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

Abstract

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 of mtaA1, mtaB1, and mtaC1, the methanol methyltransferase (Mta) genes, in 30°C versus 15°C culture, while ackA and pta 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. The in vivo half-lives of mtaA1 and mtaC1B1 mRNAs were similar in 30°C and 15°C cultures. However, the ptaackA 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 for mtaA1 and mtaC1B1 mRNAs, while only a 27-nt 5= UTR was present in the pta-ackA transcript. Removal of the 5= UTRs significantly reduced the in vitro half-lives of mtaA1 and mtaC1B1 mRNAs. Remarkably, fusion of the mtaA1 or mtaC1B1 5= UTRs to pta-ackA mRNA increased its in 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-adaptive M. mazei zm-15.

Genome-wide gene expression and RNA half-life measurements allow predictions of regulation and metabolic behavior in Methanosarcina acetivorans

“…The similarity of mRNA degradation rates among related species grown under similar conditions—as evidenced by comparison of our results to the limited studies on M. mazei [66] and findings of other studies in other organisms [12, 14]—suggests that metabolic control points and regulatory modes identified in one organism could be inferred in the metabolism of similar organisms. From an evolutionary stand-point related organisms subjected to similar environments would likely retain regulatory controls that optimize their fitness.…”

“…6 b and Additional file 1: Figure S13 and Additional file 3). Transcript counts also matched those measured in M. mazei [66] suggesting these Methanosarcina species could have similar transcription and degradation characteristics, similarly to the two Sulfolobus species. In general some of the conclusions drawn from this study might hold for evolutionarily related methanogens (e.g.…”

“…These studies show that RNA degradation is evolutionarily conserved and suggest that our study of RNA stability in M. acetivorans may be extended to related organisms such as M. mazei or M. barkeri . We compared our measured half-lives to five that had been previously measured in M. mazei [66] and found them to have similar values (Fig. 6 a).…”

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Fig. 6Comparison of Transcripts with Methanosarcina mazei [66] a ) A comparison of mRNA half lives measured via our RNAseq data compared with a previous study using qtRT-PCR in the related organism Methanosarcina mazei growing in methanol or acetate. Cao et al measured methyltransferase ( mtaA1 , mtaCB1 ) half-lives from methanol grown cells, while they measured acetoclastic gene ( pta , ack ) half-lives for acetate grown cells.

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Global mapping transcriptional start sites revealed both transcriptional and post-transcriptional regulation of cold adaptation in the methanogenic archaeon Methanolobus psychrophilus

“…This unique architecture of the cspA mRNA allows it to function as a RNA thermometer9. Our recent work demonstrated that the mRNAs for mtaA and mtaC , which encode the methylcobalamin:coenzyme M methyltransferase and methanol corrinoid proteins that are the key enzymes in methanol-derived methanogenesis of a cold-adaptive methanogen, all have long 5′ UTRs, that contribute to the transcripts' stability at low temperatures10.…”