Abstract
Covariation analysis is used to identify those positions with similar patterns of sequence variation in an alignment of RNA sequences. These constraints on the evolution of two positions are usually associated with a base pair in a helix. While mutual information (MI) has been used to accurately predict an RNA secondary structure and a few of its tertiary interactions, early studies revealed that phylogenetic event counting methods are more sensitive and provide extra confidence in the prediction of base pairs. We developed a novel and powerful phylogenetic events counting method (PEC) for quantifying positional covariation with the Gutell lab’s new RNA Comparative Analysis Database (rCAD). The PEC and MI-based methods each identify unique base pairs, and jointly identify many other base pairs. In total, both methods in combination with an N-best and helix-extension strategy identify the maximal number of base pairs. While covariation methods have effectively and accurately predicted RNAs secondary structure, only a few tertiary structure base pairs have been identified. Analysis presented herein and at the Gutell lab’s Comparative RNA Web (CRW) Site reveal that the majority of these latter base pairs do not covary with one another. However, covariation analysis does reveal a weaker although significant covariation between sets of nucleotides that are in proximity in the three-dimensional RNA structure. This reveals that covariation analysis identifies other types of structural constraints beyond the two nucleotides that form a base pair.
Highlights
Covariation analysis, one form of comparative analysis, identifies the positions in the RNA molecule that have similar patterns of variation, or covariation, for all or a subset of the sequences within the same RNA family
While the earliest covariation analysis methods searched for G:C, A:U, and G:U base pairs that occur within a regular secondary structure helix [1,20,21,22], newer more mathematically and computational rigorous methods primarily searched for columns in an alignment of sequences for similar patterns of variation, based on their nucleotide frequencies, regardless of the type of base pair and the location of each putative base pair in relation to the other base pairs [23,24,25,26]
The covariation between two positions is determined by calculating the Covariation Percentage of Events (CPE), the ratio of positive events to the total number of events (Details in Method section)
Summary
Covariation analysis, one form of comparative analysis, identifies the positions in the RNA molecule that have similar patterns of variation, or covariation, for all or a subset of the sequences within the same RNA family. The vast majority of all base pair types were canonical - G:C, A:U, and G:U, and these base pairs were consecutive and antiparallel to form a regular helix This structure agnostic method for the identification of positional covariation had independently identified two of the most fundamental principles of RNA structure – the two base pair types initially determined by Chargaff [27,28], and Watson and Crick [29], and the arrangement of these base pair types into regular nucleic acid helical structures [29]. While the vast majority of the nucleotide positions with a very strong covariation form a canonical base pair within a standard helix, a small number of significant covariations are not part of a regular helix and do not exchange solely between canonical base pair types
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