Novel selenoproteins identified in silico and in vivo by using a conserved RNA structural motif

Selenocysteine is incorporated into selenoproteins by an in-frame UGA codon whose readthrough requires the selenocysteine insertion sequence (SECIS), a conserved hairpin in the 3'-untranslated region of eukaryotic selenoprotein mRNAs. To identify new selenoproteins, we developed a strategy that obviates the need for prior amino acid sequence information. A computational screen was used to scan nucleotide sequence data bases for sequences presenting a potential SECIS secondary structure. The computer-selected hairpins were then assayed in vivo for their functional capacities, and the cDNAs corresponding to the SECIS winners were identified. Four of them encoded novel selenoproteins as confirmed by in vivo experiments. Among these, SelZf1 and SelZf2 share a common domain with mitochondrial thioredoxin reductase-2. The three proteins, however, possess distinct N-terminal domains. We found that another protein, SelX, displays sequence similarity to a protein involved in bacterial pilus formation. For the first time, four novel selenoproteins were discovered based on a computational screen for the RNA hairpin directing selenocysteine incorporation.     

Revisiting the structure/function relationships of H/ACA(-like) RNAs: a unified model for Euryarchaea and Crenarchaea

A structural and functional classification of H/ACA and H/ACA-like motifs is obtained from the analysis of the H/ACA guide RNAs which have been identified previously in the genomes of Euryarchaea (Pyrococcus) and Crenarchaea (Pyrobaculum). A unified structure/function model is proposed based on the common structural determinants shared by H/ACA and H/ACA-like motifs in both Euryarchaea and Crenarchaea. Using a computational approach, structural and energetic rules for the guide:target RNA-RNA interactions are derived from structural and functional data on the H/ACA RNP particles. H/ACA(-like) motifs found in Pyrococcus are evaluated through the classification and their biological relevance is discussed. Extra-ribosomal targets found in both Pyrococcus and Pyrobaculum might support the hypothesis of a gene regulation mediated by H/ACA(-like) guide RNAs in archaea.     

Inferring the conformation of RNA base pairs and triples from patterns of sequence variation.

The success of comparative analysis in resolving RNA secondary structure and numerous tertiary interactions relies on the presence of base covariations. Although the majority of base covariations in aligned sequences is associated to Watson-Crick base pairs, many involve non-canonical or restricted base pair exchanges (e.g. only G:C/A:U), reflecting more specific structural constraints. We have developed a computer program that determines potential base pairing conformations for a given set of paired nucleotides in a sequence alignment. This program (ISOPAIR) assumes that the base pair conformation is maintained through sequence variation without significantly affecting the path of the sugar-phosphate backbone. ISOPAIR identifies such 'isomorphic' structures for any set of input base pair or base triple sequences. The program was applied to base pairs and triples with known structures and sequence exchanges. In several instances, isomorphic structures were correctly identified with ISOPAIR. Thus, ISOPAIR is useful when assessing non-canonical base pair conformations in comparative analysis. ISOPAIR applications are limited to those cases where unusual base pair exchanges indeed reflect a non-canonical conformation.