Non-coding RNA (ncRNA) genes play important, but incompletely understood, roles in various cellular processes, notably translation and gene regulation. A recent report on the detection of the ncRNA Signal Recognition Particle gene in the nuclear ribosomal internal transcribed spacer region of several species of three genera of ectomycorrhizal basidiomycetes prompted a more thorough bioinformatics search for additional ncRNA genes in the full fungal ribosomal operon. This study reports on the detection of three ncRNA genes hitherto not known from the fungal ribosomal region: nuclear RNase P RNA, RNase MRP RNA, and a possible snoRNA U14 in a total of five species of Auricularia and Inocybe. We verified their presence through resequencing of independent specimens. Two completed Auricularia genomes were found to lack these ncRNAs elsewhere than in the ribosomal operon, suggesting that these are functional genes. It seems clear that ncRNA genes play a larger role in fungal ribosomal genetics than hitherto thought.

Basidiomycetes, IGS, ITS, MRP, non-coding RNA, RNase MRP, RNase P, SRP

Non-coding RNA (ncRNA) are stretches of RNA – typically thought of as genes – that are not translated into proteins through translation. A range of functions has been ascribed to the various groups of ncRNAs known to date, including important roles in translation, gene regulation, and chromosome inactivation (Cech and Steitz 2014). The number of ncRNA genes in the human genome alone is believed to run in the thousands, although relatively few have been characterised to any satisfactory level (Li and Liu 2019). Knowledge of ncRNAs in fungal genomes is rudimentary by comparison, but ncRNAs appear to play important cellular roles in the relatively few fungi examined to date (Li et al. 2021). The ncRNAs identified in the present study are, with few exceptions, all ubiquitous in eukaryotes and there are as yet no examples of them missing in fungi. They play important roles in tRNA processing (RNase P RNA), rRNA maturation (RNase MRP RNA), and ER targeting of proteins (SRP RNA), and they constitute the RNA component of the respective ribonucleoprotein (RNP) particles (López et al. 2009; Akopian et al. 2013).

Alm Rosenblad et al. (2016) unexpectedly found sequence analysis-derived evidence of the ncRNA Signal Recognition Particle (SRP) RNAs in the nuclear ribosomal ITS1 region of 11 species in three ectomycorrhizal genera of the Basidiomycota: Astraeus, Russula, and Lactarius. Indirect evidence furthermore suggested that these SRP RNAs could be functional. Queries in the GenBank (Sayers et al. 2021) and UNITE (Nilsson et al. 2019; Abarenkov et al. 2022) databases failed to produce any other fungi with SRP RNA in their ITS region, raising questions as to why such a rare genetic element would have been gained several times independently in the ITS1 region of a set of three relatively closely related genera.

The recent trend of employing various high-throughput sequencing technologies to generate longer stretches of the fungal ribosomal operon than just the ITS region (Wurzbacher et al. 2019) offers a possibility to extend the search for fungal ncRNAs beyond the ITS region. The present study reports on a broadening of the search of Alm Rosenblad et al. (2016) to include a wider selection of ncRNAs and to cover the full nuclear ribosomal operon. We recovered and verified three to four different ncRNAs from the ribosomal intergenic spacer 1 (IGS1) region of a set of Auricularia and Inocybe species, and we submit that ncRNAs are elements that can no longer be disregarded in the context of fungal ribosomal biology.

Alm Rosenblad et al. (2016) provided the first observation of an ncRNA other than the standard SSU, 5.8S, LSU, and 5S genes – namely the SRP RNA – in the ITS region of fungi. We expand on those findings by highlighting not only the SRP RNA gene but also an additional three ncRNA genes – nuclear RNase P RNA, RNase MRP RNA, and a possible U14 – in the IGS region of several Auricularia species. We verified the presence of these IGS ncRNAs through DNA sequencing of conspecific specimens. We furthermore used sequencing to recover the SRP RNA and a putative copy of the U14 gene in the IGS region of three Inocybe species. Our findings suggest that the SRP RNA results of Alm Rosenblad et al. (2016) were not isolated occurrences of limited interest to mycology and RNA biology. On the contrary, the SRP RNA gene seems to have been independently and repeatedly incorporated into the ribosomal operon of numerous fungi. This certainly warrants further investigation.

In addition to the SRP RNA, we also found strong evidence for two other ncRNAs in the IGS1 of both Auricularia specimens we sequenced – Auricularia delicata (MFLU16-2118) and Auricularia cornea (MFLU16-2108) – namely RNase P RNA and RNase MRP RNA. These genes correspond to important components for the maturation of tRNAs and rRNAs, respectively (López et al. 2009). While there are examples of a different type of nuclear RNase P in some organisms, all fungi use the standard RNP-type RNase P (Klemm et al. 2016). For the RNase MRP, there are, as yet, no examples of species lacking this RNP, although the exact composition of its protein subunits is unclear in some groups (Alm Rosenblad et al. 2021). Regarding the U14 snoRNA, which also plays a part in the rRNA maturation process, the prediction score did pass the Rfam threshold, but since a thorough analysis of this ncRNA gene has not yet been made for basidiomycetes, we consider the predictions to be interesting candidates pending further analysis.

The fact that draft genome assemblies of Auricularia heimuer and A. cornea contain these RNAs in their ribosomal operon, but not elsewhere in the genome, suggests that these ncRNA genes are functional. Our approach does not enable us to prove that these ncRNAs indeed are functional, but the case for them as functional must be considered strengthened. The RNase P RNA and the RNase MRP RNA genes have been identified in introns of protein coding genes in metazoans such as Caenorhabditis and Drosophila (López et al. 2009), but there is no example of them from ribosomal operons. However, it has been shown in insects that the RNase P RNA, which is usually transcribed by pol III, is dependent on the recipient gene’s pol II promoter and that splicing is not required for producing a mature RNase P RNA (Manivannan et al. 2015).

Interestingly, whereas our former study found SRP RNAs in the ITS1 region of strictly ectomycorrhizal species, this study reveals the presence of ncRNAs – including the SRP RNA – also in the non-ectomycorrhizal (but instead saprotrophic) basidiomycete genus Auricularia. This suggests that fungal nutritional mode may not determine or require the presence of these ncRNAs in the ribosomal operon, something that would be interesting to pursue in light of further data. It should nevertheless be pointed out that all five fungal genera from which ribosomal operon ncRNAs have been reported - Astraeus, Russula, Lactarius, Inocybe, and Auricularia – belong to the class Agaricomycetes of the Basidiomycota. The significance of this is unclear, but even a cursory glance at the finer levels of the Basidiomycota phylogeny shows that multiple independent gains/losses are needed to explain the observed ncRNA distribution. The fact that a single fungal class has seen a multitude of these events, whereas no other fungal class seems to have seen even a single one, certainly calls for an explanation.

It seems clear that ncRNAs must be taken into consideration in fungal ribosomal genetics. Four different ncRNAs are now known from the fungal ribosomal operon, and further research should screen genome and RNA operon sequences to determine how widespread ncRNAs are in fungi. Indeed, as databases accumulate a steadily increasing number of fungal ribosomal sequences that go far beyond the ITS region, there is every reason to think that additional ncRNAs will be recovered, presumably from non-Agaricomycetes fungi at that. The ribosomal operon is routinely excluded from many genome assemblies due to assembly difficulties (Hibbett et al. 2016), but our findings stress the importance of including it to enable research efforts like the present one. This study seeks to alert our fellow mycologists and RNA biologists to the presence of these ncRNAs in genetic regions where they up until recently were not expected to be, and we certainly hope that the evolutionary history of these ncRNAs and their presence in the fungal ribosomal operon will prove amenable to scientific explanation within not too long. Auricularia mycology primarily relies on genetic markers and genes such as the ITS region, nLSU, and RPB2 (Yuan et al. 2018; Wu et al. 2021), but several studies have explored the Auricularia IGS region for mycological usefulness (e.g., Li et al. 2011 and Li et al. 2019). The present study suggests that caution is warranted when aligning the IGS region of Auricularia and possibly also other fungi due to the potential presence of these ncRNAs. Uncritical alignments may violate homology assumptions and may give rise to noisy multiple sequence alignments and skewed phylogenetic signals.

This study reports on the detection of three non-coding RNA genes hitherto not known from the fungal ribosomal region: nuclear RNase P RNA, RNase MRP RNA, and a possible snoRNA U14 in a total of five species of Auricularia and Inocybe. This expands on the recent finding of another non-coding RNA gene – the Signal Recognition Particle (SRP) RNA – in the internal transcribed spacer (ITS) region of three ectomycorrhizal genera of basidiomycetes. There are indications that these are functional genes rather than pseudogenes. The occurrence of these non-coding RNAs and their distribution in the fungal tree of life calls for further research attention but also caution in, e.g., multiple sequence alignment-based phylogenetic inference efforts involving the ribosomal regions of these fungi.

CW gratefully acknowledge funding from the German Research Foundation (DFG: WU890/2–1), and MAR gratefully acknowledges funding from Wilhelm and Martina Lundgrens Vetenskapsfond.