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Position-dependent termination and widespread obligatory frameshifting in Euplotes translation


The ribosome can change its reading frame during translation in a process known as programmed ribosomal frameshifting. These rare events are supported by complex mRNA signals. However, we found that the ciliates Euplotes crassus and Euplotes focardii exhibit widespread frameshifting at stop codons. 47 different codons preceding stop signals resulted in either +1 or +2 frameshifts, and +1 frameshifting at AAA was the most frequent. The frameshifts showed unusual plasticity and rapid evolution, and had little influence on translation rates. The proximity of a stop codon to the 3′ mRNA end, rather than its occurrence or sequence context, appeared to designate termination. Thus, a 'stop codon' is not a sufficient signal for translation termination, and the default function of stop codons in Euplotes is frameshifting, whereas termination is specific to certain mRNA positions and probably requires additional factors.

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Figure 1: Frequent frameshifting in Euplotes.
Figure 2: Identification of amino acids inserted at frameshift sites.
Figure 3: Distribution of codons upstream of stop codons at the frameshift sites and at the sites of translation termination.
Figure 4: Metagene analysis of ribosome profiling and distribution of frameshifting according to transcript levels.
Figure 5: Comparison of ribosomal frameshifting at AAA versus non-AAA frameshifting sites and TAA versus TAG frameshifting sites.
Figure 6: Cross-species comparison and frequency of nucleotide deletions in different hexamers.

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Supported by NIH GM061603 and GM065402 to V.N.G. S.M.H. and P.V.B. are supported by the grants from Wellcome Trust (094423) and Science Foundation Ireland (12/IA/1335). Portions of this research were also supported by NIH GM103493 and the W.R. Wiley Environmental Molecular Science Laboratory (sponsored by DOE and located at Pacific Northwest National Laboratory). Pacific Northwest National Laboratory is operated by the Battelle Memorial Institute under the DOE contract DE-AC05-76RLO-1830. C.M. acknowledges the Italian PNRA and the COST action BM1102 for supporting a part of this work.

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A.V.L., S.M.H., P.V.B. and V.N.G. analyzed the data and wrote the paper with advice from D.L.H. and J.F.A.; A.A.T. and M.V.G. prepared samples for sequencing; S.P., R.R.D., C.M. and L.A.K. performed cell culture maintenance and growth, F.X., V.A.P. and R.D.S. conducted MS analysis. All authors discussed the results and implications and commented on the manuscript at all stages.

Corresponding authors

Correspondence to Pavel V Baranov or Vadim N Gladyshev.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Features of Euplotes genomes.

(a) Comparison Euplotes genomes in comparison with the genomes of other representative eukaryotes. The tree was constructed based on the sequences of 18S rRNA genes, and archaeal 16S rRNA gene (from Pyrococcus furiosis) was used as an outgroup. *number of contigs with telomeric repeats at both ends. (b) Distribution of telomeric repeat lengths in E. crassus (red) and E. focardii (black) macronuclear genomes. The X axis indicates the observed telomeric repeat number and the Y axis their frequencies. As expected, Euplotes genomes consist of gene-sized chromosomes capped by telomeres. The length of terminal repeats slightly varies; however, most chromosomes in both organisms have a double-stranded telomere length of 3.5 repeats (c) Sequence logo of subtelomeric regions at the 3’ end of E. crassus nanochromosomes. 1000 randomly selected chromosome sequences with telomeric repeat GGGGTTTTGGGGTTTTGGGGTTTTGGGG were chosen for constructing the logo. The logo detects a conserved position-specific sequence motif associated with telomeric repeats. Abundance of high-quality telomeric sequences allowed an unbiased screen for motifs and patterns associated with telomere function. A previously described TCAA motif (Baird S. E. & Klobutcher L. A., Genes Dev 3, 585-597, 1989; Klobutcher, L. A. et al., Proc Natl Acad Sci USA 78, 3015-3019,1981) was readily detected with Weblogo (Crooks, G. E. et al, Genome Res 14, 1188-1190, 2004) in the subtelomeric region due to its conserved position relative to the telomere repeats. An analysis of sequences in the vicinity of telomeres with a pattern discovery suite MEME (Bailey, T. L. et al., Nucl Acids Res 34, W369-373, 2006) did not reveal additional common motifs.

Supplementary Figure 2 Features of the Euplotes transcriptome.

(a) Euplotes Sec and Cys tRNAs that decode TGA codons. Cys tRNA with the GCA anticodon and mitochondrial Trp tRNA with TCA anticodon are shown for comparison. In total we identified 183 tRNA genes in E. crassus and 337 genes in E. focardii based on their genomes analysis. (b) Frequency of introns of different lengths. The X axis indicates the length of introns in nucleotides, and the Y axis shows how many times they are found in the transcriptomes (log scale). Short introns (~25 nucleotides) is a characteristic feature of Euplotes transcriptomes. (c) Frequency of chromosomes with different numbers of RNA molecules transcribed from them. The X axis shows a number of transcripts per chromosome, and the Y axis how many such chromosomes are found in the genome. (d) E. crassus splice sites. Nucleotide conservation around exon-intron junction and intron-exon junctions. E. crassus. Transcriptomes were assembled de novo using Trinity (Haas, B. J. et al., Nature Protoc, 8, 1494-1512, 2013); no genomic template was used for the assembly of the transcriptome to ensure independence of the analysis. The assembly procedure produced 33,701 unique transcripts with an average length of 573 nucleotides in E. crassus. We obtained the E. focardii RNA-seq reads from (Keeling, P. J. et al., PLoS Biol, 12, e1001889, 2014).; this assembly produced 28,869 unique transcripts with an average length of 667 nucleotides. To identify introns we carried out pairwise alignments between the genome and the transcriptome for each species using FASTA (Pearson, W. Curr Protoc Bioinf, Chapter 3, Unit3 9, 2004) In total, we identified 21,798 introns in E. crassus and 18,747 in E. focardii. The most frequent intron length was 25 nucleotides in both E. crassus and E. focardii with 2,895 and 2,631 occurrences, respectively. Using 10,000 intron sequences from E. crassus, we characterized sequence features of the exon-intron donor and intron-exon acceptor sites. We further aligned 32,350 E. crassus transcripts or their fragments (96%) to 18,032 genomic contigs, and similarly aligned 21,233 E. focardii transcripts (74%) to 16,950 genomic contigs. The majority of chromosomes had a single transcript aligning to them, 10,495 in E. crassus and 14,082 in E. focardii. Some chromosomes contained two or more predicted transcripts, which could be, at least in part, due to insufficient sequence coverage. Low coverage can result in missassembly of a single transcript as two or more, when reads matching internal positions are missing.

Supplementary Figure 3 Termination at AAATAA and two mRNAs with long 3′ UTRs.

In each panel ribosome footprints (top) and mRNA-seq reads (middle) are shown for a transcript whose ORF organization is shown at the bottom (red lines correspond to stop codons, and green lines to ATG codons). Identity of stop codons and adjacent 5’ codons is indicated for the site of termination. Translated segments of ORFs are highlighted in blue. (a) An example of mRNA with termination at AAATAA. (b) mRNA of selenoprotein P22. The position of UGA Sec codon is shown in dark blue. (c) A single detected example of an mRNA with a long 3’UTR not containing SECIS structure.

Supplementary Figure 4 Metagene analysis of RNA-seq density surrounding frameshifting sites.

First nucleotide of a stop codon is shown as a zero coordinate. Only minor alteration of density associated with sequencing biases at specific nucleotides of frameshift sites can be seen.

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Lobanov, A., Heaphy, S., Turanov, A. et al. Position-dependent termination and widespread obligatory frameshifting in Euplotes translation. Nat Struct Mol Biol 24, 61–68 (2017).

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