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

Abstract

In all three domains of life, tRNA genes contain introns that must be removed to yield functional tRNA. In archaea and eukarya, the first step of this process is catalyzed by a splicing endonuclease. The consensus structure recognized by the splicing endonuclease is a bulge-helix-bulge (BHB) motif which is also found in rRNA precursors. So far, a systematic analysis to identify all biological substrates of the splicing endonuclease has not been carried out. In this study, we employed CRISPRi to repress expression of the splicing endonuclease in the archaeon Haloferax volcanii to identify all substrates of this enzyme. Expression of the splicing endonuclease was reduced to 1% of its normal level, resulting in a significant extension of lag phase in H. volcanii growth. In the repression strain, 41 genes were down-regulated and 102 were up-regulated. As an additional approach in identifying new substrates of the splicing endonuclease, we isolated and sequenced circular RNAs, which identified excised introns removed from tRNA and rRNA precursors as well as from the 5′ UTR of the gene HVO_1309. In vitro processing assays showed that the BHB sites in the 5′ UTR of HVO_1309 and in a 16S rRNA-like precursor are processed by the recombinant splicing endonuclease. The splicing endonuclease is therefore an important player in RNA maturation in archaea.

Ribosome Biogenesis in Archaea

“…As in bacteria, the sequences flanking the rRNA genes have extended complementarity and pair, forming double-helical stems that are the target of certain endonucleases starting rRNA maturation. However, although, in bacteria, these stems are cleaved by RNAse III, in most archaea, they typically contain Bulge-helix-Bulge (BHB) motifs that are recognized and cleaved by the archaeal-specific endA splicing endonuclease (Tang et al, 2002;Ferreira-Cerca, 2017;Clouet-d'Orval et al, 2018;Qi et al, 2020;Schwarz et al, 2020; Figure 2). Consequently, the pre-16S and pre-23S rRNAs are ligated and first released in a circular pre-rRNA form, which is subsequently opened and matured by other enzymes that have not yet been characterized (Tang et al, 2002;Ferreira-Cerca, 2017;Clouetd'Orval et al, 2018;Jüttner et al, 2020;Qi et al, 2020;Schwarz et al, 2020).…”

“…However, although, in bacteria, these stems are cleaved by RNAse III, in most archaea, they typically contain Bulge-helix-Bulge (BHB) motifs that are recognized and cleaved by the archaeal-specific endA splicing endonuclease (Tang et al, 2002;Ferreira-Cerca, 2017;Clouet-d'Orval et al, 2018;Qi et al, 2020;Schwarz et al, 2020; Figure 2). Consequently, the pre-16S and pre-23S rRNAs are ligated and first released in a circular pre-rRNA form, which is subsequently opened and matured by other enzymes that have not yet been characterized (Tang et al, 2002;Ferreira-Cerca, 2017;Clouetd'Orval et al, 2018;Jüttner et al, 2020;Qi et al, 2020;Schwarz et al, 2020). For a comprehensive review of the rRNA maturating/modifying enzymes, see Clouet-d'Orval et al (2018) and Ferreira-Cerca (2017).…”

“…Last, the endonuclease endA known to be involved in the maturation of intron-containing tRNAs (Clouet-d'Orval et al, 2018) has been recently implicated in rRNA processing, thereby indicating a functional coordination of tRNA and rRNA maturation in archaea (Qi et al, 2020;Schwarz et al, 2020; Figure 2).…”

Insights into rRNA processing and modification mapping in Archaea using Nanopore-based RNA sequencing

“…Recently, we determined the functional requirement of the bulge-helix-bulge motifs for the formation of circ-pre-rRNAs in H. volcanii (11). Moreover, in analogy to intron containing-tRNA splicing, the rRNA bhb motifs are cleaved by the tRNA splicing endonuclease (endA) prior to covalent circularization (8, 9). Although intact circular RNA cannot be directly sequenced by the Nanopore approach, we noticed the presence of permuted transcripts by analysing clipping values, indicative of sequences that do not match to a linear template.…”

“…In Euryarchaeota, the 16S and 23S rRNAs are additionally separated by the presence of an internal tRNA. In most archaea, the 16S and 23S rRNA processing stems contain a bulge-helix-bulge (bhb) motif, which is, in the context of intron-containing tRNAs, recognised by the splicing endonuclease endA (6)(7)(8)(9). Similar to intron-containing tRNA maturation, processing at the bhb motifs is followed by the covalent ligation of the resulting extremities, thereby generating the archaeal-specific circular pre-16S and circular pre-23S rRNAs (1,5,7,10,11).…”