A versatile cis-acting element reporter system to study the function, maturation and stability of ribosomal RNA mutants in archaea

Abstract

General molecular principles of ribosome biogenesis have been well explored in bacteria and eukaryotes. Collectively, these studies have revealed important functional differences and few similarities between these processes. Phylogenetic studies suggest that the information processing machineries from archaea and eukaryotes are evolutionary more closely related than their bacterial counterparts. These observations raise the question of how ribosome synthesis in archaea may proceed in vivo. In this study, we describe a versatile plasmid-based cis-acting reporter system allowing to analyze in vivo the consequences of ribosomal RNA mutations in the model archaeon Haloferax volcanii. Applying this system, we provide evidence that the bulge-helix-bulge motif enclosed within the ribosomal RNA processing stems is required for the formation of archaeal-specific circular-pre-rRNA intermediates and mature rRNAs. In addition, we have collected evidences suggesting functional coordination of the early steps of ribosome synthesis in H. volcanii. Together our investigation describes a versatile platform allowing to generate and functionally analyze the fate of diverse rRNA variants, thereby paving the way to better understand the cis-acting molecular determinants necessary for archaeal ribosome synthesis, maturation, stability and function.

Exploring prokaryotic transcription, operon structures, rRNA maturation and modifications using Nanopore-based native RNA sequencing

“…Moreover, we have hypothesized that ligation of the 16S rRNA leader/trailer resulting from the 16S rRNA bulge-helix-bulge maturation process generates a putative new pre-rRNA intermediate which we could observe for the first time in Pyrococcus using native RNA sequencing. In addition, the presence of intermediates (1) and (3) support the idea that the maturation of the co-transcribed internal tRNA is inefficient or inhibited and may preferentially occur after processing of the 16S and 23S rRNA bulge-helix-bulge [suggested in 76,78 ].…”

“…15f). In addition, the majority of the expected processing sites, notably at the predicted or known bulge-helix-bulge motifs 78 , were faithfully observed in P. furiosus and H. volcanii (see Supplementary Fig. 14,15).…”

“…9b). Nevertheless, we could confirm the presence of processing sites and pre-rRNA intermediates in the different organisms analysed, with the exception of the archaea specific circular-pre-rRNA intermediates 73,76,78,79 , which escape analysis due to the lack of 5’/3’ extremities. Among the identified pre-rRNA intermediates, precursor (4) observed in P. furiosus , which include ligation at the bhb motif of the upstream region of the 16S leader and downstream region of the 16S trailer sequences continuous to the downstream tRNA/23S sequences is of particular interest.…”

“…In most archaea, the 16S and 23S rRNA processing stems contain a bulge-helix-bulge motif which is, in the context of intron-containing tRNA, recognized by the splicing endonuclease endA 75–77 . Similar to intron-containing tRNA maturation, processing at the bulge-helix-bulge motifs results in the ligation of the resulting extremities, thereby generating the archaeal specific circular pre-16S and circular pre-23S rRNAs 73,76,78,79 . The molecular mechanisms by which the circular pre-rRNA intermediates are further processed into linear mature rRNA are unknown 73,76 .…”

Splicing Endonuclease Is an Important Player in rRNA and tRNA Maturation in Archaea

“…In another study (Tang et al, 2002), it was suggested that circular 16S and 23S pre-rRNAs may be generated upon cleavage at the BHB site and subsequent ligation of exposed ends. Indeed, the presence of circular pre-rRNAs was confirmed in subsequent studies (Danan et al, 2012;Jüttner et al, 2020); however the enzyme catalyzing the reaction in vivo was unknown. More recently, using in vitro methods, it was shown that a splicing endonuclease catalyses this reaction in methanoarchaea (Qi et al, 2020).…”