A novel spliceosome-mediated trans-splicing can change our view on genome complexity of the divergent eukaryote Giardia intestinalis

Abstract

Although spliceosomal introns are an abundant landmark in eukaryotic genomes, the nuclear genome of the divergent eukaryote Giardia intestinalis, the causative agent of giardiasis, has been considered as “intron-poor” with only five canonical (cis-spliced) introns. However, three research groups (including ours) have independently reported a novel class of spliceosomal introns in the G. intestinalis genome. Three protein-coding genes are split into pieces in the G. intestinalis genome, and each of the partial coding regions was independently transcribed into polyadenylated premature mRNAs (pre-mRNAs). The two pre-mRNAs directly interact with each other by an intermolecular-stem structure formed between their non-coding portions, and are then processed into mature mRNAs by spliceosome-mediated trans-splicing. Here, we summarize the recently published works on split introns (“splintrons”) in the G. intestinalis genome, and then provide our speculation on the functional property of the Giardia spliceosomes based on the putative ratio of splintrons to canonical introns. Finally, we discuss a scenario for the transition from typical GT-AG boundaries to non-typical AT-AC boundaries in a particular splintron of Giardia.

Introduction

Protein coding sequences in eukaryotic nuclear genomes are often intervened by one or more introns that are excised by the spliceosome, which comprises several small nuclear RNAs and hundreds of proteins (Jurica and Moore 2003). The chemical mechanism for the excision of spliceosomal introns from pre-mRNAs is identical to that for the excision of group II introns, a class of introns in the prokaryotic and organellar genomes (Lambowitz and Zimmerly 2010). Therefore, it is generally believed that the ancestral spliceosomal intron was derived from a group II intron that resided in the ancestral mitochondrial genome (Lambowitz and Zimmerly 2004; Roger and Doolittle 2000). Because mitochondrial endosymbiosis is believed to have taken place at a very early stage of eukaryotic evolution, the “root” of spliceosomal introns can be as deep as that of mitochondria in eukaryotic evolution (Embley and Martin 2006; Martin and Koonin 2006), and these “spliceosomal” introns have undoubtedly contributed to shape the modern eukaryotic genomes. In other words, if a particular lineage diverged from the main trunk of the eukaryotic lineages before the mitochondrial endosymbiosis, then these cells are anticipated to possess a genome with no or few spliceosomal introns.

Giardia intestinalis is an intestinal parasite in humans and various animals. Apart from its significance in public health, this organism has drawn the attention of evolutionary biologists, as its cellular and genome architectures match those of the hypothesized ancestral eukaryotic cells. For a long time, it was believed that G. intestinalis possesses neither a typical mitochondrion with respiratory function nor spliceosomal introns (Adam 2000). We now know that G. intestinalis secondarily lost its mitochondria (Roger et al. 1998; Hashimoto et al. 1998; Tovar et al. 2003), and five genes had been found to bear canonical (cis-spliced) spliceosomal introns in its genome (Nixon et al. 2002; Morrison et al. 2007; Roy et al. 2011). These findings suggest that this organism does not necessarily represent the ancestral eukaryotes; rather, they lost the typical mitochondria because of their anaerobic lifestyle, and spliceosomal introns in the G. intestinalis genome are merely more sparse than those in other eukaryotic genomes. Furthermore, recent studies have identified a novel class of spliceosomal introns in the G. intestinalis genome, which challenge our conventional view on G. intestinalis and its genome complexity (Kamikawa et al. 2011a).

In the following sections, we briefly introduce spliceosomal introns in a split form (so-called splintrons; Kamikawa et al. 2011a; Kamikawa et al. 2011b), and then, explore the putative properties of G. intestinalis spliceosomes. Finally, we discuss the evolution of a particular splintron with non-typical AT-AC boundaries in G. intestinalis.

What are splintrons?

We recently identified three splintrons in two functionally indispensable genes in G. intestinalis (Kamikawa et al. 2011a, b). For instance, the N-terminal and C-terminal portions of the heat shock protein 90 (HSP90) are encoded in two distinct loci in the same chromosome of G. intestinalis, and spliceosome-mediated trans-splicing produces the mRNA molecule that encodes the entire HSP90. Mechanisms of hsp90 gene expression in G. intestinalis are shown in Fig. 1. The premature mRNAs (pre-mRNAs) for the N-terminal and C-terminal portions, which bear poly-A tails at their 3′ ends, are transcribed independently from the two loci (N-terminal and C-terminal pre-mRNAs). The two pre-mRNAs are likely in close proximity by forming an intermolecular stem structure between the sequence stretch on the 3′ non-coding region of N-terminal pre-mRNA and that on the 5′ non-coding region of C-terminal pre-mRNA. Furthermore, we noticed that the intron-like motifs conserved among the Giardia canonical spliceosomal introns were also present in the non-coding regions of the two pre-mRNAs. These observations strongly indicate that the two pre-mRNAs can behave as a single, continuous pre-mRNA molecule with a spliceosomal intron, and that they are trans-spliced into the mature mRNA by spliceosomes. Indeed, we successfully identified both the mature mRNAs and the “Y-shaped” RNA molecules excised by spliceosomes. In addition, Nageshan et al. (2011) independently investigated the splintron in the hsp90 gene and experimentally confirmed that no partial polypeptide corresponding to N-terminal pre-mRNA existed in the G. intestinalis cells. Furthermore, we and Roy et al. found two splintrons in the gene encoding the outer arm dynein β subunit (OADβ), which requires the processing of three pre-mRNAs into a single mature mRNA by trans-splicing (Kamikawa et al. 2011a, b; Roy et al. 2011). In addition, Roy et al. (2011) found a fourth splintron in the gene encoding the outer arm dynein γ chain (OADγ) and a fifth cis-spliced intron. Splintron trans-splicing is apparently indispensable for the regular expression of the four G. intestinalis genes, which is distinctive from the major classes of trans-splicing known to date (Kamikawa et al. 2011a).

Fig. 1.

A schematic representation of trans-splicing of the splintron in the Giardia intestinalis hsp90 gene. The N-terminal and C-terminal portions are encoded in two distant loci in the genome. “N-terminal” and “C-terminal” pre-mRNAs with polyA + tails are independently transcribed from the two loci, and physically interact with each other via the intermolecular stem structure between the “left intron piece” and the “right intron piece.” Spliceosome-mediated trans-splicing generates mature mRNAs encoding the entire amino acid sequence of an HSP90; the excised intron pieces connect to each other in a “Y-shape.” The “Y-shaped” RNA molecules likely stay in the nucleus and are eventually degraded, while the mature mRNAs are exported from the nucleus to cytoplasm and then subjected to translation

Prior to our work, a single splintron had been identified in the nematode Caenorhabditis elegans (Fischer et al. 2008). The mature mRNA encoding ERI-6/7 is generated through spliceosomal trans-splicing of two pre-mRNAs, which are separately transcribed from eri-6 and eri-7 loci. Importantly, eri-6 and eri-7 pre-mRNAs most likely interact with each other via an intramolecular stem structure during the trans-splicing reaction, as seen in G. intestinalis splintrons. As G. intestinalis and C. elegans are phylogenetically distantly related to each other, splintrons should have been evolved independently on the branches leading to the two species. Alternatively, as proposed by Blumenthal (2011), there is the possibility of splintrons having emerged anciently in eukaryotic evolution. For better understanding of the evolution of splintrons, we need to examine whether G. intestinalis and C. elegans are the sole organisms performing splintron splicing in mRNA maturation. Especially, intron data from close relatives of G. intestinalis, as well as those from close relatives of C. elegans, are indispensable to predict when and how splintrons emerged in eukaryotic phylogeny. Intriguingly, besides C. elegans, the genome data are available for several members of the genus Caenorhabditis, but no splintron has been identified in their genomes (e.g., C. briggsae; Fischer et al. 2008). Thus, splintron trans-splicing was most likely established after divergence of the extant Caenorhabditis species. On the other hand, the genome data of close relatives of G. intestinalis are not sufficient to propose any scenarios for splintron evolution on the branch leading to G. intestinalis. Therefore, it is important to conduct a stringent survey of canonical introns and splintrons in the sequence data from the genome project of the diplomonad Spironucleus vortens, the close relative of Giardia (Joint Genome Institute: http://genome.jgi-psf.org/).

Putative properties of the G. intestinalis spliceosomes

In light of the three studies demonstrating splintrons, our views on the evolutionary dynamics of spliceosomal introns and spliceosomes in the G. intestinalis genome need to be revised. In the G. intestinalis genome, the number of splintrons (four; see Kamikawa et al. 2011a, b; Nageshan et al. 2011; Roy et al. 2011) almost equals that of canonical introns (five; see Nixon et al. 2002; Morrison et al. 2007; Roy et al. 2011). Therefore, we predict that splintrons occupy a large fraction of the entire spliceosomal introns in G. intestinalis, albeit the precise numbers of the two types of spliceosomal introns are currently uncertain. We also anticipate that the biochemical properties of spliceosomes in G. intestinalis are quite different from those of the well-characterized spliceosomes in yeast and human cells, as the G. intestinalis complex needs to carry out both cis- and trans-splicing with an almost equal efficiency.

The spliceosomes of the nematode C. elegans include several components that are exclusively required for trans-splicing (Denker et al. 2002). Similarly, spliceosomes of trypanosomes were proposed to contain unique components that are responsible for “spliced leader” trans-splicing—spliceosomal transfer of a short non-coding RNA molecule (spliced leader) to the 5′ end of pre-mRNA molecules (Günzl 2010). Therefore, Giardia spliceosomes may have several components that are specific for splintron trans-splicing. Otherwise, some (and, potentially, many) components that are highly conserved among the spliceosomes that predominantly excise canonical introns may be divergent or even replaced by unrelated proteins in the G. intestinalis complexes in order to work for both cis- and trans-splicing. For instance, the G. intestinalis spliceosomes may contain unique components dedicated for splintron trans-splicing, such as chaperon-like components that facilitate the physical interaction between two pre-mRNAs transcribed from the two loci in the genome. This conjecture would be, to some extent, consistent with the result presented by Collins and Penny (2005) that many of the spliceosomal components conserved in the yeast and/or human complexes failed to be identified in the survey of the G. intestinalis genome.

Evolution of the OADγ splintron with non-typical boundaries

OADγ splintron possesses non-typical AT-AC boundaries (Fig. 2a; see also Roy et al. 2011). It is well known that some of the AT-AC introns are spliced by non-canonical U12-type spliceosomes. However, the Giardia OADγ splintron was predicted to be spliced by canonical U2-type spliceosomes (Roy et al. 2011), allowing us to postulate that this splintron once bore the typical GT-AG boundaries. Most simply, two nucleotide substitutions—one at the 5′ boundary (GT → AT) and the other at the 3′ boundary (AG → AC)—are sufficient to yield the AT-AC boundaries. However, in this case, it is difficult to assume that the two independent substitutions occurred simultaneously. Instead, here, we propose a scenario of how this “AT-AC” splintron was evolved from an ancestral splintron with GT-AG boundaries through neutral or semi-neutral steps, as schematically presented in Fig. 2b.

Fig. 2.

Characterization of the AT-AC splintron in Giardia intestinalis. a An alignment of the conserved 5′ and 3′ portions of the five introns and four splintrons in Giardia intestinalis. The nucleotides shared among all introns/splintrons are shaded. The branch point adenine is highlighted with an asterisk. Dinucleotides at the 5′ and 3′ boundaries are shown in bold italics. All introns/splintrons, except that in the gene encoding the outer arm dynein γ subunit (OADγ), possess the dinucleotide, AG, at the 3′ end. The OADγ splintron also retains the AG dinucleotide (highlighted by closed arrowheads) that is homologous to the dinucleotides at the 3′ end of the other introns/splintrons, but it bears an extra trinucleotide CAC (highlighted by dots). Abbreviations: Fd ferredoxin; Rlp7a ribosomal large subunit protein 7a; URF unassigned reading frame; DLC dynein light chain; PNA4 26S proteosome non-ATPase subunit 4; HSP90 heat shock protein 90; OADβ outer arm dynein β subunit; OADγ outer arm dynein γ subunit. b A scenario for the transition from the splintron with the GT-AG boundaries to that with the AT-AC boundaries found in the OADγ gene. Exon and splintron sequences are shown in black and gray, respectively. For the exon portion, the putative amino acid sequence is shown above the nucleotide sequence. The dinucleotides at the 5′ and 3′ ends of the splintron are highlighted in bold italics. We hypothesize that an ancestral form of the OADγ splintron exists with GT-AG boundaries (top). Subsequently, a trinucleotide CAC, highlighted in white, was inserted to the downstream exon (center). Corresponding to the CAC insertion in the exon sequence, a single histidine was inserted into the protein in this intermediate state. Finally, a G-to-A substitution (shown with an open arrowhead) at the 5′ boundary, coupled with a slide of the 3′ boundary from AG to a neighboring AC (an arrow), occurred simultaneously (bottom)

The 3′ portions of the OADγ splintron and those of the other introns/splintrons share apparent sequence homology (Fig. 2a). Intriguingly, the former retains a dinucleotide, AG, which is likely to be homologous to the 3′ ends of other introns/splintrons (highlighted by closed arrowheads; Fig. 2a). It is also noteworthy that the 3′ boundary of the OADγ splintron is longer by three nucleotides than those of other introns/splintrons (highlighted by dots; Fig. 2a). Considering these aspects, we hypothesize an intermediate state where the splintron has the GT-AG boundaries and an insertion of CAC right after the 3′ boundary of the splintron (center in Fig. 2b). Luukkonen and Séraphin (1997) experimentally displayed that spliceosomes can excise AT-AC introns more efficiently than AT-AG introns. Consequently, we propose that, in the last step, (1) a G-to-A substitution at the 5′ boundary (open arrowhead) and (2) a slide of the 3′ boundary from AG to a neighboring AC (arrow), occurred simultaneously to yield the AT-AC boundaries and to remove the histidine residue inserted in step 2.

G. intestinalis has been considered as a divergent eukaryote with respect to its cellular architecture, molecular machineries, and genome organization. Nevertheless, the finding of splintrons in the G. intestinalis genome implies that we have drastically underestimated the complexity of this organism. It is also important to stress that the knowledge from miscellaneous organisms for major research communities in molecular biology and biochemistry, such as G. intestinalis, can be significant for understanding the molecular mechanisms ubiquitously found in the Tree of Life.

Abbreviations

HSP90

Heat shock protein 90

OAD-β

Outer arm dynein beta chain

OAD-γ

Outer arm dynein gamma chain

pre-mRNA

Premature mRNA