TERribly Difficult: Searching for Telomerase RNAs in Saccharomycetes

Abstract

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

Extraordinary diversity of telomeres, telomerase RNAs and their template regions in Saccharomycetaceae

“…Rather, the maximum putative template always forms at least a substantial core part of the experimentally verified template region. Our predictions of basic telomere repeats and maximum putative template regions were not identical, but consistent with the repeats and template regions identified previously in biochemical experiments 29 , 32 – 36 , 43 – 47 . Thus, for most of them, we put the new coordinates of the maximum putative template region according to the consensus telomere from TRFi results in the Supplementary Table S2 .…”

“…A modified setting of Tandem Repeats Finder parameters with final reduction in number from five to two units of detected motifs of at least 20 nt in tandem uncovered the proper telomere sequence in L. kluyveri but not in K. lactis . For the latter, we were not able to detect the telomere sequences in the available datasets despite the fact that the motif (25 nt), TR, and template region had been reported previously 34 , 36 , 43 . In comparison, the reads of L. kluyveri (100 nt) were not long enough to detect at least five adjacent tandem repeats, which illustrated how this parameter could act as a bottleneck of the method.…”

“…TBE template boundary element, TWJ three-way junction, CEH core enclosed helix. Adapted from Waldl et al 36 The template region is located between the EST1 binding site and Ku binding hairpins. (Figure created using Adobe Photoshop 22.2.0).…”

“…Nevertheless, predicting promising TRs by using BLAST searches, as recently applied in land plants 27 , is a challenging task even among relatively closely related taxa. In the case of considerable evolutionary divergence among species, such as in yeasts, the BLAST strategy is not feasible to start with, given the low sequence conservation among TRs 36 . Although TRs are variable in the primary structure and are known only in a limited number of organisms, innovative bioinformatics tools using phylogenetics and covariance models 33 , 37 and conserved secondary structures in TRs 38 – 40 have led to discoveries even in more distant eukaryotic kingdoms such as protostomes 41 .…”

“…A recent survey aimed to provide yeast TR sequences, identifying 46 cases, from which 27 telomerase RNAs were found for the first time in the family Saccharomycetaceae. Several strategies were used, including the comparison of common sequence features of known TRs in this family, synteny-based homology searches, the use of known telomere template sequences, or employing a set of known TR regions for covariance model-based searches and BLAST queries (including all template regions available from Saccharomycetaceae at that time) 36 . However, these authors failed to find template regions in 13 of the TRs that they attempted to analyse, mostly due to the lack of knowledge of specific telomere sequences.…”