Mitochondrial Retroprocessing Promoted Functional Transfers of rpl5 to the Nucleus in Grasses

Abstract

Functional gene transfers from the mitochondrion to the nucleus are ongoing in angiosperms and have occurred repeatedly for all 15 ribosomal protein genes, but it is not clear why some of these genes are transferred more often than others nor what the balance is between DNA- and RNA-mediated transfers. Although direct insertion of mitochondrial DNA into the nucleus occurs frequently in angiosperms, case studies of functional mitochondrial gene transfer have implicated an RNA-mediated mechanism that eliminates introns and RNA editing sites, which would otherwise impede proper expression of mitochondrial genes in the nucleus. To elucidate the mechanisms that facilitate functional gene transfers and the evolutionary dynamics of the coexisting nuclear and mitochondrial gene copies that are established during these transfers, we have analyzed rpl5 genes from 90 grasses (Poaceae) and related monocots. Multiple lines of evidence indicate that rpl5 has been functionally transferred to the nucleus at least three separate times in the grass family and that at least seven species have intact and transcribed (but not necessarily functional) copies in both the mitochondrion and nucleus. In two grasses, likely functional nuclear copies of rpl5 have been subject to recent gene conversion events via secondarily transferred mitochondrial copies in what we believe are the first described cases of mitochondrial-to-nuclear gene conversion. We show that rpl5 underwent a retroprocessing event within the mitochondrial genome early in the evolution of the grass family, which we argue predisposed the gene towards successful, DNA-mediated functional transfer by generating a "pre-edited" sequence.

Keywords: endosymbiotic gene transfer; intracellular gene transfer; mtDNA; pseudogene; reverse transcription.

Fig. 1.

rpl5 gene status in 53 genera of Poales. The status of rpl5 in the mitochondrial and nuclear genomes is indicated as follows: A dark “+” indicates that rpl5 is an intact open reading frame over the region sequenced. A light “+” in the nuclear column indicates that a nuclear rpl5 sequence was not obtained by PCR amplification but is inferred to be present because the only mitochondrial copy of rpl5 found is a pseudogene. A “Ψ” indicates the presence of an rpl5 pseudogene. A “−” indicates a confirmed absence of an intact copy of rpl5 in a sequenced mitochondrial or nuclear genome. A “?” indicates that a copy of rpl5 was not found but its absence cannot be confirmed due to the lack of a genome sequence. Genera with intact copies of rpl5 in both genomes are highlighted with gray boxes, and those cases in which transcription of both copies has been shown are marked with arrowheads. In some cases, multiple species were sampled from the same genus (supplementary table S1, Supplementary Material online). The asterisk indicates that the inferred presence of intact genes in both genomes in Elymus is based on mitochondrial data from one species and nuclear data from another (supplementary table S1, Supplementary Material online). Additional outgroup sequences were surveyed, all of which showed evidence for an intact mitochondrial copy only (supplementary table S1, Supplementary Material online). Poaceae subfamily designations and phylogenetic relationships are based on published sources (Bouchenak-Khelladi et al. 2008; Cotton et al. 2015; Grass Phylogeny Working Group II 2012; Wysocki et al. 2015).

Fig. 2.

Bayesian phylogenetic analysis of rpl5 sequences. Included are 101 mitochondrial and nuclear rpl5 sequences from 84 taxa. Shading indicates Poaceae subfamilies (see bottom right of figure) and is shown only for intact nuclear genes, i.e., all unshaded sequences are mitochondrial (pseudo)genes or numt pseudogenes. Tips are labeled to indicate sequences from complete mitogenomes (M), sequenced nuclear genomes (N), and the NCBI EST database (EST). Accession numbers are specified for GenBank, 1KP, and EST sequences. For species with sequenced nuclear genomes, the gene/locus name is also noted in parentheses. Pseudogenes are labeled as “Ψ”, and for pseudogenes from sequenced nuclear genomes, the chromosome/contig/scaffold name is noted in parentheses. Bayesian posterior probability values >0.75 are shown. The scale bar indicates number of substitutions per site.

Fig. 3.

N-terminal sequences of nuclear-encoded rpl5 proteins. The sequences are ordered according to the three groups of grasses with distinct N-terminal sequences, indicative of independent functional transfers (also see fig. 1). The asterisk indicates the typical position of the initiator methionine when the rpl5 gene is in the mitogenome. The shaded amino acids correspond to sequence that is upstream of the mitochondrial gene. In many cases, only partial sequences were available, and no start methionine was identified. Other taxa were omitted because no N-terminal sequence was available. All sequences were aligned with MAFFT.

Fig. 4.

Nucleotide alignment showing a chimeric rpl5 sequence in Lolium perenne. The region shared by the mitochondrial sequence and the chimeric nuclear sequence is highlighted. All three sequences shown are from L. perenne.

Fig. 5.

RNA editing of mitochondrial rpl5 in grasses and other angiosperms. RNA editing sites are indicated by vertical lines. Numbering indicates the position of each editing site in a multiple alignment of these sequences, whereas the position with respect to the Oryza sativa sequence is given in parentheses. Asterisks indicate synonymous editing sites. The species for which editing was empirically determined (see Materials and Methods) are shown in bolded text. Dashed lines indicate missing data. Two predicted editing sites (alignment position 208 in all species and alignment position 257 in Costus speciosus) that were not validated in any of the empirical data sets were excluded from the figure. The triangle indicates the timing of the inferred retroprocessing event(s).

Fig. 6.

Conceptual model of the effect of retroprocessing on the relative frequency of DNA- and RNA-mediated functional gene transfers. (A) Functional transfer of an organelle gene with many nonsynonymous edit sites and/or introns may be facilitated by a two-step mechanism, in which the gene is first reverse-transcribed and reintegrated into the mitochondrial genome (i.e., retroprocessing) before being physically moved to the nucleus. Introns are drawn as lines between exons (boxes), and sites that require RNA editing are marked with “C.” These features are removed by the initial retroprocessing step. The processed gene is then more amenable to DNA-mediated functional transfer to the nucleus, but acquisition of nuclear-specific elements such as an N-terminal targeting sequence (gray box) is still required in most cases. (B) Prior to retroprocessing, the frequency of functional transfer to the nucleus is expected to be low and dominated by directly RNA-mediated events, i.e., events in which an edited and/or spliced mitochondrial cDNA is transferred to the nucleus. After wholesale retroprocessing of such a gene, the frequency of functional transfer is expected to increase significantly and be dominated by DNA-mediated events. Note that this figure is meant only to illustrate a conceptual model. The actual proportions of functional versus nonfunctional and DNA- versus directly RNA-mediated transfers have not been quantified.