ABSTRACT
To investigate if the microRNA (miRNA) pathway is required for dendritic cell (DC) development, we assessed the effect of ablating Drosha and Dicer, the two enzymes central to miRNA biogenesis. We found that while Dicer deficiency had some effect, Drosha deficiency completely halted DC development and halted myelopoiesis more generally. This indicated that while the miRNA pathway did have a role, it was a non-miRNA function of Drosha that was particularly critical. Drosha repressed the expression of two mRNAs encoding inhibitors of myelopoiesis in early hematopoietic progenitors. We found that Drosha directly cleaved stem-loop structure within these mRNAs and that this mRNA degradation was necessary for myelopoiesis. We have therefore identified a mechanism that regulates the development of DCs and other myeloid cells.
Subject(s)
Dendritic Cells/immunology , Dendritic Cells/metabolism , Myelopoiesis/immunology , RNA, Messenger/metabolism , Ribonuclease III/immunology , Animals , Base Sequence , Cell Differentiation/genetics , Cell Differentiation/immunology , DEAD-box RNA Helicases/deficiency , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/immunology , Dendritic Cells/cytology , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/metabolism , Inflammation/immunology , Inflammation/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs/genetics , MicroRNAs/metabolism , Molecular Sequence Data , Myelopoiesis/genetics , Myosin Light Chains/antagonists & inhibitors , Myosin Light Chains/genetics , Myosin Light Chains/metabolism , Nucleic Acid Conformation , RNA, Messenger/chemistry , RNA, Messenger/genetics , Ribonuclease III/deficiency , Ribonuclease III/geneticsABSTRACT
We identified Mrpl44 in a search for mammalian proteins that contain RNase III domains. This protein was previously found in association with the mitochondrial ribosome of bovine liver extracts. However, the precise Mrpl44 localization had been unclear. Here, we show by immunofluorescence microscopy and subcellular fractionation that Mrpl44 is localized to the matrix of the mitochondria. We found that it can form multimers, and confirm that it is part of the large subunit of the mitochondrial ribosome. By manipulating its expression, we show that Mrpl44 may be important for regulating the expression of mtDNA-encoded genes. This was at the level of RNA expression and protein translation. This ultimately impacted ATP synthesis capability and respiratory capacity of cells. These findings indicate that Mrpl44 plays an important role in the regulation of the mitochondrial OXPHOS capacity.
Subject(s)
Mitochondrial Proteins/metabolism , Adenosine Triphosphate/biosynthesis , Animals , Cattle , Cell Line , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Gene Expression Regulation , Gene Knockdown Techniques , HEK293 Cells , Humans , Mice , Mitochondria, Liver/metabolism , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Mitochondrial Ribosomes/metabolism , NIH 3T3 Cells , Oxidative Phosphorylation , Oxygen Consumption , Protein Multimerization , Ribonuclease III/chemistry , Ribonuclease III/genetics , Ribonuclease III/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolismABSTRACT
Roquin is an RNA-binding protein that prevents autoimmunity and inflammation via repression of bound target mRNAs such as inducible costimulator (Icos). When Roquin is absent or mutated (Roquin(san)), Icos is overexpressed in T cells. Here we show that Roquin enhances Dicer-mediated processing of pre-miR-146a. Roquin also directly binds Argonaute2, a central component of the RNA-induced silencing complex, and miR-146a, a microRNA that targets Icos mRNA. In the absence of functional Roquin, miR-146a accumulates in T cells. Its accumulation is not due to increased transcription or processing, rather due to enhanced stability of mature miR-146a. This is associated with decreased 3' end uridylation of the miRNA. Crystallographic studies reveal that Roquin contains a unique HEPN domain and identify the structural basis of the 'san' mutation and Roquin's ability to bind multiple RNAs. Roquin emerges as a protein that can bind Ago2, miRNAs and target mRNAs, to control homeostasis of both RNA species.
Subject(s)
Argonaute Proteins/metabolism , Homeostasis , MicroRNAs/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Crystallography, X-Ray , HEK293 Cells , Half-Life , Humans , Mice, Inbred C57BL , MicroRNAs/genetics , Protein Binding/genetics , Protein Structure, Tertiary , RNA Processing, Post-Transcriptional , RNA Stability , Ribonuclease III/metabolism , T-Lymphocytes/metabolism , Ubiquitin-Protein Ligases/chemistryABSTRACT
Micro RNAs (miRNAs) are â¼22 nt small RNAs that function as posttranscriptional negative regulators of gene expression. Thousands of these molecules have so far been identified across the metazoan and plant kingdoms. Although most miRNAs share the same biogenesis pathway, numerous noncanonical miRNAs and other small RNAs have also been identified. In this review, we will discuss these alternate small RNA biogenesis pathways as well as recent discoveries of non-miRNA functions of the miRNA biogenesis machinery. This review will focus only on metazoan pathways.
