Funding: Marsden Fund New Zealand Allan Wilson Centre The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.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
