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Objectives - Organelle gene expression & signaling:

Objectives - Organelle gene expression & signaling:. List the molecular processes involved in going from organelle gene to functional organelle protein complex Describe the technical approaches used to investigate each of these processes

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Objectives - Organelle gene expression & signaling:

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  1. Objectives - Organelle gene expression & signaling: List the molecular processes involved in going from organelle gene to functional organelle protein complex Describe the technical approaches used to investigate each of these processes Compare and contrast organelle gene expression processes with those of bacterial and eukaryotic gene expression systems Discuss molecular mechanisms that adapt organelle gene expression to environmental signals Define retrograde regulation and discuss possible organelle signals that alter nuclear gene expression Describe the plant pentatricopeptide repeat (PPR) gene/protein family with respect to the nature and functions of PPR proteins Discuss why PPR proteins are well-suited to be central in multiple organelle gene expression Discuss the ways in which various organelle gene expression steps can be inter-dependent and give examples

  2. Plastid gene expression overview Translation (del Campo Gene Reg & Syst Biol 3:31)

  3. Organelle DNA copy number can regulate gene expression • Cytoplasmic male sterility (CMS) in Phaseolusvulgaris • CMS gene (orf239) on a mitochondrial subgenomicmolecule • The nuclear fertility restoration gene Fr • Depresses copy number of orf239 • sub-genome • Decreased accumulation of orf239 • transcripts • Prevents expression of CMS • (Mackenzie and Chase Plant Cell 2:905)

  4. RNA Polymerases and promoters

  5. Differential plastid gene expression based upon recognition of distinct promoters by NEP and PEP (from Hajdukiewicz et al. EMBO J 16:4041

  6. Organelle transcripts - initiated vs. processed 5’ ends initiated 5’ end * *processed 5’ end PPP PPP

  7. 5’PPP P Organelle transcript initiated vs. processed 5’ ends • Processed transcripts have 5’ mono-phosphate • Substrate for ligation • e.g. RNA oligo nucleotide for 5’ RACE • e.g. Self-ligation -> Circularization • Polymerase-initiated transcripts have 5’ PP or 5’PPP termini • Substrate only after de-phosphorylation w/ • tobacco acid pyrophosphatese (TAP) • Compare 5’ RACE products +/—TAP initiated transcript –not a ligation substrate 3’ naturally processed or TAP-treated transcript Ligate RNA Adaptor cDNA 3’ Adaptor primer Gene primer Products containing initiated 5’ ends appear only after TAP treatment

  8. 5’PPP Organelle transcript initiated vs. processed 5’ ends • Processed transcripts have 5’ mono-phosphate • Substrate for ligation • e.g. RNA oligo nucleotide for 5’ RACE • e.g. Circularization • Initiated transcripts have 5’ PP or 5’PPP termini • Substrate only after de-phosphorylation w/ • Tobacco acid pyrophosphatese (TAP) • Compare PCR products +/-TAP 3’ Initiated transcript –not a ligation substrate Dilute, self – ligate & reverse transcribe a naturally processed or TAP-treated transcript cDNA 3’ 3’ 5’P Gene primer 1 Gene primer 2 Gene primer 1 5’P Gene primer 2 Amplify and sequence across ligation junction to identify 5’ and 3’ end sequences

  9. Identification of promoters in Arabidopsis plastids + T = + tobacco acid pyrophosphatase treatment - T = without pyrophosphatase treatment g = green tissue w = white tissue (seedlings grown on spectinomycin) [Swiatecka-HagenbruchMol Genet Genomics 277:725]

  10. Diversity of promoters in Arabidopsis plastids [Swiatecka-HagenbruchMol Genet Genomics 277:725]

  11. Plasticity of promoters in Arabidopsis mitochondria -TAP + TAP [Kühn et al. Nucleic Acids Res. 33:337]

  12. Plasticity of promoters in Arabidopsis mitochondria [Kühn et al. Nucleic Acids Res. 33:337]

  13. Differential plastid gene expression based upon polymerases and sigma subunits [from Lopez-Juez and Pyke Intl J Dev Biol 49:557] [Lopez-Juez& Pyke,  Int. J. Dev. Biol. 49: 557 ]

  14. Multiple sigma factors of A. thalianawith different plastid promoter targets in vivo • SIG2 and SIG6 are essential in Arabidopsis • – knock outs are chlorophyll deficient [Lysenko, Plant Cell Rep. 26:845]

  15. Light II PSII most efficient PSI less efficient Additional PSI subunits needed PQ highly reduced (as in + DBMIB) Light I PSI most efficient PSII less efficient Additional PSII subunits needed PQ highly oxidized (as in + DCMU) Redox regulation of photosynthetic gene expression is adaptive PSI PSII PET [Surpin, Plant Cell Supplement 2002:S327]

  16. Regulation of plastid transcription through plastid redox signals PSII PSI Why do the curves for relative transcript amounts and relative transcription activity differ? What do these two things measure? • Complementary changes in transcription rate and mRNA abundance forpsaAB (photosystem I) and psbA(photosystem II) during acclimation to light I or light II • [Pfannschmidt et al. Nature 397:625]

  17. Regulation of nucleargene transcription through plastid redox signals • PSI or PETE nuclear gene promoters • Fused to GUS reporter gene • GUS activity measured in response to light changes • [Pfannschmidt et al. J Biol Chem. 276:36125]

  18. Possible transduction pathways of photosynthetic redox signals [Pfannschmidt et al. Ann Bot 103:599]

  19. Plant organelle RNA metabolism • Plant organelle genes are often co-transcribed • Plastid operons • Mitochondria – di-cistronic transcripts • In contrast to prokaryotic transcripts, plant organelle transcripts: • Are processed to di or mono-cistronic • transcripts • Frequently contain introns • Must undergo RNA editing

  20. psbBoperon processing in maize [Barkan et al. EMBOJ 13:3170]

  21. Plant organelle RNA processing • Polycistronic transcripts undergo extensive, complex processing prior to translation • e.g. psbB operon in maize, encoding subunits of two different plastid protein complexes: • psbB / psbH / petB / petD • The nuclear mutation crp1 disrupts processing of the polycistronic message and consequently, PETB and PETD protein accumulation

  22. High chlorophyll fluorescence (hcf) mutants (maize and arabidopsis) • Mutants in the nuclear genes required for plastid biogenesis and function • ~15% of the Aarabidopsis nuclear genome predicted to plastid function • hcf/hcf > pale-green, yellow, or albino seedlings; some fluoresce in the dark due to dysfunctional photosystems • hcf/hcf seedlings are lethal, but in maize they grow large enough for molecular analysis [Jenkins et al. Plant Cell 9:283]

  23. psbB operon processing in maize A B   missing in crp1/crp1 mutant seedlings [Barkan et al. EMBOJ13:3170]

  24. The crp1 mutant disrupts petB/petDRNAprocessing and PETD protein accumulation Which protein complexes are, and which are not, affected by the crp1 mutant? (Barkan et al. EMBOJ 13:3170)

  25. PET A,B, C& D protein translation in wild-type and crp1 mutant maize 35S-labeled leaf proteins 35S-labeled in organello synthesized proteins petB stop codon petD start codon Secondary structures of monocistronicpetD(left) and bi-cistronicpetB-petD (right) transcripts [Barkan et al. EMBOJ 13:3170]

  26. Inter-dependence of plant organelle gene expression steps • Model: • Failure to accumulate monocistronicpetD transcripts results in failure to translate petD • The petD initiation codon is buried in secondary structure in the petB / petD transcript • The petD initiation codon is free of secondary structure in the monocistronicpetD transcript • But what about • PET C • – Translated but ... • – Reduced accumulation • – What is likely mechanism here? • PETA • – Not translated ! • – What possible mechanisms here?

  27. CRP1 interacts directly at the 5’ region of the petA transcript to promote translation • Immunoprecipitate CRP1 RNA-protein complexes • Slot-blot and hybridize • Precipitated RNA (pellet) • Unbound RNA (supernatant) • PET1 protein associates with regions 5’ of petA and 5’ of psaC • ? Does this approach demonstrate direct RNA binding? [Schmitz-Linneweber et al. Plant Cell 17:2791]

  28. CRP1- RNA interactions Why is the identification of two interaction sites much more powerful than one? C – consensus RNA binging site for CRP1 based on two binding regions D - model for CRP1 protein – RNA interaction [Schmitz-Linneweber et al. Plant Cell 17:2791]

  29. Pentatricopeptide repeat (PPR) proteins • One of the largest multigene families in plants • 441 members in arabidopsisvs 7 in humans • Primarily plastid- or mitochondria-targeted • Implicated in post-transcriptional RNA metabolism through single gene/mutant analysis • e.g. crp1 locus in maize necessary for plastid petB / petD RNA processing • e.g. restorer-of-fertility loci for CMS in petunia, radish and rice all influence processing or stability of mitochondrial CMS gene transcripts • e.g. editing of plastid ndh gene transcripts

  30. Pentatricopeptide repeat (PPR) proteins • Why so many? • ? RNA editing • How do they function? • Site-specific RNA binding proteins • Recruit enzymatic protein complexes • that act on RNA • - or - • Melt RNA structures to allow processing, splicing, translation & stabilization [Lurin et al. Plant Cell 16:2089]

  31. Pentatricopeptide repeat (PPR) proteins • Motif Structure of Arabidopsis PPR Proteins • Degenerate 35 amino acid repeats • The number and order of repeats can vary in • individual proteins • The number of proteins falling into each • subgroup is shown [Lurin et al. Plant Cell 16:2089]

  32. Plant organelle introns Group I and Group II, defined by characteristic secondary structures and splicing mechanisms [from Gillham 1994 Organelle Genes and Genomes]

  33. Plant organelle introns • Group I and Group II have distinct splicing mechansims • Group II is the ancestor of the nuclear intron • Characteristic group II intron structural domains • = ancestors of the nuclear splicosomal RNAs [from Gillham 1994 Organelle Genes and Genomes]

  34. Plant organelle introns • Land plant organelle introns primarily Group II • Characteristic spoke-and-wheel structure • Necessary for splicing • Some fungal versions self-splice in vitro • Trans-acting RNA and/or protein factors required for splicing in vivo • e.g. maize nuclear genes (crs1 & crs2) • encode proteins required for splicing • Genome rearrangements have split introns • Require trans-splicing • Spoke-and-wheel structure assembled • from separate transcripts

  35. The maize crs1 and crs2 mutants disrupt the splicing of different group II introns atpF intron rps16 intron [Jenkins et al. Plant Cell 9:283]

  36. Trans-splicing Chlamydomonas psaA transcripts i1 5’ end i1 3’ end [Gillham 1994 Organelle Genes and Genomes]

  37. Plant organelle transcript stability • Plant organelle transcripts are stabilized by 3’ stem-loop structures • Removal of the stem loop (by endonuclease cleavage) makes the 3’ end accessible for polyAaddition • PPR proteins can substitute for stem loops! • In contrast to nuclear transcripts, plant organelle transcripts are destabilized by the addition of 3’ poly A tracts • 3’ polyA is also a de-stabilizing feature of bacterial transcripts • 3’ polyA enhances susceptibility of transcript to degradation by exonucleases

  38. Model for plastid mRNA turn-over [from Monde et al. Biochimie 82:573]

  39. Plant organelle RNA editing • Post transcriptional enzymatic conversion of C > U • less commonly, U > C • Given a fully sequenced organelle genome, how would the RNA editing process be detected? • genomic coding strand 5’ ....... ACG..... • unedited RNA 5’ ....... ACG..... • edited RNA 5’ ....... AUG.... • edited cDNA 5’ ....... ATG..... • Occurs in plastids and plant mitochondria • many more mitochondrial sites • Primarily in coding sequences • improves overall conservation of predicted protein • Creates initiation codons ACG > AUG • Creates termination codons CGA > UGA • Removes termination codons UGA > CGA • Changes amino acid coding CCA > CUA • (P > L) • Silent edits ATC > ATU

  40. Plant organelle RNA editing • Edit sites within the same gene vary among species • An edit site in one species may be “pre- • edited” (correctly encoded in the genomic sequence) of another species • e.g. plastid psbLgene initiation codon: • maize ATGACA..... • tobacco ACGACA..... must be edited to AUG (RNA) = ATG (cDNA) for translation initiation codon

  41. Evolution of plant organelle RNA editing Not in algae Observed in every land plant lineage except Marchantiid liverworts [Knoop , Curr Genet 46:123]

  42. RNA editing improves evolutionary conservation Table 1. Evolutionary conserved amino acid residues changed by C-to-U editing in ribosomal protein S 12 (RPS12) of plant mitochondria Amino acid residues encoded by unedited and edited maize mitochondrial transcripts compared to amino acid residues in RPS12 polypeptides from other taxa [Mulligan and Maliga (1998) pp.153-161 In A look beyond transcription J Bailey-Serres and DR Gallie (eds) ASPB]

  43. RNA editing occurs by enzymatic de-amination  32P UTP V  32P CTP > 32P CTP [Rajasekhar and Mulligan Plant Cell 5:1843] [Russell, 1995, Genetics]

  44. Short 5’ flanking sequences define plant organelle RNA editing sites [from Mulligan and Maliga (1998) pp.153-161 In A look beyond transcription J Bailey-Serres and DR Gallie (eds) ASPB]

  45. Further evidence for cis-guiding sequences in plant mitochondrial RNA editing • Editing of naturally recombinant or rearranged mitochondrial genes • Recombination breakpoint immediately 3’ to an editing site in rice atp6 did not abolish editing • Recombination breakpoint seven nucleotides 5’ to an editing site in maize rps12 did abolish editing • Recombination breakpoint 21 nucleotides 5’ to an editing site in maize rps12 did not abolish editing • Electroporation of genes into isolated mitochondria & analysis of cDNA • Editing of mutated coxII gene demonstrated sequences from –16 to +6 required for editing • What about the trans-acting editing machinery?

  46. RNA editing – genetic analysis defines a trans-acting factor [from Kotera et al. Nature 433:326]

  47. RNA editing – genetic analysis defines a trans-acting factor [from Kotera et al. Nature 433:326]

  48. RNA editing – genetic analysis defines a trans-acting factor The immunoblots implicating crr4 in NDH complex biogenesis showed loss of the NDHH subunit, but the affected editing site is in the ndhD transcript. What are some explanations for these observations? [from Kotera et al. Nature 433:326]

  49. Translation of organelle genes • A significant regulatory process in plastid gene expression • light-regulated chloroplast protein accumulation increases 50-100 fold w/out changes in mRNA accumulation • 5’ UTR is key in regulating translation • ~ 1/2 of plastid transcripts have a 5’ Shine-Delgarno sequence (GGAG) homologous to small subunit rRNA in this region • nuclear-encoded translation factors bind 5’ untranslated region (UTR) (and in some cases also the 3’ UTR)

  50. Translation of organelle genes • Regulation of plastid gene translation by light • mediated by pH, ADP, redox signals • e.g. Translation of PSII D1 (PSBA) protein in • Chlamydomonas • Accumulation of PSBA increased in light • No change in steady-state level of mRNA • Site-directed mutagenesis of psbA 5’ UTR • 5’ SD sequence • 5’ stem-loop region • Required for translation • A set of 4 major 5’UTR binding proteins identified • Binding increased 10X in the light • PSI reduced thioredoxinrequired for binding • Binding abolished by oxidation • Binding decreased by ADP-dependent • phosphorylation • (ADP accumulates in the dark) • The details of this mechanism do not appear to be conserved in angiosperms

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