The methanogenic archaeon Methanopyrus kandleri grows near the upper temperature limit for life. Genome analyses revealed strategies to adapt to these harsh conditions and elucidated a unique transfer RNA (tRNA) C-to-U editing mechanism at base 8 for 30 different tRNA species. Here, RNA-Seq deep sequencing methodology was combined with computational analyses to characterize the small RNome of this hyperthermophilic organism and to obtain insights into the RNA metabolism at extreme temperatures. A large number of 132 small RNAs were identified that guide RNA modifications, which are expected to stabilize structured RNA molecules. The C/D box guide RNAs were shown to exist as circular RNA molecules. In addition, clustered regularly interspaced short palindromic repeats RNA processing and potential regulatory RNAs were identified. Finally, the identification of tRNA precursors before and after the unique C8-to-U8 editing activity enabled the determination of the order of tRNA processing events with termini truncation preceding intron removal. This order of tRNA maturation follows the compartmentalized tRNA processing order found in Eukaryotes and suggests its conservation during evolution.

The prokaryotic adaptive immune system is based on the incorporation of genome fragments of invading viral genetic elements into clusters of regulatory interspaced short palindromic repeats (CRISPRs). The CRISPR loci are transcribed and processed into crRNAs, which are then used to target the invading nucleic acid for degradation. The large family of CRISPR-associated (Cas) proteins mediates this interference response. We have characterized Methanopyrus kandleri Csm3, a protein of the type III-A CRISPR-Cas complex. The 2.4 angstrom resolution crystal structure shows an elaborate four-domain fold organized around a core RRM-like domain. The overall architecture highlights the structural homology to Cas7, the Cas protein that forms the backbone of type I interference complexes. Csm3 binds unstructured RNAs in a sequence non-specific manner, suggesting that it interacts with the variable spacer sequence of the crRNA. The structural and biochemical data provide insights into the similarities and differences in this group of Cas proteins

A significant number of post-transcriptional changes occur during the generation of mature transfer RNAs (tRNAs). These changes within precursor-tRNA molecules include the processing of 5' and 3' termini, the introduction of modifications, and also RNA editing. In this review, we will detail the reported cases of A-to-I and C-to-U tRNA editing. The most widespread example is the A-to-I conversion of the tRNA anticodon wobble base mediated by TadA in prokaryotes and the heterodimeric ADAT2-ADAT3 complex in eukaryotes. Recently, the plant chloroplast adenosine-to-inosine tRNA editing enzyme has been discovered. The editing of C-to-U is much less prevalent within tRNA and is currently only known to occur in few organellar tRNA species and the cytoplasmic threonyl-tRNA in trypanosomatids. The responsible editing enzyme remains to be identified. Finally, an unusually widespread C-to-U editing scenario was discovered in the archaeon Methanopyrus kandleri. This editing is mediated by CDAT8, which is responsible for the restoration of the proper folding of thirty different tRNA species. The evolution of CDAT8 will be discussed.