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List the molecular processes or steps involved in going from organelle gene to functional organelle protein complex and briefly describe a technical approach.

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Presentation on theme: "List the molecular processes or steps involved in going from organelle gene to functional organelle protein complex and briefly describe a technical approach."— Presentation transcript:

1 List the molecular processes or steps involved in going from organelle gene to functional organelle protein complex and briefly describe a technical approach that can be used to assay each of these steps Discuss the ways in which various organelle gene expression steps can be inter-dependent and give examples Describe molecular mechanisms that adapt plastid gene expression to different light environments Define retrograde regulation and describe the nature of retrograde signaling molecules Describe the nature and functions of plant pentatricopeptide repeat (PPR) proteins Discuss the reasons that PPR proteins are well- suited to be a central player in multiple organelle gene expression processes Design a genetic screen to identify nuclear genes that function in plastid gene expression and explain how you will analyze mutants to determine which plastid genes are affected Objectives - Organelle gene expression & signaling:

2 What are the processes needed to take us from gene to fully functional, multi-subunit, organelle protein complex?

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

4 1 – Consider RUBISCO – the most abundant protein on earth! 2 – Mitochondrial orf239 in Phaseolus vulgaris cytoplasmic male sterility (CMS) gene locates on a subgenomic molecule high copy number > CMS reduced copy number > pollen fertility copy number mediated by nuclear gene – Fr (Mackenzie and Chase Plant Cell 2:905) Organelle DNA copy number can influence gene expression levels

5 RNA Polymerases and promoters PolymeraseSubunitsConsensus promoter Bacterial αββ’ β’’& σ 70 -35/-10 GTGTTGACA/TATAA TG Plastid – encoded (PEP) αββ’ & nuclear- encoded σ specificity -35/-10 -TTGACA/TATAAT Phage T7 single core no σ overlaps initiation ATACGACTCACTATA GGGAGA 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

6 Differential plastid gene expression based upon recognition of distinct promoters by NEP and PEP (from Hajdukiewicz et al. EMBO J 16:4041) Most plastid genes have promoters for both polymerases Genes encoding expression machinery (e.g. rpo, rrn, rps, rpl) primarily transcribed by NEP Photosynthetic genes primarily transcribed by PEP

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

8 Processed transcripts 5’ mono-phosphate Substrate for ligation e.g. RNA adapter for 5’ RACE e.g. Self-ligation -> Circularization Initiated transcripts 5’ tri-phosphate Ligate only after de-phosphorylation tobacco acid pyrophosphatese (TAP) Compare 5’ RACE products +/—TAP processed or TAP-treated transcript PCR products containing initiated 5’ ends appear only after TAP treatment Organelle transcripts - initiated vs. processed 5’ ends Adaptor primer RNA adaptor P 3’ Gene primer cDNA 3’ PCR product RNA 5’PPP initiated transcript –not a ligation substrate 3’ RNA

9 Identification of promoters in Arabidopsis plastids [Swiatecka-Hagenbruch Mol Genet Genomics 277:725] + T: with tobacco acid pyrophosphatase treatment - T: no 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] Consensus of 11 sequences supporting initiation at G Consensus for 20 sequences supporting initiation at A

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

14 I ( ↓ ) under expression II( ↑ ) over expression −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 [Lysenko, Plant Cell Rep. 26:845] SIG2 and SIG6 are essential – 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 PET nuclear gene promoters Fused to GUS reporter gene GUS activity measured in response to light changes

18 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 [Barkan Plant Physiol 155:1524] Plant organelle RNA metabolism: psbB operon processing in maize Plastid operons Processed to di or mono-cistronic forms endo- and exo-nucleases termini stabilized by stem-loops termini stabilized by PPR protein binding

21 Plant organelle RNA metabolism: psbB operon processing in maize Plastid operons Frequently contain introns Splicing mediated by different sub-sets of nuclear-encoded RNA binding proteins [Barkan Plant Physiol 155:1524]

22 Plant organelle RNA metabolism: psbB operon processing in maize [Barkan Plant Physiol 155:1524] RNA processing factors are discovered through forward genetics!!!!!!!!!!!! APO1, APO2 CAF1, CAF2 CFM3 CRP1 HCF107, HCF152 RNC1 WTF1

23 Mutants in the nuclear genes required for plastid biogenesis and 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)

24 Nuclear mutation crp1 [Barkan et al. EMBOJ13:3170]  missing in crp1/crp1 mutant seedlings: 1.1 and 0.75 kb petB RNA 0.75 kb petD RNA How do we see this experimentally? Disrupts processing of the psbB operon

25 Which proteins are reduced in the crp1 mutant? We saw RNA processing effects for petB & petD transcripts. Why might PSAA/B be affected? Why are ALL of the PET protein subunits missing? (Hint, there must be 50 ways to lose a protein, name two!) Disrupts processing of the psbB-psbH- petB-petD operon (Barkan et al. EMBOJ 13:3170) Nuclear mutation crp1

26 [Barkan et al. EMBOJ 13:3170] 35 S-labeled leaf proteins immunoprecipitated 35 S-labeled in organello synthesized proteins immunoprecipitated PET A,B,C,D protein translation studies Nuclear mutation crp1 Which proteins are translated in the crp1 mutant? Which are not? We saw PETA, B,C & D proteins did not accumulate in this mutant. What explains the difference between translation and accumulation?

27 [Barkan et al. EMBOJ 13:3170] Secondary structures of monocistronic petD (left) and bi-cistronic petB-petD (right) transcripts petD start codon petB stop codon PET A,B,C,D protein translation studies Nuclear mutation crp1 Propose a model: How does and RNA processing defect interfere with protein synthesis?

28 No monocistronic petD transcripts and no PETD translation 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 PETB and PETC – Translated but no accumulation – What is likely mechanism here? PETA – Not translated ! – What possible mechanisms here? Inter-dependence of plant organelle gene expression steps

29 CRP1 associates w/ the 5’ region of the petA transcript [Schmitz-Linneweber et al. Plant Cell 17:2791] Immunoprecipitate CRP1 RNA-protein complexes Slot-blot and hybridize Immunoprecipitated RNA (pellet) Unbound RNA (supernatant) PET1 protein associates with regions 5’ of petA and 5’ of psaC Does this show direct RNA binding?

30 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

31 One of the largest multigene families in plants 441 members in arabidopsis vs 7 in humans Plastid- or mitochondria-targeted Most aspects of post-transcriptional RNA metabolism 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 CRP1 is a Pentatricopeptide repeat (PPR) protein

32 Why so many? ? RNA editing How do they function? Site-specific RNA binding proteins Endo and Exonucleases Recruit enzymatic protein complexes Simply melt RNA structures to allow interaction with processing, splicing, translation & editing factors Pentatricopeptide repeat (PPR) proteins

33 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]

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

35 Group II intron structural domains are the ancestor of the nuclear splicosomal RNAs splicosomal RNAs Organelle introns [from Gillham 1994 Organelle Genes and Genomes]

36 In land plants almost all are group II Spoke-and-wheel structure Necessary for splicing Some fungal group IIs self-splice in vitro RNA &/or protein factors required in vivo e.g. maize nuclear genes crs1 & crs2 encode proteins required for splicing Genome rearrangements have split some group II introns Require trans-splicing Spoke-and-wheel structure can be assembled from separate transcripts! Organelle introns

37 How do we see whether introns are spliced or not? There are lots of ways! Reverse transcribe + PCR (RT-PCR) Others you may see: Ribonuclease protection Poison primer RT-PCR RNA blot hybridization Organelle introns DNA/ un-spliced RNA cDNA 3’ 5’ spliced RNA 3’ cDNA { "@context": "http://schema.org", "@type": "ImageObject", "contentUrl": "http://images.slideplayer.com/13/3922738/slides/slide_37.jpg", "name": "How do we see whether introns are spliced or not.There are lots of ways.", "description": "Reverse transcribe + PCR (RT-PCR) Others you may see: Ribonuclease protection Poison primer RT-PCR RNA blot hybridization Organelle introns DNA/ un-spliced RNA cDNA 3’ 5’ spliced RNA 3’ cDNA

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

39 Plant organelle intron splicing requires multiple nuclear-encoded splicing factors [Watkins et al. Plant Cell 23:1082

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

41 [Barkan Plant Physiol 155:1524] Plant organelle RNA metabolism: psbB operon processing in maize What two features confer RNA stability? For nuclear-encoded transcripts 3’ poly A stabilizes For organelle-encoded transcripts 3’ poly A tract DE-stabilizes Also a de-stabilizing feature of bacterial transcripts Enhances susceptibility to degradation by exonucleases

42 Plant organelle RNA editing Post transcriptional enzymatic conversion of C > U, or 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 conserves 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 CTT > CTC (L > L)

43 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

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

45 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 S12 (RPS12) of plant mitochondria

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

47 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]

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

49 Genetic analysis defines a PPR- motif RNA editing factor

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

51 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

52 Regulation of plastid gene translation by light - mediated by pH, ADP, redox signals e.g. translation of PSII D1 (PSBA) in Chlamydomonas Accumulation of PSBA increased in light No change in steady-state level of mRNA Site-directed mutagenesis of 5’ UTR 5’ SD sequence 5’ stem-loop region Required for translation 5’UTR binding proteins identified Binding increased 10X in the light Reduced thioredoxin required for binding Binding abolished by oxidation Binding decreased by ADP-dependent phosphorylation (ADP accumulates in the dark) The details of this mechanism are NOT conserved in angiosperms Translation of organelle genes

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

54 Translation of organelle genes- PPR protein RNA re-modeling enhances ATPH translation

55 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 Down regulates translation Translation of organelle genes

56 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

57 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


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