Mechanism for Stabilizing mRNAs Involved in Methanol-Dependent Methanogenesis of Cold-Adaptive Methanosarcina mazei zm-15

Methylotrophic methanogenesis predominates at low temperatures in the cold Zoige wetland in Tibet. To elucidate the basis of cold-adapted methanogenesis in these habitats,Methanosarcina mazei zm-15 was isolated, and the molecular basis of its cold activity was studied. For this strain, aceticlastic methanogenesis was reduced 7.7-fold during growth at 15°C versus 30°C. Methanol-derived methanogenesis decreased only 3-fold under the same conditions, suggesting that it is more cold adaptive. Reverse transcription-quantitative PCR (RT-qPCR) detected <2-fold difference in the transcript abundances ofmtaA1 ,mtaB1 , andmtaC1 , the methanol methyltransferase (Mta) genes, in 30°C versus 15°C culture, whileackA andpta mRNAs, encoding acetate kinase (Ack) and phosphotransacetylase (Pta) in aceticlastic methanogenesis, were 4.5- and 6.8-fold higher in 30°C culture than in 15°C culture. Thein vivo half-lives ofmtaA1 andmtaC1B1 mRNAs were similar in 30°C and 15°C cultures. However, thepta-ackA mRNA half-life was significantly reduced in 15°C culture compared to 30°C culture. Using circularized RNA RT-PCR, large 5′ untranslated regions (UTRs) (270 nucleotides [nt] and 238 nt) were identified formtaA1 andmtaC1B1 mRNAs, while only a 27-nt 5′ UTR was present in thepta-ackA transcript. Removal of the 5′ UTRs significantly reduced thein vitro half-lives ofmtaA1 andmtaC1B1 mRNAs. Remarkably, fusion of themtaA1 ormtaC1B1 5′ UTRs topta-ackA mRNA increased itsin vitro half-life at both 30°C and 15°C. These results demonstrate that the large 5′ UTRs significantly enhance the stability of the mRNAs involved in methanol-derived methanogenesis in the cold-adaptiveM. mazei zm-15.