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Knowing when an RNA sequence includes a conserved RNA structure that could lead to new biology is not trivial and depends on clues left behind by conservation in the form of covariation and variation. For that purpose, the R-scape statistical test was created to identify from alignments of RNA sequences, the base pairs that significantly covary above phylogenetic expectation. R-scape treats base pairs as independent units. However, RNA base pairs do not occur in isolation. The Watson-Crick (WC) base pairs stack together forming helices that constitute the scaffold that facilitates the formation of the non-WC base pairs, and ultimately the complete 3D structure. The helix-forming WC base pairs carry most of the covariation signal in an RNA structure. Here, I introduce a new measure of statistically significant covariation at helix-level by aggregation of the covariation significance and covariation power calculated at base-pair-level resolution. Performance benchmarks show that helix-level aggregated covariation increases sensitivity in the detection of evolutionarily conserved RNA structure without sacrificing specificity. This additional helix-level sensitivity reveals an artifact that results from using covariation to build an alignment for a hypothetical structure and then testing the alignment for whether its covariation significantly supports the structure. Helix-level reanalysis of the evolutionary evidence for a selection of long non-coding RNAs (lncRNAs) reinforces the evidence against these lncRNAs having a conserved secondary structure.<\/jats:p>","DOI":"10.1371\/journal.pcbi.1011262","type":"journal-article","created":{"date-parts":[[2023,7,14]],"date-time":"2023-07-14T13:47:02Z","timestamp":1689342422000},"page":"e1011262","update-policy":"https:\/\/doi.org\/10.1371\/journal.pcbi.corrections_policy","source":"Crossref","is-referenced-by-count":15,"title":["RNA covariation at helix-level resolution for the identification of evolutionarily conserved RNA structure"],"prefix":"10.1371","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2084-269X","authenticated-orcid":true,"given":"Elena","family":"Rivas","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2023,7,14]]},"reference":[{"key":"pcbi.1011262.ref001","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1038\/nmeth.4066","article-title":"A statistical test for conserved RNA structure shows lack of evidence for structure in lncRNAs","volume":"14","author":"E Rivas","year":"2017","journal-title":"Nature Methods"},{"key":"pcbi.1011262.ref002","doi-asserted-by":"crossref","first-page":"3072","DOI":"10.1093\/bioinformatics\/btaa080","article-title":"Estimating the power of sequence covariation for detecting conserved RNA structure","volume":"36","author":"E Rivas","year":"2020","journal-title":"Bioinformatics"},{"key":"pcbi.1011262.ref003","doi-asserted-by":"crossref","first-page":"2988","DOI":"10.1093\/bioinformatics\/btl514","article-title":"Measuring covariation in RNA alignments: physical realism improves information measures","volume":"22","author":"S Lindgreen","year":"2006","journal-title":"Bioinformatics"},{"key":"pcbi.1011262.ref004","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1186\/s13059-018-1419-z","article-title":"Gene-level differential analysis at transcript-level resolution","volume":"19","author":"L Yi","year":"2018","journal-title":"Genome Biology"},{"key":"pcbi.1011262.ref005","unstructured":"Fisher RA. 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