While many aspects of archaeal cell biology remain relatively unexplored, systems biology approaches like mass spectrometry (MS) based proteomics offer an opportunity for rapid advances. Unfortunately, the enormous amount of MS data generated often remains incompletely analyzed due to a lack of sophisticated bioinformatic tools and field-specific biological expertise for data interpretation. Here we present the initiation of the Archaeal Proteome Project (ArcPP), a community-based effort to comprehensively analyze archaeal proteomes. Starting with the model archaeon Haloferax volcanii, we reanalyze MS datasets from various strains and culture conditions. Optimized peptide spectrum matching, with strict control of false discovery rates, facilitates identifying > 72% of the reference proteome, with a median protein sequence coverage of 51%. These analyses, together with expert knowledge in diverse aspects of cell biology, provide meaningful insights into processes such as Nterminal protein maturation, N-glycosylation, and metabolism. Altogether, ArcPP serves as an invaluable blueprint for comprehensive prokaryotic proteomics.

Eukaryotic ribosome biogenesis is a complex dynamic process which requires the action of numerous ribosome assembly factors. Among them, the eukaryotic Rio protein family members (Rio1, Rio2 and Rio3) belong to an ancient conserved atypical protein kinase/ ATPase family required for the maturation of the small ribosomal subunit (SSU). Recent structure–function analyses suggested an ATPase-dependent role of the Rio proteins to regulate their dynamic association with the nascent pre-SSU. However, the evolutionary origin of this feature and the detailed molecular mechanism that allows controlled activation of the catalytic activity remained to be determined. In this work we provide functional evidence showing a conserved role of the archaeal Rio proteins for the synthesis of the SSU in archaea. Moreover, we unravel a conserved RNA-dependent regulation of the Rio ATPases, which in the case of Rio2 involves, at least, helix 30 of the SSU rRNA and the P-loop lysine within the shared RIO domain. Together, our study suggests a ribosomal RNA-mediated regulatory mechanism enabling the appropriate stimulation of Rio2 catalytic activity and subsequent release of Rio2 from the nascent pre-40S particle. Based on our findings we propose a unified release mechanism for the Rio proteins.

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.

Ribosomes are intricate molecular machines ensuring proper protein synthesis in every cell. Ribosome biogenesis is a complex process which has been intensively analyzed in bacteria and eukaryotes. In contrast, our understanding of the in vivo archaeal ribosome biogenesis pathway remains less characterized. Here, we have analyzed the in vivo role of the almost universally conserved ribosomal RNA dimethyltransferase KsgA/Dim1 homolog in archaea. Our study reveals that KsgA/Dim1-dependent 16S rRNA dimethylation is dispensable for the cellular growth of phylogenetically distant archaea. However, proteomics and functional analyses suggest that archaeal KsgA/Dim1 and its rRNA modification activity (i) influence the expression of a subset of proteins and (ii) contribute to archaeal cellular fitness and adaptation. In addition, our study reveals an unexpected KsgA/Dim1-dependent variability of rRNA modifications within the archaeal phylum. Combining structure-based functional studies across evolutionary divergent organisms, we provide evidence on how rRNA structure sequence variability (re-)shapes the KsgA/Dim1-dependent rRNA modification status. Finally, our results suggest an uncoupling between the KsgA/Dim1-dependent rRNA modification completion and its release from the nascent small ribosomal subunit. Collectively, our study provides additional understandings into principles of molecular functional adaptation, and further evolutionary and mechanistic insights into an almost universally conserved step of ribosome synthesis.

Archaea are widespread organisms colonizing almost every habitat on Earth. However, the molecular biology of archaea still remains relatively uncharacterized. RNA metabolism is a central cellular process, which has been extensively analyzed in both bacteria and eukarya. In contrast, analysis of RNA metabolism dynamic in archaea has been limited to date. To facilitate analysis of the RNA metabolism dynamic at a system-wide scale in archaea, we have established non-radioactive pulse labeling of RNA, using the nucleotide analog 4-thiouracil (4TU) in two commonly used model archaea: the halophile Euryarchaeota Haloferax volcanii, and the thermo-acidophile Crenarchaeota Sulfolobus acidocaldarius. In this work, we show that 4TU pulse labeling can be efficiently performed in these two organisms in a dose- and time-dependent manner. In addition, our results suggest that uracil prototrophy had no critical impact on the overall 4TU incorporation in RNA molecules. Accordingly, our work suggests that 4TU incorporation can be widely performed in archaea, thereby expanding the molecular toolkit to analyze archaeal gene expression network dynamic in unprecedented detail.