ABSTRACT
N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m(6)A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m(6)A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m(6)A modification has fundamental and broad functions in mammalian cells.
Subject(s)
Dioxygenases/metabolism , Membrane Proteins/metabolism , Oxidoreductases, N-Demethylating/metabolism , RNA Processing, Post-Transcriptional , RNA, Messenger/metabolism , AlkB Homolog 5, RNA Demethylase , Animals , Base Sequence , Cell Nucleus/metabolism , Dioxygenases/chemistry , Dioxygenases/genetics , Gene Knockdown Techniques , HeLa Cells , Humans , Infertility, Male/enzymology , Male , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Mice, Knockout , Organ Size , Oxidoreductases, N-Demethylating/chemistry , Oxidoreductases, N-Demethylating/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Transport , RNA Interference , RNA, Messenger/chemistry , Spermatogenesis/genetics , Testis/enzymology , Testis/pathology , TranscriptomeABSTRACT
Supramolecular split-enzyme complementation restores enzymatic activity and allows for on-off switching. Split-luciferase fragment pairs were provided with an N-terminal FGG sequence and screened for complementation through host-guest binding to cucurbit[8]uril (Q8). Split-luciferase heterocomplex formation was induced in a Q8 concentration dependent manner, resulting in a 20-fold upregulation of luciferase activity. Supramolecular split-luciferase complementation was fully reversible, as revealed by using two types of Q8 inhibitors. Competition studies with the weak-binding FGG peptide revealed a 300-fold enhanced stability for the formation of the ternary heterocomplex compared to binding of two of the same fragments to Q8. Stochiometric binding by the potent inhibitor memantine could be used for repeated cycling of luciferase activation and deactivation in conjunction with Q8, providing a versatile module for inâ vitro supramolecular signaling networks.
Subject(s)
Bridged-Ring Compounds/pharmacology , Enzyme Inhibitors/pharmacology , Imidazoles/pharmacology , Luciferases/antagonists & inhibitors , Bridged-Ring Compounds/chemistry , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Imidazoles/chemistry , Luciferases/metabolism , Macromolecular Substances/chemistry , Macromolecular Substances/pharmacology , Models, Molecular , Structure-Activity RelationshipABSTRACT
Protein immobilization on surfaces, and on lipid bilayers specifically, has great potential in biomolecular and biotechnological research. Of current special interest is the immobilization of proteins using supramolecular noncovalent interactions. This allows for a reversible immobilization and obviates the use of harsh ligation conditions that could denature fragile proteins. In the work presented here, reversible supramolecular immobilization of proteins on lipid bilayer surfaces was achieved by using the host-guest interaction of the macrocyclic molecule cucurbit[8]uril. A fluorescent protein was successfully immobilized on the lipid bilayer by making use of the property of cucurbit[8]uril to host together a methylviologen and the indole of a tryptophan positioned on the N-terminal of the protein. The supramolecular complex was anchored to the bilayer through a cholesterol moiety that was attached to the methylviologen tethered with a small polyethylene glycol spacer. Protein immobilization studies using a quartz crystal microbalance (QCM) showed the assembly of the supramolecular complexes on the bilayer. Specific immobilization through the protein N-terminus is more efficient than through protein side-chain events. Reversible surface release of the proteins could be achieved by washing with cucurbit[8]uril or buffer alone. The described system shows the potential of supramolecular assembly of proteins and provides a method for site-specific protein immobilization under mild conditions in a reversible manner.
Subject(s)
Bridged-Ring Compounds/chemistry , Imidazoles/chemistry , Immobilized Proteins/chemistry , Lipid Bilayers/chemistry , Polyethylene Glycols/chemistry , Molecular StructureABSTRACT
Supramolecular assembly of a beta-barrel protein via cucurbit[8]uril results in compact z-shaped protein dimers. SAXS data reveal the formation of a well ordered protein dimer, notwithstanding being connected by a reversible and flexible peptide linker, and highlight the supramolecular induced interplay of the proteins, analogous to covalently linked proteins.