The archaeal RNA chaperone TRAM0076 shapes the transcriptome and optimizes the growth of Methanococcus maripaludis.

Jie Li,William B Whitman,Lei Yue,Wenting Zhang,Liguang Zhou,Huicai Cheng,Xiuzhu Dong,Lei Qi,Bo Zhang,Lennart Randau

doi:10.1371/journal.pgen.1008328

Abstract

TRAM is a conserved domain among RNA modification proteins that are widely distributed in various organisms. In Archaea, TRAM occurs frequently as a standalone protein with in vitro RNA chaperone activity; however, its biological significance and functional mechanism remain unknown. This work demonstrated that TRAM0076 is an abundant standalone TRAM protein in the genetically tractable methanoarcheaon Methanococcus maripaludis. Deletion of MMP0076, the gene encoding TRAM0076, markedly reduced the growth and altered transcription of 55% of the genome. Substitution mutations of Phe39, Phe42, Phe63, Phe65 and Arg35 in the recombinant TRAM0076 decreased the in vitro duplex RNA unfolding activity. These mutations also prevented complementation of the growth defect of the MMP0076 deletion mutant, indicating that the duplex RNA unfolding activity was essential for its physiological function. A genome-wide mapping of transcription start sites identified many 5′ untranslated regions (5′UTRs) of 20–60 nt which could be potential targets of a RNA chaperone. TRAM0076 unfolded three representative 5′UTR structures in vitro and facilitated the in vivo expression of a mCherry reporter system fused to the 5′UTRs, thus behaving like a transcription anti-terminator. Flag-tagged-TRAM0076 co-immunoprecipitated a large number of cellular RNAs, suggesting that TRAM0076 plays multiple roles in addition to unfolding incorrect RNA structures. This work demonstrates that the conserved archaeal RNA chaperone TRAM globally affects gene expression and may represent a transcriptional element in ancient life of the RNA world.

Highlights

RNA chaperones impact mRNA metabolism including transcript synthesis, processing and degradation as well as translation
Physiological, biochemical and transcriptomic studies, we demonstrate that the standalone TRAM protein MMP0076 from M. maripaludis acts as an RNA chaperone and is required for the normal expression of more than half of the genome and normal growth
To confirm that the growth reduction phenotype was not due to a mutation at a second site, the ΔMMP0076::pac deletion mutant (Δ0076) mutant was complemented by expressing TRAM0076 from the replicative plasmid pMEV2-MMP0076 carrying the MMP0076 gene (MMP0076-com strain)

Summary

Introduction

RNA chaperones impact mRNA metabolism including transcript synthesis, processing and degradation as well as translation They play an important role in controlling gene expression, especially in response to environmental perturbations such as cold shock [1]. The distinctive features of RNA chaperones include the absence of an energy requirement for activity, transient interactions with their RNA targets without obvious sequence specificity, and the ability to melt kinetically trapped RNA hairpin structures [2, 3]. These characteristics enable RNA chaperones to interact with many classes of RNAs and through lowering the energetic barriers to assist RNA folding into thermodynamically favorable conformations [3]. Only a few archaeal RNA chaperones have been reported, and little is known about their physiological functions

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Results

Conclusion