A spliceosomal intron in Giardia lamblia

Abstract

Short introns occur in numerous protist lineages, but there are no reports of intervening sequences in the protists Giardia lamblia and Trichomonas vaginalis, which may represent the deepest known branches in the eukaryotic line of descent. We have discovered a 35-bp spliceosomal intron in a gene encoding a putative [2Fe-2S] ferredoxin of G. lamblia. The Giardia intron contains 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). We have also identified several G. lamblia genes with spliceosomal peptides, including homologues of eukaryote-specific spliceosomal peptides (Prp8 and Prp11), several DExH-box RNA-helicases that have homologues in eubacteria, but serve essential functions in the splicing of introns in eukaryotes, and 11 predicted archaebacteria-like Sm and like-Sm core peptides, which coat small nuclear RNAs. Phylogenetic analyses show the Giardia Sm core peptides are the products of multiple, ancestral gene duplications followed by divergence, but they retain strong similarity to Sm and like-Sm peptides of other eukaryotes. Although we have documented only a single intron in Giardia, it likely has other introns and fully functional, spliceosomal machinery. If introns were added during eukaryotic evolution (the introns-late hypothesis), then these results push back the date of this event before the branching of G. lamblia.

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.

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.