesirable phenotypes in host cells, such as changes in cell morphology or in lysosomal activity. With the platform, we screened â¼900K compounds and triaged hits, thereby identifying potential candidate compounds carrying broad-spectrum activity with limited off-target effects. The flexibility and early-stage identification of compounds with limited host cell effects provided by this high-content imaging platform can facilitate coronavirus drug discovery. We anticipate that its rapid deployability and fast turnaround can also be applied to combat future pandemics.
Assuntos
Infecções por Coronavirus , Coronavirus , Humanos , Antivirais/farmacologia , Antivirais/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Ensaios de Triagem em Larga Escala/métodos , Corantes/farmacologia , Corantes/uso terapêutico , PandemiasRESUMO
Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.
Assuntos
Proteômica , Fatores de Transcrição , Humanos , Proteômica/métodos , Cisteína/metabolismo , LigantesRESUMO
The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partly under control by vaccination. However, highly potent and safe antiviral drugs for SARS-CoV-2 are still needed to avoid development of severe COVID-19. We report the discovery of a small molecule, Z-Tyr-Ala-CHN2, which was identified in a cell-based antiviral screen. The molecule exerts sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. Time-of-addition studies reveal that Z-Tyr-Ala-CHN2 acts at the early phase of the infection cycle, which is in line with the observation that the molecule inhibits cathepsin L. This results in antiviral activity against SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but not in Caco-2 cells or primary human nasal epithelial cells since the latter two cell types also permit entry via transmembrane protease serine subtype 2 (TMPRSS2). Given their cell-specific activity, cathepsin L inhibitors still need to prove their value in the clinic; nevertheless, the activity profile of Z-Tyr-Ala-CHN2 makes it an interesting tool compound for studying the biology of coronavirus entry and replication.
RESUMO
Adult-stem-cell-derived organoids model human epithelial tissues ex vivo, which enables the study of host-microbe interactions with great experimental control. This protocol comprises methods to coculture organoids with microbes, particularly focusing on human small intestinal and colon organoids exposed to individual bacterial species. Microinjection into the lumen and periphery of 3D organoids is discussed, as well as exposure of organoids to microbes in a 2D layer. We provide detailed protocols for characterizing the coculture with regard to bacterial and organoid cell viability and growth kinetics. Spatial relationships can be studied by fluorescence live microscopy, as well as scanning electron microscopy. Finally, we discuss considerations for assessing the impact of bacteria on gene expression and mutations through RNA and DNA sequencing. This protocol requires equipment for standard mammalian tissue culture, or bacterial or viral culture, as well as a microinjection device.
Assuntos
Intestinos , Organoides , Técnicas de CoculturaRESUMO
Canonical Wnt signaling plays a key role during organ development, homeostasis and regeneration and these processes are conserved between invertebrates and vertebrates. Mutations in Wnt pathway components are commonly found in various types of cancer. Upon activation of canonical Wnt signaling, ß-catenin binds in the nucleus to members of the TCF-LEF family and activates the transcription of target genes. Multiple Wnt target genes, including Lgr5/LGR5 and Axin2/AXIN2, have been identified in mouse models and human cancer cell lines. Here we set out to identify the transcriptional targets of Wnt signaling in five human tissues using organoid technology. Organoids are derived from adult stem cells and recapitulate the functionality as well as the structure of the original tissue. Since the Wnt pathway is critical to maintain the organoids from the human intestine, colon, liver, pancreas and stomach, organoid technology allows us to assess Wnt target gene expression in a human wildtype situation. We performed bulk mRNA sequencing of organoids immediately after inhibition of Wnt pathway and identified 41 genes as commonly regulated genes in these tissues. We also identified large numbers of target genes specific to each tissue. One of the shared target genes is TEAD4, a transcription factor driving expression of YAP/TAZ signaling target genes. In addition to TEAD4, we identified a variety of genes which encode for proteins that are involved in Wnt-independent pathways, implicating the possibility of direct crosstalk between Wnt signaling and other pathways. Collectively, this study identified tissue-specific and common Wnt target gene signatures and provides evidence for a conserved role for these Wnt targets in different tissues.
Assuntos
Sistema Digestório/citologia , Regulação da Expressão Gênica no Desenvolvimento , Organoides/metabolismo , Via de Sinalização Wnt , Adulto , Sistema Digestório/embriologia , Sistema Digestório/metabolismo , Endoderma , Perfilação da Expressão Gênica , Humanos , Especificidade de ÓrgãosRESUMO
Cryptosporidium parvum is one of the major causes of human diarrheal disease. To understand the pathology of the parasite and develop efficient drugs, an in vitro culture system that recapitulates the conditions in the host is needed. Organoids, which closely resemble the tissues of their origin, are ideal for studying host-parasite interactions. Organoids are three-dimensional (3D) tissue-derived structures which are derived from adult stem cells and grow in culture for extended periods of time without undergoing any genetic aberration or transformation. They have well defined polarity with both apical and basolateral surfaces. Organoids have various applications in drug testing, bio banking, and disease modeling and host-microbe interaction studies. Here we present a step-by-step protocol of how to prepare the oocysts and sporozoites of Cryptosporidium for infecting human intestinal and airway organoids. We then demonstrate how microinjection can be used to inject the microbes into the organoid lumen. There are three major methods by which organoids can be used for host-microbe interaction studies-microinjection, mechanical shearing and plating, and by making monolayers. Microinjection enables maintenance of the 3D structure and allows for precise control of parasite volumes and direct apical side contact for the microbes. We provide details for optimal growth of organoids for either imaging or oocyst production. Finally, we also demonstrate how the newly generated oocysts can be isolated from the organoid for further downstream processing and analysis.
Assuntos
Cryptosporidium parvum/fisiologia , Microinjeções , Organoides/citologia , Técnicas de Cultura de Tecidos/métodos , Humanos , Intestinos/citologia , Intestinos/microbiologiaRESUMO
The deubiquitinating enzyme BAP1 is a tumor suppressor, among others involved in cholangiocarcinoma. BAP1 has many proposed molecular targets, while its Drosophila homolog is known to deubiquitinate histone H2AK119. We introduce BAP1 loss-of-function by CRISPR/Cas9 in normal human cholangiocyte organoids. We find that BAP1 controls the expression of junctional and cytoskeleton components by regulating chromatin accessibility. Consequently, we observe loss of multiple epithelial characteristics while motility increases. Importantly, restoring the catalytic activity of BAP1 in the nucleus rescues these cellular and molecular changes. We engineer human liver organoids to combine four common cholangiocarcinoma mutations (TP53, PTEN, SMAD4, and NF1). In this genetic background, BAP1 loss results in acquisition of malignant features upon xenotransplantation. Thus, control of epithelial identity through the regulation of chromatin accessibility appears to be a key aspect of BAP1's tumor suppressor function. Organoid technology combined with CRISPR/Cas9 provides an experimental platform for mechanistic studies of cancer gene function in a human context.
Assuntos
Colangiocarcinoma/genética , Cromatina/metabolismo , Células Epiteliais/fisiologia , Neoplasias Hepáticas/genética , Fígado/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Bioengenharia , Carcinogênese , Células Cultivadas , Cromatina/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Citoesqueleto/metabolismo , Feminino , Humanos , Mutação com Perda de Função/genética , Camundongos , Camundongos SCID , Organoides , Transplante Heterólogo , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genéticaRESUMO
Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Fibrose Cística/patologia , Células Epiteliais/patologia , Técnicas de Cultura de Órgãos/métodos , Organoides/patologia , Infecções por Vírus Respiratório Sincicial/patologia , Sistema Respiratório/patologia , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Células Cultivadas , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Células Epiteliais/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Organoides/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/isolamento & purificação , Sistema Respiratório/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Stem-cell-derived organoids recapitulate in vivo physiology of their original tissues, representing valuable systems to model medical disorders such as infectious diseases. Cryptosporidium, a protozoan parasite, is a leading cause of diarrhoea and a major cause of child mortality worldwide. Drug development requires detailed knowledge of the pathophysiology of Cryptosporidium, but experimental approaches have been hindered by the lack of an optimal in vitro culture system. Here, we show that Cryptosporidium can infect epithelial organoids derived from human small intestine and lung. The parasite propagates within the organoids and completes its complex life cycle. Temporal analysis of the Cryptosporidium transcriptome during organoid infection reveals dynamic regulation of transcripts related to its life cycle. Our study presents organoids as a physiologically relevant in vitro model system to study Cryptosporidium infection.
Assuntos
Criptosporidiose/genética , Cryptosporidium/patogenicidade , Perfilação da Expressão Gênica/métodos , Organoides/parasitologia , Criptosporidiose/parasitologia , Cryptosporidium/crescimento & desenvolvimento , Regulação da Expressão Gênica , Humanos , Intestino Delgado/parasitologia , Pulmão/parasitologia , Modelos Biológicos , Técnicas de Cultura de Órgãos , Análise de Sequência de RNA , Análise Espaço-TemporalRESUMO
Organoids are 3D in vitro culture systems derived from self-organizing stem cells. They can recapitulate the in vivo architecture, functionality, and genetic signature of original tissues. Thus, organoid technology has been rapidly applied to understanding stem cell biology, organogenesis, and various human pathologies. The recent development of human patient-derived organoids has enabled disease modeling with precision, highlighting their great potential in biomedical applications, translational medicine, and personalized therapy. In light of recent breakthroughs using organoids, it is only apt that we appreciate the advantages and shortcomings of this technology to exploit its full potential. We discuss recent advances in the application of organoids in studying cancer and hereditary diseases, as well as in the examination of host cell-microorganism interactions.
Assuntos
Organoides/metabolismo , Organoides/fisiologia , Animais , Modelos Animais de Doenças , Humanos , Neoplasias/metabolismo , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologiaRESUMO
The RNA-binding protein TRBP is a central component of the Dicer complex. Despite a decade of biochemical and structural studies, the essential functionality of TRBP in microRNA (miRNA) biogenesis remains unknown. Here we show that TRBP is an integral cofactor for time-efficient Dicer processing in RNA-crowded environments. We competed for Dicer processing of pre-miRNA with a large amount of cellular RNA species and found that Dicer-TRBP, but not Dicer alone, remains resilient. To apprehend the mechanism of this substrate selectivity, we use single-molecule fluorescence. The real-time observation reveals that TRBP acts as a gatekeeper, precluding Dicer from engaging with pre-miRNA-like substrates. TRBP acquires the selectivity using the PAZ domain of Dicer, whereas Dicer moderates the RNA-binding affinity of TRBP for fast turnover. This coordinated action between TRBP and Dicer accomplishes an efficient way of discarding pre-miRNA-like substrates.
Assuntos
MicroRNAs/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/fisiologia , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/metabolismo , Coativadores de Receptor Nuclear , Ligação Proteica , Domínios Proteicos , RNA , Proteínas de Ligação a RNA/genética , Transcrição GênicaRESUMO
Culturing intestinal stem cells into 3D organoids results in heterogeneous cell populations, reflecting the in vivo cell type diversity. In a recent paper published in Nature, Wang et al. established a culture condition for a highly homogeneous population of intestinal stem cells.
Assuntos
Intestinos/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , HumanosRESUMO
We have solved two families of crystal structures of the human Dicer "platform-PAZ-connector helix" cassette in complex with small interfering RNAs (siRNAs). The structures possess two adjacently positioned pockets: a 2 nt 3'-overhang-binding pocket within the PAZ domain (3' pocket) and a phosphate-binding pocket within the platform domain (phosphate pocket). One family of complexes contains a knob-like α-helical protrusion, designated "hDicer-specific helix," that separates the two pockets and orients the bound siRNA away from the surface of Dicer, which could be indicative of a product release/transfer state. In the second complex, the helical protrusion is melted/disordered and the bound siRNA is aligned toward the surface of Dicer, suggestive of a cleavage-competent state. These structures allow us to propose that the transition from the cleavage-competent to the postulated product release/transfer state may involve release of the 5'-phosphate from the phosphate pocket while retaining the 3' overhang in the 3' pocket.
Assuntos
RNA Helicases DEAD-box/química , Ribonuclease III/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Cristalografia por Raios X , RNA Helicases DEAD-box/metabolismo , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Fosfatos/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Ribonuclease III/metabolismo , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de SuperfícieRESUMO
Single murine and human intestinal stem cells can be expanded in culture over long time periods as genetically and phenotypically stable epithelial organoids. Increased cAMP levels induce rapid swelling of such organoids by opening the cystic fibrosis transmembrane conductor receptor (CFTR). This response is lost in organoids derived from cystic fibrosis (CF) patients. Here we use the CRISPR/Cas9 genome editing system to correct the CFTR locus by homologous recombination in cultured intestinal stem cells of CF patients. The corrected allele is expressed and fully functional as measured in clonally expanded organoids. This study provides proof of concept for gene correction by homologous recombination in primary adult stem cells derived from patients with a single-gene hereditary defect.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Intestinos/patologia , Organoides/metabolismo , Células-Tronco/metabolismo , Adulto , Células-Tronco Adultas/metabolismo , Animais , Sequência de Bases , Terapia Genética , Humanos , Camundongos , Dados de Sequência Molecular , Edição de RNA/genéticaRESUMO
RNase III Drosha initiates microRNA (miRNA) maturation by cleaving a primary miRNA transcript and releasing a pre-miRNA with a 2 nt 3' overhang. Dicer recognizes the 2 nt 3' overhang structure to selectively process pre-miRNAs. Here, we find that, unlike prototypic pre-miRNAs (group I), group II pre-miRNAs acquire a shorter (1 nt) 3' overhang from Drosha processing and therefore require a 3'-end mono-uridylation for Dicer processing. The majority of let-7 and miR-105 belong to group II. We identify TUT7/ZCCHC6, TUT4/ZCCHC11, and TUT2/PAPD4/GLD2 as the terminal uridylyl transferases responsible for pre-miRNA mono-uridylation. The TUTs act specifically on dsRNAs with a 1 nt 3' overhang, thereby creating a 2 nt 3' overhang. Depletion of TUTs reduces let-7 levels and disrupts let-7 function. Although the let-7 suppressor, Lin28, induces inhibitory oligo-uridylation in embryonic stem cells, mono-uridylation occurs in somatic cells lacking Lin28 to promote let-7 biogenesis. Our study reveals functional duality of uridylation and introduces TUT7/4/2 as components of the miRNA biogenesis pathway.
Assuntos
Proteínas de Ligação a DNA/metabolismo , MicroRNAs/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , RNA Nucleotidiltransferases/metabolismo , Processamento Pós-Transcricional do RNA , Uridina Monofosfato/metabolismo , Sequência de Bases , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas de Ligação a RNA/metabolismo , Fatores de Poliadenilação e Clivagem de mRNARESUMO
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 (â¼22 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.
Assuntos
RNA Helicases DEAD-box/metabolismo , MicroRNAs/metabolismo , Ribonuclease III/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Proteínas de Drosophila/metabolismo , Células-Tronco Embrionárias/metabolismo , Evolução Molecular , Giardia/enzimologia , Células HEK293 , Humanos , MicroRNAs/biossíntese , MicroRNAs/química , MicroRNAs/genética , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Fosfatos/metabolismo , Fosforilação , RNA Helicases/metabolismo , Ribonuclease III/deficiência , Ribonuclease III/genética , Especificidade por Substrato/genéticaRESUMO
Single-molecule techniques have been used for only a subset of biological problems because of difficulties in studying proteins that require cofactors or post-translational modifications. Here, we present a new method integrating single-molecule fluorescence microscopy and immunopurification to study protein complexes. We used this method to investigate Lin28-mediated microRNA uridylation by TUT4 (terminal uridylyl transferase 4, polyU polymerase), which regulates let-7 microRNA biogenesis. Our real-time analysis of the uridylation by the TUT4 immunoprecipitates suggests that Lin28 functions as a processivity factor of TUT4. Our new technique, SIMPlex (single-molecule approach to immunoprecipitated protein complexes), provides a universal tool to analyse complex proteins at the single-molecule level.
Assuntos
Bioensaio , Imunoprecipitação/métodos , MicroRNAs/metabolismo , Complexos Multiproteicos/metabolismo , RNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Proteínas de Ligação a RNARESUMO
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.
Assuntos
Interferência de RNA , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Vias Biossintéticas , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Plantas/genética , Plantas/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Pequeno RNA não Traduzido/genéticaRESUMO
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 (Paroo et al., 2009), now reveal the posttranslational modifications that take part in the regulation of the core RNA silencing factors, Ago, Piwi, and TRBP.
Assuntos
Chaperonas Moleculares/metabolismo , Processamento de Proteína Pós-Traducional , Interferência de RNA , RNA não Traduzido/metabolismo , Animais , HumanosRESUMO
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.