A spliceosomal intron in Giardia lamblia

The G. lamblia [2Fe-2S] Ferredoxin Gene Contains a 35-bp Intron.

We discovered the first example of an intron in the G. lamblia genome from blast analyses of >54,000 single-pass shotgun sequences that identified a putative hydrogenosome-like [2Fe-2S] ferredoxin gene (30, 31). Inspection of sequence alignments in the blast report revealed the occurrence of an in-frame stop codon in the Giardia hydrogenosome-like [2Fe-2S] ferredoxin-coding region. We designed primers to test the possibility that the in-frame stop-codon resided within an intron. RT-PCR products from Giardia mRNA, with primers spanning the coding region of the [2Fe-2S] ferredoxin gene, were composed of two bands, the larger of which matched the sequences of the PCR product from G. lamblia genomic DNA and the shotgun sequences (Fig. 1A). In contrast, the smaller RT-PCR product lacked the 35-bp intron from the [2Fe-2S] ferredoxin mRNA. The Giardia intron contained a canonical splice site at its 3′ end (AG), a noncanonical splice site at its 5′ end (CT), and a branch point sequence that fits the yeast consensus sequence of TACTAAC except for the first nucleotide (AACTAAC) (1). In contrast, there was no polypyrimidine tract upstream of the 3′ AG. The resulting [2Fe-2S] ferredoxin ORF encoded a 133-aa protein, which contained four conserved Cys residues that form the iron–sulfur binding site (Fig. 1B) (30, 31). No G. lamblia intron has been identified previously (14, 29).

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Figure 1

Demonstration of an intron in the G. lamblia [2Fe-2S] ferredoxin gene by RT-PCR and the sequencing of the RT-PCR product. (A) Lane 1, PCR with G. lamblia RNA with no RT reaction (negative control) using primers flanking the [2Fe-2S] ferredoxin gene. Lane 2, RT-PCR using primers for the alcohol dehydrogenase E gene of G. lamblia (positive control). Lane 3, PCR product from G. lamblia genomic DNA with primers flanking the [2Fe-2S] ferredoxin gene. Lane 4, RT-PCR product from G. lamblia RNA with primers flanking the [2Fe-2S] ferredoxin gene. The lower band is the spliced product, whereas the upper band is the unspliced product. (B) Nucleotide sequence and predicted amino acid sequence in single-letter code of G. lamblia [2Fe-2S] ferredoxin gene. Start and stop codons are marked in purple, whereas locations of the primers to obtain the PCR and RT-PCR products are underlined. The 35-bp intron is red, whereas the branch point sequence is marked in green. Conserved cysteines in the iron-sulfur binding site are marked in turquoise.

The G. lamblia Genome Predicts Spliceosomal Peptides That Are Eukaryote-Specific or Are Homologous to Eubacterial Peptides.

The discovery of an intron in the Giardia hydrogenosome-like [2Fe-2S] ferredoxin prompted a search for G. lamblia genes encoding spliceosomal peptides that work in concert to remove introns from mRNAs (15–33). We identified a putative G. lamblia Prp8p, which is a eukaryote-specific spliceosomal peptide used to argue for the presence of introns in trichomonads (27, 28). The predicted G. lamblia Prp8p showed a 27% identity with the S. cerevisiae Prp8p. The predicted G. lamblia Prp11p, which is another eukaryote-specific spliceosomal peptide, showed a 30% identity with the S. cerevisiae Prp11p (32). Searches of the G. lamblia shotgun sequences identified six DExH-box RNA helicases (the expected number) that are present in spliceosomes and have homologues in ribosomes of eukaryotes and eubacteria (24–26). Two predicted G. lamblia Brr2p, which each contain two DExH-box RNA helicases fused head-to-tail with each other, showed ≈30% identity with S. cerevisiae Brr2p (26). Four predicted G. lamblia DExH-box helicases, each of which contained a single helicase domain, showed 25–31% identity with the most similar S. cerevisiae Prp2p, Prp16p, Prp22p, or Prp43p (24, 25). Because the predicted G. lamblia DExH-box RNA helicases were relatively divergent from those of other eukaryotes, it was not possible to use phylogenetic methods to infer the identity of each Giardia helicase. In contrast, the G. lamblia Sm core peptides, which appear to result from extensive duplication and divergence of archaeal genes, retain sufficient phylogenetic signal for inferring evolutionary history.

G. lamblia Sm Spliceosomal Peptides Resemble Those of Other Eukaryotes.

Sm and Lsm peptides form heptameric rings, which surround spliceosomal snRNAs and mRNAs (19–23, 33). The shotgun sequences of G. lamblia contained 11 putative Sm or Lsm peptides, whereas the E. histolytica sequences (positive control) contained 10 putative Sm/Lsm peptides (of 14 theoretical peptides total) (10, 11, 14, 32, 35). Six putative G. lamblia Sm peptides (all except SmG or SmE) that are aligned with those of S. cerevisiae in Fig. 2A contained the conserved Sm domain (pfam01423) (19–21, 36). In addition, G. lamblia SmB, SmD1, and SmD3 peptides had C-terminal tails with numerous basic amino acids, which are important for binding snRNA and targeting snRNP complexes from the cytosol to the nucleus (22, 23). Phylogenetic analyses, which included Sm peptides of G. lamblia, E. histolytica, S. cerevisiae, Trypanosoma brucei, Drosophila melanogaster, and Homo sapiens, supported our tentative identification of G. lamblia Sm peptides with the exception of a putative Sm peptide that could correspond to either GlSmE or GlSmG. This Sm peptide failed to branch with any particular clade (Fig. 2B) (37–40). Classifications of G. lamblia SmD2, SmD3, and SmF were supported by bootstrap analyses, as were placements of most of the amoebic and trypanosome Sm peptides (20, 37). These results suggest that Sm peptides were reasonably well-developed at the time that Giardia and other protists branched from the main eukaryotic trunk (13, 19–21). In the same way, chaperonin peptides, which result from duplications and divergence of heat-shock protein genes called CCT, are well developed in G. lamblia (41).

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Figure 2

(A) Alignment of Sm peptides of G. lamblia (Gl) with those of S. cerevisiae (Sc). Green marks where the sequences are strongly similar, whereas red indicates weaker similarity. The basic residues at the C termini of SmB, SmD1 and SmD3 peptides are marked in pink. (B) Phylogenetic tree, inferred by distance methods, of Sm peptides of G. lamblia (Gl), S. cerevisiae (Sc), E. histolytica (Eh), T. brucei (Tb), D. melanogaster (Dm), and H. sapiens (Hs). Bootstrap values at nodes derive from distance and parsimony methods, respectively. An asterisk indicates where a bootstrap value was below 50%. Nodes were left blank when both distance and parsimony analyses failed to identify the same bifurcating branch in over 50% of the bootstrap replicates. The sequences of the G. lamblia genes encoding Sm peptides have been assigned accession numbers as follows: SmB, AC042725; SmD1, AC029597; SmD2, AC071828; SmD3, AC036637; SmE, AC046581; and SmF, AC039972. The sequences of the E. histolytica genes encoding Sm peptides have been assigned accession numbers as follows: SmB, AZ670740; SmD1, AZ678368; SmD2, AZ546815; SmD3, AZ668476; and SmG, AZ690577.

Origin of Introns in Eukaryotes.

The debate concerning the origins of spliceosomal introns has focused on which organisms have introns and what the introns look like (1–9). The occurrence of introns in Giardia and its basal position in molecular evolution studies—e.g., phylogenetic analyses of rRNA genes (42), translation initiation factors (43), and elongation factors (44)—suggests that spliceosomal machinery evolved very early, possibly in the last common eukaryotic ancestor (5–9, 13). But alternative interpretations of molecular trees argue that deep-branching protists are merely artifacts of “long branch attraction” between rapidly evolving basal eukaryotic branches and the distantly related archaeal and bacterial outgroups (45, 46). This has led to the “Big Bang hypothesis,” which states that all extant eukaryotes are descendants of a sudden evolutionary radiation that occurred ≈1,000 million years ago (Mya). This interpretation is not without problems. The paleontological record offers evidence of 1,800- to 2,100-Mya eukaryotic microfossils (47, 48) and 2,700-Mya archaean molecular signatures in the form of steranes that are attributed to eukaryotes (49). To explain the disparity between the Big Bang hypothesis and the paleontological record, Philippe et al. (46) hypothesize that only a single early eukaryotic lineage survived extinction events that preceded the ≈1,000-Mya evolutionary radiation of plants, animals, fungi, and all other protists. If this scenario is correct, the thread of life leading to contemporary eukaryotes is incredibly thin and difficult to explain. In molecular phylogenies, long, unbroken basal branches are characteristic of extinction events. This phenomenon is well known for mammals, birds, and angiosperms, but has not been documented for protists. Eukaryotic microbial communities consist of associations of thousands of species separated by large evolutionary distances relative to that of angiosperms, birds, or mammals. Their mutual extinction would require global events encompassing an enormous variety of the earth's environments and ecological diversity. Finally, analyses of protein clocks do not support the Big Bang hypothesis. Comparisons of 57 sets of amino acid sequences suggest a 2,000- to 3,500-Mya divergence between eukaryotes and prokaryotes followed by the ≈1,500-Mya divergence of protists and the ≈1,200-Mya separation of plants from animals plus fungi (50, 51).

Our discovery of an intron in the Giardia putative [2Fe-2S] ferredoxin and the occurrence of proteins involved in spliceosomal machinery cannot resolve which of these theories about eukaryotic evolutionary history is correct. However, it would appear that spliceosomal machinery was present in the last common ancestor to extant eukaryotes. To date, only a single intron has been identified and demonstrated from a G. lamblia shotgun sequence database that includes ≈5,800 ORFs (G. Olsen, H.G.M., and M.L.S., unpublished data). Because it is unlikely that Giardia has carried all of the spliceosomal peptide machinery required for processing a single intron, we expect to find other examples of introns in the G. lamblia genome. Although fragmentary and preliminary, our results suggest that the spliceosomal machinery is conserved across eukaryotes and may, in some cases (e.g., Sm core peptides), be used to study the evolution of introns (15–28). Because spliceosomal peptides are encoded by genes that derive from eubacteria and archaebacteria and by genes that are eukaryote-specific, it is unlikely that all of these genes were present in prokaryotic ancestors and subsequently lost, as suggested by the introns-early hypothesis (2, 3).