Computational identification of four spliceosomal snRNAs from the deep-branching eukaryote Giardia intestinalis

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.

Figure 1. Identification of a Giardia U1-snRNA candidate.

A. Proposed structure for writing the U1 descriptor file. The content in the U-1 descriptor cell can be visualized in this figure. “s” stands for strand and “h” stands for helix. The elements within the proposed U-1 structure are marked in order from the 5′-end to the 3′-end. The two stem-loops drawn as dotted lines are not compulsory in the proposed structure of Giardia U-1 candidate; therefore they are marked as a free-folding strand s4. B. The structures of Human, T. brucei and Giardia-candidate U1-snRNAs. The conserved loops among the human, Giardia and Trypanosome U1-snRNAs are indicated by the circles. The Sm-protein-binding sites are boxed. C. RT-PCR test showing high expression the of the Giardia U1-snRNA candidate. + control: PCR with genomic DNA. − control: PCR with total RNA without reverse transcription.

Figure 2. Identification of a Giardia U2-snRNA candidate.

A. RT-PCR test for expression of the Giardia U2-snRNA candidates. The highly expressed candidate 2 was analysed further. − control: PCR with total RNA without reverse transcription. + control: PCR with genomic DNA. B. Structure of Giardia U2-snRNA candidate and its interaction with the branch-point intron region.

Figure 3. Identification of Giardia U6 and U4 snRNA candidates.

A1. Structure of U2-U6-snRNA base pairing in S. cerevisiae. A2. Structure of U6-U4-snRNA base pairing in Human. B1. Visualization of the model for searching a U6-snRNA candidate. B2. RT-PCR test for expression of the U6 candidate. + control: PCR with genomic DNA. − control: PCR with total RNA without reverse transcription. B3. Interaction between Giardia U6 and U2 snRNA candidates. C1. RT-PCR test for expression of the U4 candidate. + control: PCR with genomic DNA. − control: PCR with total RNA without reverse transcription. C2. Interaction between Giardia U6 and U4 snRNA candidates.