Identification of Obelisk-like covalently closed circular RNA replicon in hot springs by double-stranded RNA sequencing and expansion of the diversity of the Obelisk superfamily

Urayama, S.-i.; Fukudome, A.; Mutz, P.; Matsushita, Y.; Takaki, Y.; Nishimura, Y.; Medvedeva, S.; Krupovic, M.; Koonin, E. V.; Nunoura, T.

doi:10.1101/2025.09.03.673927

bioRxiv · 10.1101/2025.09.03.673927

Identification of Obelisk-like covalently closed circular RNA replicon in hot springs by double-stranded RNA sequencing and expansion of the diversity of the Obelisk superfamily

Urayama, S.-i.·Fukudome, A.·Mutz, P.·Matsushita, Y.·Takaki, Y.·Nishimura, Y.·Medvedeva, S.·Krupovic, M.·Koonin, E. V.·Nunoura, T.

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Abstract

Extensive metatranscriptome mining has recently vastly expanded the range of covalently closed circular (ccc) RNA replicons. A notable group of such replicons are Obelisks, cccRNAs of about 1 kilobase (kb) encoding a protein with a unique fold, Oblin-1, and detected in a broad variety of metatranscriptomes, in particular, those from the human gastrointestinal tract. We adopted Fragmented and primer-Ligated DsRNA Sequencing (FLDS) method to selectively sequence double-stranded (ds) RNAs, the replicative intermediates of RNA replicons, and to identify cccRNAs among the resulting sequences. From these data, we selected cccRNAs with predicted extensive intramolecular base-pairing, a hallmark of viroid-like elements. ch We employed FLDS to explore metatranscriptomes from acidic hot springs in Japan and discovered a distinct family of Obelisks probably associated with thermoacidophilic bacteria (Hot spring Obelisks, HsObs). The proteins encoded by HsObs, HsOblins, show no significant sequence similarity to previously identified Oblin-1 proteins, but are predicted to adopt a closely similar structure. A comprehensive search of metagenomes for Oblin-1 and HsOblin homologs substantially expanded this family of Obelisk-encoded proteins revealing several distinct subfamilies that share the same core fold. A cccRNA encoding an HsOblin homolog was also detected in a Yellowstone hot spring metatranscriptome. Apart from Oblin-1, some subfamilies of Obelisks were predicted to encode additional small proteins with simple alpha-helical folds.

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Urayama, S.-i., Fukudome, A., Mutz, P., Matsushita, Y., Takaki, Y., Nishimura, Y., Medvedeva, S., Krupovic, M., Koonin, E. V., Nunoura, T.. 2025-09-03. Identification of Obelisk-like covalently closed circular RNA replicon in hot springs by double-stranded RNA sequencing and expansion of the diversity of the Obelisk superfamily. https://doi.org/10.1101/2025.09.03.673927

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