Massive programmed translational jumping in mitochondria

Abstract

Programmed translational bypassing is a process whereby ribosomes "ignore" a substantial interval of mRNA sequence. Although discovered 25 y ago, the only experimentally confirmed example of this puzzling phenomenon is expression of the bacteriophage T4 gene 60. Bypassing requires translational blockage at a "takeoff codon" immediately upstream of a stop codon followed by a hairpin, which causes peptidyl-tRNA dissociation and reassociation with a matching "landing triplet" 50 nt downstream, where translation resumes. Here, we report 81 translational bypassing elements (byps) in mitochondria of the yeast Magnusiomyces capitatus and demonstrate in three cases, by transcript analysis and proteomics, that byps are retained in mitochondrial mRNAs but not translated. Although mitochondrial byps resemble the bypass sequence in the T4 gene 60, they utilize unused codons instead of stops for translational blockage and have relaxed matching rules for takeoff/landing sites. We detected byp-like sequences also in mtDNAs of several Saccharomycetales, indicating that byps are mobile genetic elements. These byp-like sequences lack bypassing activity and are tolerated when inserted in-frame in variable protein regions. We hypothesize that byp-like elements have the potential to contribute to evolutionary diversification of proteins by adding new domains that allow exploration of new structures and functions.

Keywords: heterologous expression; mitochondrial genome; proteome analysis; ribosome hopping.

Fig. 1.

Magnusiomyces growth on nonfermentable carbon sources and phylogeny. (A) Phylogeny of yeast species based on mitochondrial protein sequences. The tree (PhyloBayes and the CAT/GTR model) was built using 13 mtDNA-encoded proteins. All divergence points are supported by posterior probability values of 1.0, except where indicated. The fungal outgroup consists of Rhizopus oryzae, Aspergillus niger, Podospora anserina, Fusarium oxysporum, Cantharellus cibarius, and Ustilago maydis. Species containing byps or byp-like insertion elements (ins) analyzed in this study are colored red. Note that GC clusters in mtDNAs of many more yeast species may be byp-related. Species for which mtDNAs were sequenced in the context of this work are labeled by gray shading. (B) Yeast cells were cultivated on synthetic medium with glucose, glycerol, ethanol, or lactate as the sole carbon source. See Materials and Methods for details.

Fig. 2.

Representative byps and byp-like elements. Upper- and lowercase nucleotides, putatively translated and untranslated sequence, respectively. Dotted lines, continuity of protein sequence. Blue and purple, translation demonstrated by proteomics data. Highlighting in yellow, takeoff and landing sites; orange, unused codon following takeoff site; gray, in-frame stop codons. (A–E) Byps in M. capitatus mtDNA. (A–C) Byps representing the three groups, CGA, UCC, and ACC, (A and B) in standard protein-coding genes and (C) in an intron ORF. (B, D, and E) Byps for which translational bypassing has been demonstrated experimentally. (F) Byp-like element in mitochondrion-encoded rps3 of S. cerevisiae that, in contrast to A–E, is translated. The two tryptic peptides encoded by the purple and blue sequence, respectively, are MLNNNNMNPAGANPVVHR and IGPAGNINNK (our data and data at http://gpmdb.thegpm.org/seq.html) (43).

Fig. 3.

Transcript processing and translation of byp-containing genes in M. capitatus. (A) Analysis of mitochondrial transcripts indicates that mRNAs contain byps. PCR reactions were carried out using primers specific for M. capitatus genes rps3, cob, nad3, nad2, and cox1 and the following templates: lane 1, genomic DNA; lane 2, RNA extracted from purified mitochondria; lane 3, cDNA synthesized from RNase A-treated mitochondrial RNA; and lane 4, cDNA synthesized from mitochondrial RNA (for details see Materials and Methods). Amplification products were separated by electrophoresis. As molecular weight marker (M) served λ DNA digested with PstI endonuclease. The sizes of PCR and RT-PCR products are identical for rps3, nad3, and nad2 but differ for cob and cox1, which contain introns removed from the mRNA by splicing. The retention of byps and elimination of introns from cob and cox1 cDNAs was confirmed by DNA sequencing. The cox1 gene fragment was not amplified from genomic DNA due to its large size (∼10.1 kbp). (B) List of peptides corresponding to byp-containing genes and intron ORFs. Peptides spanning byps are indicated in bold, and amino acids in red correspond to the takeoff codon. Mass spectrometry profiles of peptides spanning byps are shown in Fig. S1. (C–F) Diagram of the translational bypassing process in M. capitatus mitochondria. Purple-, sky blue-, and mint green-filled boxes indicate individual exons at the gene and transcript level and the corresponding portions in the protein. (C) Gene structure of the cob gene. The multicolored narrow box within exon 2 represents the byp element. (D) Mature cob transcript after intron removal by RNA splicing. (E) Messenger RNA magnified in size. Dark purple rectangle, takeoff codon; red rectangle, unused codon; orange hairpin, folded RNA sequence; bright blue rectangle, landing codon. (F) Protein sequence of apocytochrome b that contains an amino acid corresponding to the takeoff codon whereas the other components of byps are not translated.