The existence of sense overlapping transcripts that share regulatory and coding information in the same genomic sequence shows an additional level of prokaryotic gene expression complexity. Here we report the discovery of ncRNAs associated with IS1341-type transposase (tnpB) genes, at the 3'-end of such elements, with examples in archaea and bacteria. Focusing on the model haloarchaeon Halobacterium salinarum NRC-1, we show the existence of sense overlapping transcripts (sotRNAs) for all its IS1341-type transposases. Publicly available transcriptome compendium show condition-dependent differential regulation between sotRNAs and their cognate genes. These sotRNAs allowed us to find a UUCA tetraloop motif that is present in other archaea (ncRNA family HgcC) and in a H. salinarum intergenic ncRNA derived from a palindrome associated transposable elements (PATE). Overexpression of one sotRNA and the PATE-derived RNA harboring the tetraloop motif improved H. salinarum growth, indicating that these ncRNAs are functional.

Antisense RNAs (asRNAs) are present in diverse organisms and play important roles in gene regulation. In this work, we mapped the primary antisense transcriptome in the halophilic archaeon Halobacterium salinarum NRC-1. By reanalyzing publicly available data, we mapped antisense transcription start sites (aTSSs) and inferred the probable 3′ ends of these transcripts. We analyzed the resulting asRNAs according to the size, location, function of genes on the opposite strand, expression levels and conservation. We show that at least 21% of the genes contain asRNAs in H. salinarum. Most of these asRNAs are expressed at low levels. They are located antisense to genes related to distinctive characteristics of H. salinarum, such as bacteriorhodopsin, gas vesicles, transposases and other important biological processes such as translation. We provide evidence to support asRNAs in type II toxin–antitoxin systems in archaea. We also analyzed public Ribosome profiling (Ribo-seq) data and found that ~10% of the asRNAs are ribosome-associated non-coding RNAs (rancRNAs), with asRNAs from transposases overrepresented. Using a comparative transcriptomics approach, we found that ~19% of the asRNAs annotated in H. salinarum belong to genes with an ortholog in Haloferax volcanii, in which an aTSS could be identified with positional equivalence. This shows that most asRNAs are not conserved between these halophilic archaea.

NAD is a coenzyme central to metabolism that also serves as a 5′-terminal cap for bacterial and eukaryotic transcripts. Thermal degradation of NAD can generate nicotinamide and ADP-ribose (ADPR). Here, we use LC-MS/MS and NAD captureSeq to detect and identify NAD-RNAs in the thermophilic model archaeon Sulfolobus acidocaldarius and in the halophilic mesophile Haloferax volcanii. None of the four Nudix proteins of S. acidocaldarius catalyze NAD-RNA decapping in vitro, but one of the proteins (Saci_NudT5) promotes ADPR-RNA decapping. NAD-RNAs are converted into ADPR-RNAs, which we detect in S. acidocaldarius total RNA. Deletion of the gene encoding the 5′−3′ exonuclease Saci-aCPSF2 leads to a 4.5-fold increase in NAD-RNA levels. We propose that the incorporation of NAD into RNA acts as a degradation marker for Saci-aCPSF2. In contrast, ADPR-RNA is processed by Saci_NudT5 into 5′-p-RNAs, providing another layer of regulation for RNA turnover in archaeal cells.

Advances in genome-wide sequence technologies allow for detailed insights into the complexity of RNA landscapes of organisms from all three domains of life. Recent analyses of archaeal transcriptomes identified interaction and regulation networks of noncoding RNAs in this understudied domain. Here, we review current knowledge of small, noncoding RNAs with important functions for the archaeal lifestyle, which often requires adaptation to extreme environments. One focus is RNA metabolism at elevated temperatures in hyperthermophilic archaea, which reveals elevated amounts of RNA-guided RNA modification and virus defense strategies. Genome rearrangement events result in unique fragmentation patterns of noncoding RNA genes that require elaborate maturation pathways to yield functional transcripts. RNA-binding proteins, e.g., L7Ae and LSm, are important for many posttranscriptional control functions of RNA molecules in archaeal cells. We also discuss recent insights into the regulatory potential of their noncoding RNA partners.