Abstract
Non-coding RNAs (ncRNAs) have diverse essential biological functions in all organisms, and in eukaryotes, two such classes of ncRNAs are the small nucleolar (sno) and small nuclear (sn) RNAs. In this study, we have identified and characterized a collection of sno and snRNAs in Giardia lamblia, by exploiting our discovery of a conserved 12 nt RNA processing sequence motif found in the 3′ end regions of a large number of G. lamblia ncRNA genes. RNA end mapping and other experiments indicate the motif serves to mediate ncRNA 3′ end formation from mono- and di-cistronic RNA precursor transcripts. Remarkably, we find the motif is also utilized in the processing pathway of all four previously identified trans-spliced G. lamblia introns, revealing a common RNA processing pathway for ncRNAs and trans-spliced introns in this organism. Motif sequence conservation then allowed for the bioinformatic and experimental identification of additional G. lamblia ncRNAs, including new U1 and U6 spliceosomal snRNA candidates. The U6 snRNA candidate was then used as a tool to identity novel U2 and U4 snRNAs, based on predicted phylogenetically conserved snRNA–snRNA base-pairing interactions, from a set of previously identified G. lamblia ncRNAs without assigned function. The Giardia snRNAs retain the core features of spliceosomal snRNAs but are sufficiently evolutionarily divergent to explain the difficulties in their identification. Most intriguingly, all of these snRNAs show structural features diagnostic of U2-dependent/major and U12-dependent/minor spliceosomal snRNAs.
Highlights
Eukaryotic precursorRNA processing often requires ribonucleoprotein (RNP) complexes consisting of conserved and essential non-codingRNAs
We examined the genomic context of previously biochemically identified Giardia non-coding RNAs (ncRNAs) searching for conserved sequence elements that may be involved in their expression and/or processing
We have used bioinformatic and molecular techniques to identify novel G. lamblia ncRNAs and characterize their expression and processing strategies, which to date are largely unexplored in this organism
Summary
Eukaryotic precursor (pre-)RNA processing often requires ribonucleoprotein (RNP) complexes consisting of conserved and essential non-coding (nc)RNAs. Notable examples are the small nucleolar (sno) RNPs that participate in eukaryotic ribosome biogenesis through structural modification of specific nucleotides in ribosomal RNA (rRNA) and/or targeting cleavage of the pre-rRNA [reviewed in [1,2,3]]. Notable examples are the small nucleolar (sno) RNPs that participate in eukaryotic ribosome biogenesis through structural modification of specific nucleotides in ribosomal RNA (rRNA) and/or targeting cleavage of the pre-rRNA [reviewed in [1,2,3]] Another prevalent eukaryotic RNA processing event is mRNA splicing—the removal of intervening intron sequences from pre-mRNAs that is catalysed by the dynamic RNP complex termed the spliceosome [reviewed in [4]]. Spliceosomemediated intron recognition and excision requires intricate base-pairing interactions between the snRNAs and conserved intron boundary and internal branch-point sequences and numerous snRNA–snRNA intermolecular base pairings, dynamically changing during the splicing cycle [7]
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