ABSTRACT
RNA G-quadruplexes (G4s) play important roles in RNA biology. However, the function and regulation of mRNA G-quadruplexes in embryonic development remain elusive. Previously, we identified RHAU (DHX36, G4R1) as an RNA helicase that resolves mRNA G-quadruplexes. Here, we find that cardiac deletion of Rhau leads to heart defects and embryonic lethality in mice. Gene expression profiling identified Nkx2-5 mRNA as a target of RHAU that associates with its 5' and 3' UTRs and modulates its stability and translation. The 5' UTR of Nkx2-5 mRNA contains a G-quadruplex that requires RHAU for protein translation, while the 3' UTR of Nkx2-5 mRNA possesses an AU-rich element (ARE) that facilitates RHAU-mediated mRNA decay. Thus, we uncovered the mechanisms underlying Nkx2-5 post-transcriptional regulation during heart development. Meanwhile, this study demonstrates the function of mRNA 5' UTR G-quadruplex-mediated protein translation in organogenesis.
Subject(s)
DEAD-box RNA Helicases/metabolism , G-Quadruplexes , Homeodomain Proteins/genetics , Transcription Factors/genetics , 3' Untranslated Regions/genetics , 5' Untranslated Regions/genetics , Animals , Blotting, Northern , COS Cells , Cell Line , Chlorocebus aethiops , DEAD-box RNA Helicases/genetics , Heart/embryology , Homeobox Protein Nkx-2.5 , Humans , Mice , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolismABSTRACT
RIG-I is a DExD/H-box RNA helicase and functions as a critical cytoplasmic sensor for RNA viruses to initiate antiviral interferon (IFN) responses. Here we demonstrate that another DExD/H-box RNA helicase DHX36 is a key molecule for RIG-I signaling by regulating double-stranded RNA (dsRNA)-dependent protein kinase (PKR) activation, which has been shown to be essential for the formation of antiviral stress granule (avSG). We found that DHX36 and PKR form a complex in a dsRNA-dependent manner. By forming this complex, DHX36 facilitates dsRNA binding and phosphorylation of PKR through its ATPase/helicase activity. Using DHX36 KO-inducible MEF cells, we demonstrated that DHX36 deficient cells showed defect in IFN production and higher susceptibility in RNA virus infection, indicating the physiological importance of this complex in host defense. In summary, we identify a novel function of DHX36 as a critical regulator of PKR-dependent avSG to facilitate viral RNA recognition by RIG-I-like receptor (RLR).
Subject(s)
DEAD-box RNA Helicases/immunology , RNA Virus Infections/immunology , Signal Transduction/immunology , eIF-2 Kinase/immunology , Cytoplasmic Granules/immunology , DEAD Box Protein 58 , DEAD-box RNA Helicases/metabolism , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Gene Knockout Techniques , HEK293 Cells , HeLa Cells , Humans , Immunoprecipitation , RNA Viruses/immunology , RNA, Double-Stranded/immunology , RNA, Small Interfering/genetics , RNA, Viral/immunology , Receptors, Immunologic , Reverse Transcriptase Polymerase Chain Reaction , Stress, Physiological , TransfectionABSTRACT
The DEAH helicase RHAU (alias DHX36, G4R1) is the only helicase shown to have G-quadruplex (G4)-RNA resolvase activity and the major source of G4-DNA resolvase activity. Previous report showed RHAU mRNA expression to be elevated in human lymphoid and CD34(+) BM cells, suggesting a potential role in hematopoiesis. Here, we generated a conditional knockout of the RHAU gene in mice. Germ line deletion of RHAU led to embryonic lethality. We then targeted the RHAU gene specifically in the hematopoiesis system, using a Cre-inducible system in which an optimized variant of Cre recombinase was expressed under the control of the Vav1 promoter. RHAU deletion in hematopoietic system caused hemolytic anemia and differentiation defect at the proerythroblast stage. The partial differentiation block of proerythroblasts was because of a proliferation defect. Transcriptome analysis of RHAU knockout proerythroblasts showed that a statistically significant portion of the deregulated genes contain G4 motifs in their promoters. This suggests that RHAU may play a role in the regulation of gene expression that relies on its G4 resolvase activity.
Subject(s)
DEAD-box RNA Helicases/physiology , Hematopoiesis/genetics , Promoter Regions, Genetic/genetics , Anemia, Hemolytic, Congenital/genetics , Animals , Bone Marrow Transplantation , Cell Cycle , Crosses, Genetic , DEAD-box RNA Helicases/deficiency , DEAD-box RNA Helicases/genetics , Embryonic Development/genetics , Embryonic Development/physiology , Erythroblasts/pathology , Erythropoietin/blood , Genes, Lethal , Genes, Synthetic , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Hematopoiesis/physiology , Leukopenia/congenital , Leukopenia/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Folding , Proto-Oncogene Proteins c-vav/genetics , Radiation Chimera , Recombinases/deficiency , Recombinases/genetics , Recombinases/physiology , Thrombocytopenia/congenital , Thrombocytopenia/geneticsABSTRACT
Anticonvulsant sodium valproate interferes with brain glucose metabolism. The mechanism underlying such metabolic disturbance is unclear. We tested the hypothesis that sodium valproate interferes with cellular glucose transport with a focus on Glut1 since glucose transport across the blood-brain barrier relies on this transporter. Cell types enriched with Glut1 expression including human erythrocytes, human skin fibroblasts, and rat astrocytes were used to study the effects of sodium valproate on glucose transport. Sodium valproate significantly inhibited Glut1 activity in normal and Glut1-deficient erythrocytes by 20%-30%, causing a corresponding reduction of Vmax of glucose transport. Similarly, in primary astrocytes as well as in normal and Glut1-deficient fibroblasts, sodium valproate inhibited glucose transport by 20%-40% (P < 0.05), accompanied by an up to 60% downregulation of GLUT1 mRNA expression (P < 0.05). In conclusion, sodium valproate inhibits glucose transport and exacerbates Glut1 deficiency in vitro. Our findings imply the importance of prudent use of sodium valproate for patients with compromised Glut1 function.
