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Leaderless mRNAs are circularized in Chlamydomonas reinhardtii mitochondria

Current Genetics volume 64, pages 1321–1333 (2018)Cite this article

Abstract

The mitochondrial genome of Chlamydomonas reinhardtii encodes eight protein coding genes transcribed on two polycistronic primary transcripts. The mRNAs are endonucleolytically cleaved from these transcripts directly upstream of their AUG start codons, creating leaderless mRNAs with 3′ untranslated regions (UTR) comprised of most or all of their downstream intergenic regions. In this report, we provide evidence that these processed linear mRNAs are circularized, which places the 3′ UTR upstream of the 5′ start codon, creating a leader sequence ex post facto. The circular mRNAs were found to be ribosome associate by polysome profiling experiments suggesting they are translated. Sequencing of the 3′–5′ junctions of the circularized mRNAs found the intra-molecular ligations occurred between fully processed 5′ ends (the start AUG) and a variable 3′ terminus. For five genes (cob, cox, nd2, nd4, and nd6), some of the 3′ ends maintained an oligonucleotide addition during ligation, and for two of them, cob and nd6, these 3′ termini were the most commonly recovered sequence. Previous reports have shown that after cleavage, three untemplated oligonucleotide additions may occur on the 3′ termini of these mRNAs—adenylation, uridylylation, or cytidylation. These results suggest oligo(U) and oligo(C) additions may be part of the maturation process since they are maintained in the circular mRNAs. Circular RNAs occur in organisms across the biological spectrum, but their purpose in some systems, such as organelles (mitochondria and chloroplasts) is unclear. We hypothesize, that in C. reinhardtii mitochondria it may create a leader sequence to facilitate translation initiation, which may negate the need for an alternative translation initiation mechanism in this system, as previously speculated. In addition, circularization may play a protective role against exonucleases, and/or increase translational productivity.

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Acknowledgements

Funding was provided by the Buchanan Chair of Biology endowment at UVa-Wise. The authors wish to thank Yue Zou and lab of East Tennessee State University’s Quillen School of Medicine for the use of their ultracentrifuge and David Stern of The Boyce Thompson Institute and Sarah Zimmer and the Zimmer lab at the University of Minnesota for critically reading the manuscript and providing valuable comments.

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  1. Department of Natural Sciences, The University of Virginia’s College at Wise, Wise, VA, 24293, USA

    A. Bruce Cahoon & Ali A. Qureshi

  2. Harvard University, Cambridge, MA, USA

    Ali A. Qureshi

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  1. A. Bruce Cahoon
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  2. Ali A. Qureshi
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Correspondence to A. Bruce Cahoon.

Additional information

Communicated by M. Kupiec.

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294_2018_848_MOESM1_ESM.tiff

Supplementary material 1. Fig. S1. The 3’ termini of protein coding mRNAs are poly-cytidylated. Total RNA from C. reinhardtii (strain cc-503) was deep sequenced and aligned to its chondriome sequence (EU306622.1). A. nd6 deep sequencing reads from a previous study (Cahoon et al 2017) aligned to the DNA template and the presence of non-template poly(C) stretches present at the 3’ terminus created by endonucleolytic cleavage separating nd6 from the tRNA trnW. Colored nucleotides represent cDNA regions that disagree with the genomic reference sequence at the top. B. The 3’ terminal poly(C) additions for all eight mitochondria-encoded mRNAs. In each case, the most common site of the addition is represented but there was typically ± 1–3 nucleotide variation of the poly(C) addition site. (TIFF 14611 KB)

294_2018_848_MOESM2_ESM.tiff

Supplementary material 2. Fig. S2. RT-PCR Evidence that the 3’ and 5’ termini of each mRNA are naturally ligated to form circular transcripts. A. Schematic of gene-specific nested divergent primers designed for cRT-PCR reactions. 1° denotes primers used in the primary PCR reaction and 2° denotes primers used in the second amplification step (shown in B and C). Products should only be generated if the mRNA’s 3’ and 5’ termini were joined. B. Secondary amplicons of a PCR reaction using as template the products of a primary one-step RT-PCR reaction that uses rTth DNA polymerase for the first-strand synthesis. Unmodified mRNA was input for the primary one-step RT-PCR. C. Secondary amplicons in which the primary PCR products used as template were generated with a two-step RT-PCR approach. cDNA was produced from unmodified RNA using MMLV reverse transcriptase, which were used as template for primary PCR reactions. Primary amplicons were used as template for secondary PCR amplicons. The amplicons seen in panels B and C were purified from replicate gels and deep sequenced (Fig. S4). (TIFF 14611 KB)

294_2018_848_MOESM3_ESM.tiff

Supplementary material 3. Fig. S3. RT-PCR of DNA and cDNA. Primary and secondary amplicons. To confirm that RT-PCR secondary products shown in Figs. 2 and S2 were produced from circularized RNA and not contaminating DNA, the reactions were also performed using total DNA as template and compared to reactions in which MMLV-generated cDNA was used as template. A—Convergent nested primary and secondary primer sets were used to amplify a region within the coding region of cob to confirm that amplicons of the same size were produced when the initial DNA and cDNA templates were identical. B – Divergent nested primary and secondary primer sets were used that would produce distinctive secondary products from circularized mRNAs. For all eight coding regions, primary amplicons from cDNA appeared as a diffuse band within the size range predicted for each one. Some primary products were produced from DNA template (nd2, nd4, cox, and rtl) but these were not in the predicted size ranges and were most likely due to spurious annealing of the primers. Distinctive amplicons were produced from the cDNA-derived primary amplicons using the secondary primer set for all coding regions. Amplicons were also produced from DNA primary amplicon template using the same secondary primer sets but the products differed greatly from the cDNA-derived secondary amplicons, demonstrating they were not produced from the same template as the cDNA-derived products. (TIFF 14611 KB)

294_2018_848_MOESM4_ESM.tiff

Supplementary material 4. Fig. S4. Linear representations and proportions of circularized mRNAs. Divergent primers were used to PCR amplify the 3’–5’ junctions from circularized mRNAs of A—cob, B—cox, C—nd1, D—nd2, E—nd4, F—nd5, and G—rtl. These were gel purified and deep sequenced to determine the 3’ and 5’ termini that had been ligated. The right side of each figure represents all the possible linear mRNA sequences hypothesized to be circularized (not all were detected), AUG on the left is the start codon for each mRNA and represents the 5’ terminus and UAA or UAG (bold) the stop codons. All nucleotides to the right of stop codons are in the 3’ untranslated region, with the exception of untemplate oligo(C) and oligo(U) additions. The sequence at the top represents the longest 3’ UTR region detected. The graph on the left represents the proportion of each 3’ terminus found in the sequence reads. (TIFF 14611 KB)

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Cahoon, A.B., Qureshi, A.A. Leaderless mRNAs are circularized in Chlamydomonas reinhardtii mitochondria. Curr Genet 64, 1321–1333 (2018). https://doi.org/10.1007/s00294-018-0848-2

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Keywords

  • Mitochondria
  • Circular RNA
  • Oligocytidylation
  • Oligouridylation
  • Leaderless mRNA