Massive programmed translational jumping in mitochondria

B. F. Lang, M. Jakubkova, E. Hegedusova, R. Daoud, L. Forget, B. Brejova, T. Vinar, P. Kosa, D. Fricova, M. Nebohacova, P. Griac, L. Tomaska (+2 others)
2014 Proceedings of the National Academy of Sciences of the United States of America  
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,
more » ... e 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. ribosome hopping | mitochondrial genome | proteome analysis | heterologous expression T he traditional view of translation is that mRNA is read sequentially, one codon at a time. However, low-level nonprogrammed translational bypassing (i.e., the occasional skipping of a few nucleotides) can be triggered by various factors, including tRNA paucity, unusual codons, and homo-polymer sequence tracts (1). In addition, programmed translational bypassing of 50 nt has been demonstrated for the gene 60 transcript of bacteriophage T4 (2-4). In vitro mutagenesis experiments showed that efficient translational "jumping" or "hopping" in T4 requires matching takeoff and landing codons (most effective is the wild-type GGA), a stop codon, and both a hairpin RNA secondary structure directly downstream of the takeoff site, and a Shine-Dalgarno (SD) sequence a few nucleotides upstream of the landing codon. Finally, a particular amino acid sequence in the nascent peptide encoded upstream of the takeoff site confers highest jumping efficiency. Additional cases of programmed bypassing have been postulated but currently lack supporting evidence (e.g., ref. 5), making the T4 gene 60 expression the only confirmed instance. Here, we report the massive occurrence of translational bypassing elements in mitochondria of the opportunistic human pathogen Magnusiomyces (also known as Blastoschizomyces or Geotrichum) capitatus (6), which belongs to a deeply branching lineage of Saccharomycetales (Fig. 1A) . Our findings suggest that translational bypassing might be more widespread than previously thought. Results Protein-Coding Genes Interrupted by Dozens of Insertions. We sequenced mitochondrial DNAs (mtDNAs) from five Magnusiomyces strains (M. capitatus, M. ingens, M. magnusii (2 isolates), and M. tetrasperma (Table S1 ). The identified genes specify a standard set of highly conserved proteins involved in oxidative phosphorylation (atp6, -8, and -9; cob; cox1, -2, and -3; and nad1, -2, -3, -4, -4L, -5, and -6) and translation (rps3), two ribosomal RNAs (rnl and rns), and 25 transfer RNAs (trnA-Y). During comparative genome analysis, we identified 57 short insertions (30-55 bp) in protein-coding regions of otherwise typical genes in M. capitatus mtDNA (Fig. 2) . Using covariance models for computational searches, we detected 24 additional inserts (27-54 bp) in intron ORFs of this mtDNA (Table 1 and Table S2 ). The most "infested" gene is nad5 with 12 inserts, increasing the overall gene size by more than 20%. In total, these inserts make up 7.2% of the M. capitatus mitochondrial genome Significance During translation, ribosomes decode mRNAs in a sequential fashion. In this paper, we report the discovery of more than 80 translational bypassing elements (byps) 27-55 nt long in mitochondrial protein-coding regions of the yeast Magnusiomyces capitatus. We demonstrate experimentally that byps are retained in mRNA but not translated into protein. Byps somewhat resemble the single bypass element in bacteriophage T4 but also display unique features. We further discovered byplike sequences in other yeast species, indicating that these inserts are mobile genetic elements. In contrast to byps, byp-like sequences are not bypassed during translation. When inserted in variable protein regions, they have the potential to drive the evolutionary diversification of protein structure and function.
doi:10.1073/pnas.1322190111 pmid:24711422 pmcid:PMC4000857 fatcat:al5db5fv2jbkbnzcbtpfauy5ci