Dataset Information

ALKBH7-mediated demethylation regulates mitochondrial polycistronic RNA processing.

ABSTRACT: Members of the mammalian AlkB family are known to mediate nucleic acid demethylation^1,2. ALKBH7, a mammalian AlkB homologue, localizes in mitochondria and affects metabolism³, but its function and mechanism of action are unknown. Here we report an approach to site-specifically detect N¹-methyladenosine (m¹A), N³-methylcytidine (m³C), N¹-methylguanosine (m¹G) and N²,N²-dimethylguanosine (m₂²G) modifications simultaneously within all cellular RNAs, and discovered that human ALKBH7 demethylates m₂²G and m¹A within mitochondrial Ile and Leu1 pre-tRNA regions, respectively, in nascent polycistronic mitochondrial RNA^4-6. We further show that ALKBH7 regulates the processing and structural dynamics of polycistronic mitochondrial RNAs. Depletion of ALKBH7 leads to increased polycistronic mitochondrial RNA processing, reduced steady-state mitochondria-encoded tRNA levels and protein translation, and notably decreased mitochondrial activity. Thus, we identify ALKBH7 as an RNA demethylase that controls nascent mitochondrial RNA processing and mitochondrial activity.

SUBMITTER: Zhang LS

PROVIDER: S-EPMC8716185 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:The RNA-dependent RNA polymerase (RdRp) of influenza A virus consists of three subunits, PB2, PB1, and PA, and catalyses both viral RNA genome replication and transcription. Cotransfection of four monocistronic expression vectors for these subunits and nucleoprotein with an expression vector for viral RNA reconstitutes functional viral ribonucleoprotein complex (vRNP). However, the specific activity of reconstituted RdRp is usually very low since the expression level and the ratio of the three subunits by transfection are uncontrollable at single-cell levels. For efficient reconstitution of RdRp and vRNP, their levels need to be at least comparable. We constructed polycistronic expression vectors in which the coding sequences of the three subunits were joined with the 2A-like self-processing sequence of Thosea asigna virus (TaV2A) in various orders. The level of PB1 protein, even when it was placed at the most downstream, was comparable with that expressed from the monocistronic PB1 vector. In contrast, the levels of PB2 and PA were very low, the latter of which was most likely due to proteasomal degradation caused by the TaV2A-derived sequences attached to the amino- and/or carboxyl-terminal ends in this expression system. Interestingly, two of the constructs, in which the PB1 coding sequence was placed at the most upstream, showed much higher reporter activity in a luciferase-based mini-genome assay than that observed by cotransfection of the monocistronic vectors. When the coding sequence of selective antibiotic marker was further placed at the most downstream of the PB1-PA-PB2 open reading frame, stable cells expressing RdRp were easily established, indicating that acquisition of antibiotic resistance assured the expression of upstream RdRp. The addition of an affinity tag to the carboxyl-terminal end of PB2 allowed us to isolate reconstituted vRNP. Taken together, the polycistronic expression system for influenza virus RdRp may be available for functional and structural studies on vRNP.

Dataset Information

ALKBH7-mediated demethylation regulates mitochondrial polycistronic RNA processing.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets