1. 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 Objectives - Organelle gene expression & signaling:

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

3. Cytoplasmic male sterility (CMS) in Phaseolus vulgaris CMS gene (orf239) on a mitochondrial subgenomic molecule 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) Organelle DNA copy number can regulate gene expression

4. RNA Polymerases and promoters PolymeraseSubunitsConsensus promoter Bacterialαββ’ β’’& σ 70 -35/-10 GTGTTGACA/TATAATG Plastid – encoded (PEP) αββ’ & nuclear- encoded σ specificity -35/-10 -TTGACA/TATAAT Phage T7 single core no σ overlaps initiation ATACGACTCACTATAGG GAGA Nuclear - encoded plastid (NEP) T7-like core & +/- specificity factor overlaps initiation ATAGAAT A/G AA Nuclear – encoded mit T7-like core & +/- specificity factor overlaps initiation CRTA G/T

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

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

7. 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 Organelle transcript initiated vs. processed 5’ ends 5’PPP initiated transcript –not a ligation substrate P Ligate RNA Adaptor naturally processed or TAP-treated transcript Gene primer Adaptor primer cDNA 3’ Products containing initiated 5’ ends appear only after TAP treatment

8. 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 Organelle transcript initiated vs. processed 5’ ends Dilute, self – ligate & reverse transcribe a naturally processed or TAP-treated transcript 5’PPP Initiated transcript –not a ligation substrate 3’ cDNA Gene primer 2 5’P Gene primer 1 3’ Amplify and sequence across ligation junction to identify 5’ and 3’ end sequences 5’P Gene primer 1 3’ Gene primer 2

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

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

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

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

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

14. I ( ↓ )II( ↑ ) −Sig2−Sig4−Sig5−Sig6+Sig2+Sig5 trnEYDndhF LRP- psbD b atpBE- 2.6kb b trnEYDpsaA trnV psbA c psbA trnM psbB c psbB psaJ psbC c psbD psbA a psbD c const- psbD b psbH c psbN c psbT c rbcL c rrn16 c rrn23 c rrn5 c rrn4.5 c Multiple sigma factors of A. thaliana with different plastid promoter targets in vivo [Lysenko, Plant Cell Rep. 26:845] SIG2 and SIG6 are essential in Arabidopsis – knock outs are chlorophyll deficient

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

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

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

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

19. 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 Plant organelle RNA metabolism

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

21. 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 Plant organelle RNA processing

22. 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] High chlorophyll fluorescence (hcf) mutants (maize and arabidopsis)

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

24. The crp1 mutant disrupts petB/petD RNA processing 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 [Barkan et al. EMBOJ 13:3170] 35 S-labeled leaf proteins 35 S-labeled in organello synthesized proteins Secondary structures of monocistronic petD (left) and bi-cistronic petB-petD (right) transcripts petD start codon petB stop codon

26. Model: Failure to accumulate monocistronic petD 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 monocistronic petD transcript But what about PET C – Translated but... – Reduced accumulation – What is likely mechanism here? PETA – Not translated ! – What possible mechanisms here? Inter-dependence of plant organelle gene expression steps

27. CRP1 interacts directly at the 5’ region of the petA transcript to promote translation [Schmitz-Linneweber et al. Plant Cell 17:2791] 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?

28. CRP1- RNA interactions [Schmitz-Linneweber et al. Plant Cell 17:2791] 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

29. One of the largest multigene families in plants 441 members in arabidopsis vs 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 Pentatricopeptide repeat (PPR) proteins

30. 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 Pentatricopeptide repeat (PPR) proteins [Lurin et al. Plant Cell 16:2089]

31. 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 Pentatricopeptide repeat (PPR) proteins [Lurin et al. Plant Cell 16:2089]

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

33. 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] Plant organelle introns

34. 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 o e.g. maize nuclear genes (crs1 & crs2) encode proteins required for splicing Genome rearrangements have split introns o Require trans-splicing o Spoke-and-wheel structure assembled from separate transcripts Plant organelle introns

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 [Gillham 1994 Organelle Genes and Genomes] i1 3’ end i1 5’ end

37. Plant organelle transcripts are stabilized by 3’ stem-loop structures Removal of the stem loop (by endonuclease cleavage) makes the 3’ end accessible for polyA addition 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 Plant organelle transcript stability

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

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

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. 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? Further evidence for cis-guiding sequences in plant mitochondrial RNA editing

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

48. [from Kotera et al. Nature 433:326] 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?

49. 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) Translation of organelle genes

50. 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 o 5’ SD sequence o 5’ stem-loop region o Required for translation A set of 4 major 5’UTR binding proteins identified o Binding increased 10X in the light o PSI reduced thioredoxin required for binding o Binding abolished by oxidation o Binding decreased by ADP-dependent phosphorylation (ADP accumulates in the dark) The details of this mechanism do not appear to be conserved in angiosperms Translation of organelle genes

51. Photosynthetic redox chemistry & plastid gene expression [Pfannschmit Trends Plant Sci 8:33] Light I PSI most efficient PSII less efficient More PSII needed PQ highly oxidized (as in + DCMU) Light II PSII most efficient PSI less efficient More PSI needed PQ highly reduced (as in + DBMIB)

52. Redox regulation of PSBA protein synthesis in Chlamydomonas [from Pfannschmit (2003) Trends Plant Sci 8:33]

53. Control by Epistasy of Synthesis (CES) Regulation of protein synthesis by presence or absence of assembly partners e.g. Down-regulation of tobacco nuclear rbcS gene by antisense Decreased translation of rbcL in plastid e.g. Chlamydomonas plastid cytochrome f (PET complex) Absent other subunits, cytochrome f cannot assemble Unassembled) cytochrome f binds to its own (petA ) 5’ UTR to down regulate translation Translation of organelle genes

54. Failure to assemble a protein complex > degradation of unassembled subunits Assembly dependent upon availability of all subunits and co-factors Plastids contain several proteases that are homologues of bacterial proteases o Functions in protein turn-over o ? P rotease independent chaperone functions (as seen in bacteria) Organelle protein complex assembly and protein turn-over

55. ProteaseLocation and Function in plastid ClpP/ClpC ATP-dependent serine protease stroma degrades mis-targeted proteins and cytb6/f subunits FtsH membrane-bound, ATP- dependent metallo protease stromal face of thylakoid membranes degrades photo-damaged PSI protein D1 from stromal side DegP serine heat-shock protease lumenal side of thylakoid membranes degrades photo-damaged PSI protein D1 from lumen side Bacterial – type proteases in plastids