Abstract
RNAs processing other RNAs is very general in eukaryotes, but is not clear to what extent it is ancestral to eukaryotes. Here we focus on pre-mRNA splicing, one of the most important RNA-processing mechanisms in eukaryotes. In most eukaryotes splicing is predominantly catalysed by the major spliceosome complex, which consists of five uridine-rich small nuclear RNAs (U-snRNAs) and over 200 proteins in humans. Three major spliceosomal introns have been found experimentally in Giardia; one Giardia U-snRNA (U5) and a number of spliceosomal proteins have also been identified. However, because of the low sequence similarity between the Giardia ncRNAs and those of other eukaryotes, the other U-snRNAs of Giardia had not been found. Using two computational methods, candidates for Giardia U1, U2, U4 and U6 snRNAs were identified in this study and shown by RT-PCR to be expressed. We found that identifying a U2 candidate helped identify U6 and U4 based on interactions between them. Secondary structural modelling of the Giardia U-snRNA candidates revealed typical features of eukaryotic U-snRNAs. We demonstrate a successful approach to combine computational and experimental methods to identify expected ncRNAs in a highly divergent protist genome. Our findings reinforce the conclusion that spliceosomal small-nuclear RNAs existed in the last common ancestor of eukaryotes.
Highlights
Extant eukaryotes are marked by having RNA extensively processing other RNA molecules, whether it is RNase P on tRNAs, RNase MRP and snoRNAs on rRNAs, or snRNAs on mRNAs
We focus on the major spliceosomal snRNAs involved in mRNA splicing, and address the question whether these small snRNAs occur in all deep eukaryotic lineages; in other words, whether the early splicing mechanism in eukaryotes involved both RNA and proteins, or was initially a protein mediated process, with RNAs added later
SnRNA had been identified in Giardia, so it had appeared possible that the ancestral spliceosome was mainly protein based, and that the catalytic role of snRNAs had evolved later in eukaryotic evolution
Summary
Extant eukaryotes are marked by having RNA extensively processing other RNA molecules, whether it is RNase P on tRNAs, RNase MRP and snoRNAs on rRNAs, or snRNAs on mRNAs. In addition RNAi processes are known to inhibit or enhance mRNA expression. A major question in eukaryotic origin is the extent of RNA processing in the last common ancestor of eukaryotes. Perhaps the major question is whether much of the RNA processing traces back to the proposed RNA World [1] and how much is a later invention within eukaryotes [2]. We focus on the major spliceosomal snRNAs involved in mRNA splicing, and address the question whether these small snRNAs occur in all deep eukaryotic lineages; in other words, whether the early splicing mechanism in eukaryotes involved both RNA and proteins, or was initially a protein mediated process, with RNAs added later. We use a combination of computational techniques with experimental evaluation of the results to help test these alternatives
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