The telomerase RNA in yeasts is large, usually > 1, 000 nt, and contains functional elements 1 that have been extensively studied experimentally in several disparate species. Nevertheless, they 2 are very difficult to detect by homology-based methods and so far have escaped annotation in the 3 majority of the genomes of Saccharomycotina. This is a consequence of sequences that evolve rapidly 4 at nucleotide level, are subject to large variations in size, and are highly plastic with respect to 5 their secondary structures. Here we report on a survey that was aimed at closing this gap in RNA 6 annotation. Despite considerable efforts and the combination of a variety of different methods, it 7 was only partially successful. While 27 new telomerase RNAs were identified, we had to restrict our 8 efforts to the subgroup Saccharomycetacea because even this narrow subgroup was diverse enough to 9 require different search models for different phylogenetic subgroups. More distant branches of the 10 Saccharomycotina still remain without annotated telomerase RNA. 11

RNA molecules may be subject to independent selection pressures on sequence and structure. This can, in principle, lead to the preservation of structural features without maintaining the exact position on the conserved sequence. Consequently, structurally analogous base pairs are no longer formed by homologous bases, and homologous nucleotides do not preserve their structural context. In other words, the evolution of sequence and structure is incongruent. We model this phenomenon by introducing bi-alignments, defined as a pair of alignments, one modeling sequence homology; the other, structural homology, together with an alignment of the two alignments that models the relative shifts between conserved sequence and conserved structure. Bialignments therefore form a special class of four-way alignments. A preliminary survey of the Rfam database suggests that incongruent evolution is not a very rare phenomenon among structured ncRNAs and RNA elements.

The telomerase RNA in yeasts is large, usually >1000 nt, and contains functional elements that have been extensively studied experimentally in several disparate species. Nevertheless, they are very difficult to detect by homology-based methods and so far have escaped annotation in the majority of the genomes of Saccharomycotina. This is a consequence of sequences that evolve rapidly at nucleotide level, are subject to large variations in size, and are highly plastic with respect to their secondary structures. Here, we report on a survey that was aimed at closing this gap in RNA annotation. Despite considerable efforts and the combination of a variety of different methods, it was only partially successful. While 27 new telomerase RNAs were identified, we had to restrict our efforts to the subgroup Saccharomycetacea because even this narrow subgroup was diverse enough to require different search models for different phylogenetic subgroups. More distant branches of the Saccharomycotina remain without annotated telomerase RNA.

RNA molecules may experience independent selection pressures on their sequence and (secondary) structure. Structural features then may be preserved without maintaining their exact position along the sequence. In such cases, corresponding base pairs are no longer formed by homologous bases, leading to the incongruent evolutionary conservation of sequence and structure. In order to model this phenomenon, we introduce bi-alignments as a superposition of two alignments: one modeling sequence homology; the other, structural homology. We show that under natural assumptions on the scoring functions, bi-alignments form a special case of 4-way alignments, in which the incongruencies are measured as indels in the pairwise alignment of the two alignment copies. A preliminary survey of the Rfam database suggests that incongruent evolution of RNAs is not a very rare phenomenon. Availability: Our software is freely available at https://github.com/s-will/BiAlign

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