Leaderless mRNAs are circularized in Chlamydomonas reinhardtii mitochondria

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.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. Amberg AC, Van Ommen GJ, Van Bruggen EF, Borst P (1980) Some yeast mitochondrial RNAs are circular. Cell 19:313–319

    Article  Google Scholar 

  2. Andronescu M, Condon A, Hoos HH, Mathews DH, Murphy KP (2007) Efficient parameter estimation for RNA secondary structure prediction. Bioinformatics 23:19–28

    Article  Google Scholar 

  3. Barbrook AC, Dorrell RG, Burrows J, Plenderleith LJ, Nisbet RE, Howe CJ (2012) Polyuridylylation and processing of transcripts from multiple gene minicircles in chloroplasts of the dinoflagellate Amphidinium carterae. Plant Mol Biol 79:347–357

    CAS  Article  Google Scholar 

  4. Bell SA, Shen C, Brown A, Hunt AG (2015) Experimental genome-wide determination of RNA polyadenylation in Chlamydomonas reinhardtii. PLoS One 11(1):e0146107

    Article  Google Scholar 

  5. Blum B, Bakalara N, Simpson L (1990) A model for RNA editing in kinetoplastid mitochondria: “guide” RNA molecules transcribed from maxicircle DNA provide the edited information. Cell 60:189–198

    CAS  Article  Google Scholar 

  6. Boer PH, Gray MW (1988) Genes encoding a subunit of respiratory NADH dehydrogenase (ND1) and a reverse transcriptase-like protein (RTL) are linked to ribosomal RNA gene pieces in Chlamydomonas reinhardtii mitochondrial DNA. EMBO J 7:3501–3508

    CAS  Article  Google Scholar 

  7. Boer PH, Gray MW (1991) Short dispersed repeats localized in spacer regions of Chlamydomonas reinhardtii mitochondrial DNA. Curr Genet 19:309–312

    CAS  Article  Google Scholar 

  8. Boynton JE, Gillham NW (1996) Genetics and transformation of mitochondria in the green alga Chlamydomonas. Methods Enzymol 264:279–296

    CAS  Article  Google Scholar 

  9. Brock JE, Pourshahian S, Giliberti J, Limbach PA, Jansses GR (2008) Ribosomes bind leaderless mRNA in Escherichia coli through recognition of their 5′terminal AUG. RNA 14:2159–2169

    CAS  Article  Google Scholar 

  10. Cahoon AB, Nauss JA, Stanley CD, Qureshi A (2017) Deep transcriptome sequencing of two green algae, Chara vulgaris and Chlamydomonas reinhardtii, provides no evidence of organellar RNA editing. Genes 8:80

    Article  Google Scholar 

  11. Cardol P, Remacle C (2009) The mitochondrial genome. In: David B, Stern (eds) In The chlamydomonas sourcebook, vol 2, 2nd edn. Academic Press, Oxford

    Google Scholar 

  12. Cardol P, Matagne RF, Remacle C (2002) Impact of mutations affecting ND mitochondria-encoded subunits on the activity and assembly of complex I in Chlamydomonas. Implication for the structural organization of the enzyme. J Mol Biol 319:1211–1221

    CAS  Article  Google Scholar 

  13. Christian BE, Spremulli LL (2010) Preferential selection of the 5′-terminal start codon on leaderless mRNAs by mammalian mitochondrial ribosomes. J Biol Chem 285:28379–28386

    CAS  Article  Google Scholar 

  14. Cocquerelle C, Mascrez B, Hétuin D, Bailleul B (1993) Mis-splicing yields circular RNA molecules. FASEB J 7:155–160

    CAS  Article  Google Scholar 

  15. Colleaux L, Michel-Wolwertz MR, Matagne RF, Dujon B (1990) The apocytochrome b gene of Chlamydomonas smithii contains a mobile intron related to both Saccharomyces and Neurospora introns. Mol Gen Genet 223:288–296

    CAS  Article  Google Scholar 

  16. Darbani B, Noeparvar S, Borg S (2016) Identification of circular RNAs from parental genes involved in multiple aspects of cellular metabolism in barley. Front Plant Sci 7:776

    Article  Google Scholar 

  17. De Paepe B, Lefever S, Mestdagh P (2018) How long noncoding RNAs enforce their will on mitochondrial activity: regulation of mitochondrial respiration, reactive oxygen species production, apoptosis, and metabolic reprogramming in cancer. Curr Genet 64:163–172

    Article  Google Scholar 

  18. Dombrowski S, Brennicke A, Binder S (1997) 3′-Inverted repeats in plant mitochondrial mRNAs are processing signals rather than transcription terminators. EMBO J 16:5069–5076

    CAS  Article  Google Scholar 

  19. Dorrell RG, Klinger CM, Newby RJ, Butterfield ER, Richardson E, Dacks JB, Howe CJ, Nisbet ER, Bowler C (2016) Progressive and biased divergent evolution underpins the origin and diversification of peridinin dinoflagellate plastids. Mol Biol Evol 34:361–379

    Google Scholar 

  20. Duby F, Cardol P, Matgne RF, Remacle C (2001) Structure of the telomeric ends of mt DNA, transcriptional analysis and complex I assembly in the dum24 mitochondrial mutant of Chlamydomonas reinhardtii. Mol Genet Genomics 266:109–114

    CAS  Article  Google Scholar 

  21. Eriksson M, Gardestrom P, Samuelsson G (1995) Isolation, purification, and characterization of mitochondrial from Chlamydomonas reinhardtii. Plant Phys 107:479–483

    CAS  Article  Google Scholar 

  22. Forner J, Weber B, Thuss S, Wildum S, Binder S (2007) Mapping of mitochondrial mRNA termini in Arabidopsis thaliana: t-elements contribute to 5′ and 3′ end formation. Nuc Acids Res 35:3676–3692

    CAS  Article  Google Scholar 

  23. Gallaher SD, Fitz-Gibbon ST, Strenkert D, Purvine SO, Pellegrini M, Merchant SS (2017) High-throughput sequencing of the chloroplast and mitochondrion of Chlamydomonas reinhardtii to generate improved de novo assemblies, analyze expression patterns and transcript speciation, and evaluate diversity among laboratory strains and wild isolates. Plant J 93:545–565

    Article  Google Scholar 

  24. Gazestani VH, Hampton M, Abrahante JE, Slavati R, Zimmer SL (2016) circtTAIL-seq, a targeted method for deep analysis of RNA 3′ tails, reveals transcript-specific differences by multiple metrics. RNA 22:477–486

    CAS  Article  Google Scholar 

  25. Gray MW, Boer PH (1988) Organization and expression of algal (Chlamydomonas reinhardtii) mitochondrial DNA. Phil Trans R Soc Lon B Biol Sci 319:135–147

    CAS  Article  Google Scholar 

  26. Grimes BT, Sisay AK, Carroll HD, Cahoon AB (2014) Deep sequencing of the tobacco mitochondrial transcriptome reveals expressed ORFs and numerous editing sites outside coding regions. BMC Genom 15:31

    Article  Google Scholar 

  27. Hammani K, Giegé P (2014) RNA metabolism in plant mitochondria. Trends Plant Sci 19:380–389

    CAS  Article  Google Scholar 

  28. Hang R, Deng X, Liu C, Mo B, Cao X (2015) Circular RT-PCR assay using Arabidopsis samples. Bio Protoc 5:1533

    Article  Google Scholar 

  29. Harris EH (2009) The chlamydomonas sourcebook: introduction to chlamydomonas and its laboratory use, vol 1, 2nd edn. Academic Press, Oxford

    Google Scholar 

  30. Hossain ST, Malhotra A, Deutscher MP (2016) How RNase R degrades structured RNA, role of the helicase activity and S1 domain. J Biol Chem 291:7877–7887

    CAS  Article  Google Scholar 

  31. Jacobson A (1996) Poly(A) metabolism and translation: the closed-loop model. In: Hershey JWB, Mathews MB, Sonenberg N (eds) Translational control. Cold Spring Harbor Laboratory Press, New York, pp 451–480

    Google Scholar 

  32. Janssen GR (1993) Eubacterial, archaebacterial, and eukaryotic genes that encode leaderless mRNA. In: Baltz R et al. (ed) Industrial microorganisms: basic and applied molecular genetics. ASM Press, Washington, DC, pp 59–67

    Google Scholar 

  33. Jeck WR, Sorrentino JA, Wang K, Slevin MK, Burd CE, Liu J, Marzluff WF, Sharpless NE (2013) Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA 19:141–157

    CAS  Article  Google Scholar 

  34. Jones CN, Wilkinson KA, Hung KT, Weeks KM, Spremulli LL (2008) Lack of secondary structure characterizes the 5′ ends of mammalian mitochondrial mRNAs. RNA 14:862–871

    CAS  Article  Google Scholar 

  35. Komine Y, Kwong L, Anguera MC, Schuster G, Stern DB (2000) Polyadenylation of three classes of chloroplast RNA in Chlamydomonas reinhardtii. RNA 6:598–607

    CAS  Article  Google Scholar 

  36. Komine Y, Kikis E, Schuster G, Stern D (2002) Evidence for in vivo modulation of chloroplast RNA stability by 3′-UTR homopolymeric tails in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 99:4085–4090

    CAS  Article  Google Scholar 

  37. Lasda E, Parker R (2014) Circular RNAs: diversity of form and function. RNA 20:1829–1842

    CAS  Article  Google Scholar 

  38. Lee S-M, Kong HG, Ryu C-M (2017) Are circular RNAs new kids on the block? Trends Plant Sci 122:357–360

    Article  Google Scholar 

  39. Levy S, Schuster G (2016) Polyadenylation and degradation of RNA in the mitochondria. Biochem Soc Trans 44:1475–1482

    CAS  Article  Google Scholar 

  40. Ma D-P, Yang Y-W, King Y-T, Hasnain SE (1989) Nucleotide sequence of cloned nad4 (urf4) gene from Chlamydomonas reinhardtii. Gene 85:363–370

    CAS  Article  Google Scholar 

  41. Ma D-P, King Y-T, King TY, Hasnain SE (1990) The mitochondrial apocytochrome b gene from Chlamydomonas reinhardtii. Plant Mol Biol 15:357–359

    CAS  Article  Google Scholar 

  42. Moll I, Hirokawa G, Kiel MC, Kaji A, Blasi U (2004) Translation initiation with 70S ribosomes: an alternative pathway for leaderless mRNAs. Nucleic Acids Res 32:3354–3363

    CAS  Article  Google Scholar 

  43. Montoya J, Ojala D, Attardi G (1981) Distinctive features of the 5′-terminal sequences of the human mitochondrial mRNAs. Nature 290:465–470

    CAS  Article  Google Scholar 

  44. Nagaike T, Suzuk T, Katoh T, Ueda T (2005) Human mitochondrial mRNAs are stabilized with polyadenylation regulated by mitochondria-specific poly(A) polymerase and polynucleotide phosphorylase. J Biol Chem 280:19721–19727

    CAS  Article  Google Scholar 

  45. Nickelsen J, Kück U (2000) The unicellular green alga Chlamydomonas reinhardtii as an experimental system to study chloroplast RNA metabolism. Naturwissenschaften 87:97–107

    CAS  Article  Google Scholar 

  46. Ptashne M, Backman K, Humayun MZ, Jeffrey A, Maurer R, Meyer B, Sauer RT (1976) Autoregulation and function of a repressor in bacteriophage lambda. Science 194:156–161

    CAS  Article  Google Scholar 

  47. Salinas-Giegé T, Cavaiuolol M, Cognat V, Ubrig E, Remacle C, Duchêne A-M, Vallon O, Maréchal-Drouard L (2017) Polycytidylation of mitochondrial mRNAs in Chlamydomonas reinhardtii. Nucleic Acids Res 45:12963–12973

    Article  Google Scholar 

  48. Schuster G, Stern D (2009) RNA polyadenylation and decay in mitochondria and chloroplasts. Prog Mol Biol 85:393–422

    CAS  Google Scholar 

  49. Simpson L, Sbicego S, Aphasizhev R (2003) Uridine insertion/deletion RNA editing in trypanosome mitochondria: a complex business. RNA 9:265–276

    CAS  Article  Google Scholar 

  50. Slomovic S, Schuster G (2013) Circularized RT-PCR (cRT-PCR): Analysis of the 5′ Ends, 3′ Ends, and poly(A) tails of RNA. Meth Enzymol 530:227–251

    CAS  Article  Google Scholar 

  51. Soma A (2014) Circularly permuted tRNA genes: their expression and implications for their physiological relevance and development. Front Genet 5:63

    Article  Google Scholar 

  52. Soma A, Onodera A, Suahara J, Kanai A, Yachie N, Tomita M, Kawamura F, Sekine Y (2007) Permuted tRNA genes expressed via a circular RNA intermediate in Cyanidioschyzon merolae. Science 318:450–453

    CAS  Article  Google Scholar 

  53. Stewart JB, Beckenbach AT (2009) Characterization of mature mitochondrial transcripts in Drosophila, and the implications for the tRNA punctuation model in arthropods. Gene 445:49–57

    CAS  Article  Google Scholar 

  54. Sun X, Wang X, Ding J, Wang Y, Wang J, Zhang X, Che Y, Liu Z, Zhang X, Ye J, Wang J, Sablok G, Deng Z, Zhao H (2016) Integrative analysis of Arabidopsis thaliana transcriptomics reveals intuitive splicing mechanism for circular RNA. FEBS Lett 590:3510–3516

    CAS  Article  Google Scholar 

  55. Thompson MK, Gilbert WV (2017) mRNA length-sensing in eukaryotic translation: reconsidering the “closed-loop” and its implications for translational control. Curr Genet 63:613–620

    CAS  Article  Google Scholar 

  56. Torarinsson E, Klenk HP, Garrett RA (2005) Divergent transcriptional and translational signals in Archaea. Environ Microbiol 7:47–54

    CAS  Article  Google Scholar 

  57. Tracy RL, Stern DB (1995) Mitochondrial transcription initiation: promoter structures and RNA polymerases. Curr Genet 28:205–216

    CAS  Article  Google Scholar 

  58. Udagawa T, Shimizy Y, Ueda T (2004) Evidence for the translation initiation of leaderless mRNAs by intact 70S ribosomes without its dissociation into subunits in bacteria. J Biol Chem 279:8539–8546

    CAS  Article  Google Scholar 

  59. Wang Y, Morse D (2006) Rampant polyuridylylation of plastid gene transcripts in the dinoflagellate Lingulodinium. Nucleic Acids Res 34:613–619

    Article  Google Scholar 

  60. Zheng X, Hu G-Q, She Z-S, Zhu H (2011) Leaderless genes in bacteria: clue to the evolution of translation initiation mechanisms in prokaryotes. BMC Genom 12:361

    CAS  Article  Google Scholar 

  61. Zimmer SL, Schein A, Zipor G, Stern DB, Schuster G (2009) Polyadenylation in Arabidopsis and Chlamydomonas organelles: the input of nucleotidyltransferases, poly(A) polymerases and polynucleotide phosphorylase. Plant J 59:88–99

    CAS  Article  Google Scholar 

Download references

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.

Author information

Affiliations

Authors

Corresponding author

Correspondence to A. Bruce Cahoon.

Additional information

Communicated by M. Kupiec.

Electronic supplementary material

Below is the link to the electronic supplementary material.

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)

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)

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)

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)

Supplementary material 5 (TIFF 14611 KB)

Supplementary material 6 (TIFF 14611 KB)

Supplementary material 7 (TIFF 14611 KB)

Supplementary material 8 (TIFF 14611 KB)

Supplementary material 9 (TIFF 14611 KB)

Supplementary material 10 (TIFF 14611 KB)

Supplementary material 11 (XLSX 56 KB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Keywords

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