Spliced DNA sequences in the Paramecium germline: their properties and evolutionary potential

Abstract

Despite playing a crucial role in germline-soma differentiation, the evolutionary significance of developmentally regulated genome rearrangements (DRGRs) has received scant attention. An example of DRGR is DNA splicing, a process that removes segments of DNA interrupting genic and/or intergenic sequences. Perhaps, best known for shaping immune-system genes in vertebrates, DNA splicing plays a central role in the life of ciliated protozoa, where thousands of germline DNA segments are eliminated after sexual reproduction to regenerate a functional somatic genome. Here, we identify and chronicle the properties of 5,286 sequences that putatively undergo DNA splicing (i.e., internal eliminated sequences [IESs]) across the genomes of three closely related species of the ciliate Paramecium (P. tetraurelia, P. biaurelia, and P. sexaurelia). The study reveals that these putative IESs share several physical characteristics. Although our results are consistent with excision events being largely conserved between species, episodes of differential IES retention/excision occur, may have a recent origin, and frequently involve coding regions. Our findings indicate interconversion between somatic--often coding--DNA sequences and noncoding IESs, and provide insights into the role of DNA splicing in creating potentially functional genetic innovation.

Keywords: DNA splicing; ciliated protozoa; developmentally regulated genome rearrangements; genome evolution; internal eliminated sequences.

Fig. 1.—

IES size distribution. Size distribution (in base pairs) of the two sets of putative IESs (i.e., imperfectly excised and cryptic IESs) detected for Paramecium tetraurelia, P. biaurelia, and P. sexaurelia.

Fig. 2.—

Nucleotide composition and frequency of IES termini consensus sequences. Nucleotide composition and frequency of the consensus sequences of the 8-bp imperfect terminal inverted repeat (IR) of IESs detected for Paramecium tetraurelia, P. biaurelia, and P. sexaurelia. Nucleotide frequency is expressed as the proportion between the observed frequency relative to the expected frequency (the expected frequency is calculated on the basis of the average nucleotide composition of the species-specific set of IESs).

Fig. 3.—

Position of IESs in coding sequences. Correlation analyses between number of exon-mapping IESs (partitioned according to size and presence of PTCs) and IES distance from the gene start in Paramecium tetraurelia. Distance values are allocated in 10 bins and are equal to the ratio between the IES distance from the gene start and the total gene length.

Fig. 4.—

Conservation and variability of IES excision between Paramecium aurelia species. The BLAST program is employed to determine whether imperfectly excised IESs and cryptic IESs detected in one P. aurelia species (query species) are present or absent from the macronuclear genome assembly of the remaining species (database species). We screen the genome assembly of the database species using: 1) merged IES-flanking regions (40 nt from each end); 2) IES-flanking regions (40 nt from each end) plus the intervening IES; and 3) IES sequences only. The graph represents the cumulative number of hits in the genome assembly of the database species.

Fig. 5.—

Putative example of a mutational event triggering erroneous excision of a macronuclear DNA region. A T → A mutational change (indicated by the arrow) presumably triggers the excision of a macronuclear-destined sequence (part of the gene model GSPATG00029756001) in Paramecium tetraurelia. The alignment includes all detectable homologous sequences in the surveyed Paramecium species (BLAST E value = 10⁻⁵). The unrooted NJ-tree is based on regions that immediately flank the IES under examination (114 sites). Bootstrap values lower than 75% are not shown. Estimates of sequencing coverage are provided for each of the scaffold regions that are shown in the alignment: 13× (11 ungapped and 2 gapped reads) for P. tetraurelia scaffold 112; 10× and 19× (all ungapped reads) for P. tetraurelia scaffold 19 and scaffold 75, respectively; 6× and 12× (all ungapped reads) for P. biaurelia scaffold00658 and scaffold00308, respectively; 10× and 17× (all ungapped reads) for P. sexaurelia scaffold2033 and scaffold00049, respectively.