Spliceosomal introns in the deep-branching eukaryote Trichomonas vaginalis

Abstract

Eukaryotes have evolved elaborate splicing mechanisms to remove introns that would otherwise destroy the protein-coding capacity of genes. Nuclear premRNA splicing requires sequence motifs in the intron and is mediated by a ribonucleoprotein complex, the spliceosome. Here we demonstrate the presence of a splicing apparatus in the protist Trichomonas vaginalis and show that RNA motifs found in yeast and metazoan introns are required for splicing. We also describe the first introns in this deep-branching lineage. The positions of these introns are often conserved in orthologous genes, indicating they were present in a common ancestor of trichomonads, yeast, and metazoa. All examined T. vaginalis introns have a highly conserved 12-nt 3' splice-site motif that encompasses the branch point and is necessary for splicing. This motif is also found in the only described intron in a gene from another deep-branching eukaryote, Giardia intestinalis. These studies demonstrate the conservation of intron splicing signals across large evolutionary distances, reveal unexpected motif conservation in deep-branching lineages that suggest a simplified mechanism of splicing in primitive unicellular eukaryotes, and support the presence of introns in the earliest eukaryote.

Fig. 1.

T. vaginalis can splice a 35-nt intron. (A) CAT activity in T. vaginalis cells transfected with plasmid constructs of a CAT gene containing the wild-type Giardia intron (Int2) and mutations of this 35-nt sequence (Int1 and Int3–12). Mutations are indicated in bold and underlined. The positive control (at the top) contains a construct with an intron-less CAT gene and the negative control (at the bottom) has a CAT gene with the Int1 intron inserted in the opposite orientation. CAT activity is expressed as cpm of n-butyrylated chloramphenicol per microgram of protein. The number of independent transfections tested are designated in brackets, and standard error bars are shown. (B) RT-PCR products separated on a 2% agarose gel show two populations of CAT cDNAs corresponding to spliced and unspliced mRNA in T. vaginalis cells transfected with the Int1 (GT) and the Int4 (GC) constructs. No spliced CAT cDNA is detected in cells transfected with the Int2 construct containing wild-type Giardia intron (CT) or the Int1 construct with the intron inserted in the reverse orientation (neg). Pos, RT-PCR reaction by using cells transfected with the intronless CAT construct. (C) RNA blot analysis of total RNA from transfected cells. The blot was hybridization with a CAT gene probe showing that cells without CAT activity do transcribe the CAT RNA. A ferredoxin probe was used as an internal standard for RNA loading.

Fig. 2.

T. vaginalis poly(A) polymerase gene contains an intron with extended conserved 5′ and 3′ SS that are necessary for splicing. (A) Alignment of the only known Giardia intron and the T. vaginalis poly(A) polymerase (PAP) intron reveals identity at 5 of 6 nt at the 5′ SS and 12 or 12 at the 3′ SSs. (B) RT-PCR products generated by using T. vaginalis genomic DNA (gDNA) or mRNA (cDNA) as the template and primers flanking the putative intron in the PAP gene were separated on a 2% agarose gel. (C) CAT activity in T. vaginalis cells transfected with plasmid constructs of a CAT gene containing the Giardia 35-nt intron with mutations (Int13–25), as indicated in bold and underlined. The positive control is described in Fig. 1. CAT activity is expressed as cpm of n-butyrylated chloramphenicol per microgram of protein. The number of independent transfections assayed is shown in brackets, and bars designate standard error. A derived consensus sequence required at the 5′ and 3′ SS for CAT activity is listed in bold.

Fig. 3.

In silico analysis reveals the presence of additional intron-containing genes. (A) The sequence used to search the T. vaginalis genome database (www.tigr.org/tdb/e2k1/tvg) for introns is shown (at the top), and the 5′ and 3′ motifs and length of the 39 sequences identified are listed (at the bottom). (B) RT-PCR products generated by using T. vaginalis genomic DNA (gDNA) or mRNA (cDNA) as the template and primers flanking the putative introns in genes encoding a TATA-binding protein-associated factor (Taf6–81), a small C-terminal domain phosphatase (Scp 67), and a STK (STK 196).