RESUMO
MicroRNAs (miRNAs) are sequentially processed by two RNase III enzymes, Drosha and Dicer. miR-451 is the only known miRNA whose processing bypasses Dicer and instead relies on the slicer activity of Argonaute-2 (Ago2). miR-451 is highly conserved in vertebrates and regulates erythrocyte maturation, where it becomes the most abundant miRNA. However, the basis for the non-canonical biogenesis of miR-451 is unclear. Here, we show that Ago2 is less efficient than Dicer in processing pre-miRNAs, but this deficit is overcome when miR-144 represses Dicer in a negative-feedback loop during erythropoiesis. Loss of miR-144-mediated Dicer repression in zebrafish embryos and human cells leads to increased canonical miRNA production and impaired miR-451 maturation. Overexpression of Ago2 rescues some of the defects of miR-451 processing. Thus, the evolution of Ago2-dependent processing allows miR-451 to circumvent the global repression of canonical miRNAs elicited, in part, by the miR-144 targeting of Dicer during erythropoiesis.
Assuntos
Proteínas Argonautas/genética , Eritropoese/genética , MicroRNAs/genética , Animais , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Interferência de RNA , Ribonuclease III/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.
Assuntos
Células Epiteliais Alveolares/metabolismo , COVID-19/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , SARS-CoV-2/metabolismo , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , Animais , Antivirais , COVID-19/genética , COVID-19/patologia , Chlorocebus aethiops , Efeito Citopatogênico Viral , Citoesqueleto , Avaliação Pré-Clínica de Medicamentos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/virologia , Fosfoproteínas/genética , Transporte Proteico , Proteoma/genética , SARS-CoV-2/genética , Transdução de Sinais , Células Vero , Tratamento Farmacológico da COVID-19RESUMO
RNA provides the framework for the assembly of some of the most intricate macromolecular complexes within the cell, including the spliceosome and the mature ribosome. The assembly of these complexes relies on the coordinated association of RNA with hundreds of trans-acting protein factors. While some of these trans-acting factors are RNA-binding proteins (RBPs), others are adaptor proteins, and others still, function as both. Defects in the assembly of these complexes results in a number of human pathologies including neurodegeneration and cancer. Here, we demonstrate that Silencing Defective 2 (SDE2) is both an RNA binding protein and also a trans-acting adaptor protein that functions to regulate RNA splicing and ribosome biogenesis. SDE2 depletion leads to widespread changes in alternative splicing, defects in ribosome biogenesis and ultimately complete loss of cell viability. Our data highlight SDE2 as a previously uncharacterized essential gene required for the assembly and maturation of the complexes that carry out two of the most fundamental processes in mammalian cells.
Assuntos
Processamento Alternativo/genética , Proteínas de Ligação a DNA/genética , Splicing de RNA/genética , Ribossomos/genética , Genes Essenciais/genética , Humanos , Proteínas de Ligação a RNA/genética , Spliceossomos/genéticaRESUMO
Differentiation of stem and progenitor cells is a highly regulated process that involves the coordinated action of multiple layers of regulation. Here we show how the post-transcriptional regulatory layer instructs the level of chromatin regulation via miR-144 and its targets to orchestrate chromatin condensation during erythropoiesis. The loss of miR-144 leads to impaired chromatin condensation during erythrocyte maturation. Among the several targets of miR-144 that influence chromatin organization, the miR-144-dependent regulation of Hmgn2 is conserved from fish to humans. Our genetic probing of the miR-144/Hmgn2 regulatory axis established that intact miR-144 target sites in the Hmgn2 3'UTR are necessary for the proper maturation of erythrocytes in both zebrafish and human iPSC-derived erythroid cells while loss of Hmgn2 rescues in part the miR-144 null phenotype. Altogether, our results uncover miR-144 and its target Hmgn2 as the backbone of the genetic regulatory circuit that controls the terminal differentiation of erythrocytes in vertebrates.
RESUMO
Differentiation of stem and progenitor cells is a highly regulated process that involves the coordinated action of multiple layers of regulation. Here we show how the post-transcriptional regulatory layer instructs the level of chromatin regulation via miR-144 and its targets to orchestrate chromatin condensation during erythropoiesis. The loss of miR-144 leads to impaired chromatin condensation during erythrocyte maturation. Among the several targets of miR-144 that influence chromatin organization, the miR-144-dependent regulation of Hmgn2 is conserved from fish to humans. Our genetic probing of the miR-144/Hmgn2 regulatory axis establish that intact miR-144 target sites in the Hmgn2 3'UTR are necessary for the proper maturation of erythrocytes in both zebrafish and human iPSC-derived erythroid cells while loss of Hmgn2 rescues in part the miR-144 null phenotype. Altogether, our results uncover miR-144 and its target Hmgn2 as the backbone of the genetic regulatory circuit that controls the terminal differentiation of erythrocytes in vertebrates.
Assuntos
Cromatina , Eritropoese , MicroRNAs , Peixe-Zebra , MicroRNAs/metabolismo , MicroRNAs/genética , Eritropoese/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Humanos , Animais , Cromatina/metabolismo , Cromatina/genética , Eritrócitos/metabolismo , Regiões 3' não Traduzidas/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Diferenciação Celular/genéticaRESUMO
The proto-oncogene c-MYC is a key representative of the MYC transcription factor network regulating growth and metabolism. MML-1 (Myc- and Mondo-like) is its homolog in C. elegans. The functional and molecular cooperation between c-MYC and H3 lysine 79 methyltransferase DOT1L was demonstrated in several human cancer types, and we have earlier discovered the connection between C. elegans MML-1 and DOT-1.1. Here, we demonstrate the critical role of DOT1L/DOT-1.1 in regulating c-MYC/MML-1 target genes genome-wide by ensuring the removal of "spent" transcription factors from chromatin by the nuclear proteasome. Moreover, we uncover a previously unrecognized proteolytic activity of DOT1L, which may facilitate c-MYC turnover. This new mechanism of c-MYC regulation by DOT1L may lead to the development of new approaches for cancer treatment.