Identification of hot spring Obelisk-like RNA replicons and expanded diversity of the Obelisk superfamily

Recent extensive metatranscriptome mining vastly expanded the range of apparently covalently closed circular (ccc) RNA replicons. A notable family of such replicons is Obelisks, ~1 kilobase (kb) cccRNAs encoding a protein with a unique fold, Oblin-1, and detected in diverse metatranscriptomes. To identify potential cccRNAs in a sequence similarity–independent manner, we adopt the Fragmented and primer-Ligated DsRNA Sequencing (FLDS) method to selectively sequence double-stranded (ds) RNAs, replicative intermediates of RNA replicons. We focus on candidates with predicted extensive intramolecular base-pairing, a hallmark of viroid-like elements. Using FLDS, we explore metatranscriptomes from acidic hot springs in Japan and discover a distinct family of Obelisks apparently associated with thermoacidophilic bacteria (Hot spring Obelisks, HsObs). Despite lacking sequence similarity to known Oblins, HsObs share key features, including ~1 kb genome size, rod-like RNA secondary structure, and the predicted fold of the encoded protein, HsOblin. A comprehensive metatranscriptome search for Oblin-1 and HsOblin homologs expands Obelisk diversity about two-fold, revealing multiple subfamilies sharing the same core fold,. some of which are also predicted to encode additional small proteins with simple alpha-helical folds. These findings highlight Obelisks as widespread and overlooked components of microbial ecosystems, expanding understanding of viroid-like RNA replicon diversity and evolution.