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 examplesDescribe 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:

What are the processes needed to take us from gene to fully

functional, multi-subunit, organelle protein complex?

(del Campo Gene Reg & Syst Biol 3:31)

Plastid gene expression overview

Translation

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

RNA Polymerases and promotersPolymera

seSubunits Consensus

promoter

Bacterial αββ’ β’’& σ 70

-35/-10 GTGTTGACA/TATAAT

G

Plastid –encoded (PEP)

αββ’ & nuclear-

encoded σ specificity

-35/-10-TTGACA/TATAAT

Phage T7 single core no σ

overlaps initiation ATACGACTCACTATAG

GGAGA

Nuclear -encoded plastid (NEP)

T7-like core &

+/- specificity

factor

overlaps initiationATAGAAT A/G AA

Nuclear –encoded mit

T7-like core &

+/- specificity

factor

overlaps initiation CRTA G/T

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

initiated 5’ end -

PPP

Organelle transcripts - initiated vs.

processed 5’ ends

PPP

* processed 5’ end - P

*

Processed transcripts 5’ mono-phosphate

Substrate for ligatione.g. RNA adapter for 5’ RACEe.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

Adaptorprimer

RNAadapt

orP 3’

Geneprimer

cDNA 3’3’PCR

product

RNA

5’PPPinitiated transcript –not a ligation substrate

3’RNA

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 tissuew: white tissue (seedlings grown on spectinomycin)

Diversity of promoters in Arabidopsis plastids

[Swiatecka-Hagenbruch Mol Genet Genomics 277:725]

[Kühn et al. Nucleic Acids Res. 33:337]

Plasticity of promoters in Arabidopsis mitochondria

+TAP - TAP

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

[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 ]

I (↓) under expression II(↑) over expression

−Sig2 −Sig4 −Sig5 −Sig6 +Sig2 +Sig5trnEYD ndhF LRP-

psbDb atpBE-2.6kbb trnEYD psaA

trnV     psbAc psbA psbAtrnM     psbBc   psbBpsaJ     psbCc   psbDpsbAa     psbDc    const-psbDb     psbHc          psbNc          psbTc          rbcLc          rrn16c          rrn23c          rrn5c          rrn4.5c    

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

Light IPSI most efficientPSII less efficientAdditional PSII subunits neededPQ highly oxidized(as in + DCMU)

Light IIPSII most efficientPSI less efficientAdditional PSI subunits neededPQ highly reduced(as in + DBMIB)

[Surpin, Plant Cell Supplement 2002:S327]

Redox regulation of photosynthetic gene

expression is adaptive

PSIIPSI

PET

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 amountsand relative transcription activity differ? What do these two things measure?

PSIIPSI

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

Transduction pathways of photosynthetic redox signals

[Pfannschmidt et al. Ann Bot 103:599]

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

[Barkan Plant Physiol 155:1524]

Plant organelle RNA metabolism:psbB operon processing in maize

Plastid operonsProcessed to di or mono-cistronic forms

• endo- and exo-nucleases• termini stabilized by stem-loops• termini stabilized by PPR protein

binding

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]

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

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)

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

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

[Barkan et al. EMBOJ 13:3170]

35S-labeled leaf proteins

immunoprecipitated

35S-labeled in organello

synthesized proteins

immunoprecipitated

PET A,B,C,D protein translation studiesNuclear 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?

[Barkan et al. EMBOJ 13:3170]

Secondary structures of monocistronic petD (left) and bi-cistronic petB-petD (right) transcripts

petD startcodon

petB stopcodon

PET A,B,C,D protein translation studiesNuclear mutation crp1

Propose a model: How does and RNA processing defect interfere with protein synthesis?

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

CRP1 associates w/ the 5’ region of the petA transcript

[Schmitz-Linneweber et al. Plant Cell 17:2791]

Immunoprecipitate CRP1 RNA-protein complexesSlot-blot and hybridize

• Immunoprecipitated RNA (pellet)• Unbound RNA (supernatant)

PET1 protein associates with regions 5’ of petA and 5’ of psaCDoes this show direct RNA binding?

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

One of the largest multigene families in plants

• 441 members in arabidopsis vs 7 in humans

Plastid- or mitochondria-targetedMost 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

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

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]

Group I and Group II, defined by characteristic secondary structures and splicing mechanisms

[from Gillham 1994 Organelle Genes and Genomes]

Plant organelle introns

Group II intron structural domains are the ancestor of the nuclear splicosomal RNAs splicosomal RNAs

Organelle introns

[from Gillham 1994 Organelle Genes and Genomes]

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

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

RNAcDNA 3’5’ <RPCR

3’5’ <RF>

spliced RNA 3’cDNA <R5’

PCR

F> <R 3’5’

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

Plant organelle intron splicing requires multiple nuclear-encoded

splicing factors

[Watkins et al. Plant Cell 23:1082

Trans-splicing Chlamydomonas psaA

transcripts

[Gillham 1994 Organelle Genes and Genomes]

i1 3’ endi1 5’ end

[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 stabilizesFor organelle-encoded transcripts 3’ poly A tract DE-stabilizes

• Also a de-stabilizing feature of bacterial transcripts

• Enhances susceptibility to degradation by exonucleases

Plant organelle RNA editing Post transcriptional enzymatic

conversion of C > U, or less commonly, U > CGiven 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 sitesPrimarily in coding sequences

• conserves predicted proteinCreates initiation codons ACG > AUGCreates termination codons CGA > UGARemoves termination codons UGA > CGAChanges amino acid coding CCA > CUA (P > L) Silent edits CTT > CTC (L > L)

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

Evolution of plant organelle RNA editing

Not in algae

Observed in every land plant lineage except Marchantiid liverworts

[Knoop , Curr Genet 46:123]

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

RNA editing by enzymatic de-amination

[Rajasekhar and Mulligan Plant Cell 5:1843]

[Russell, 1995, Genetics]

32P CTP

32P CTP > 32P UTP

V

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]

RNA editing – genetic analysis defines a trans-acting factor

[from Kotera et al. Nature 433:326]

[from Kotera et al. Nature 433:326]

Genetic analysis defines a PPR-motif RNA editing factor

[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

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

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

Redox regulation of PSBA protein synthesis in Chlamydomonas

[Pfannschmit (2003) Trends Plant Sci 8:33]

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

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

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 proteaseso Functions in protein turn-overo ? Protease independent chaperone

functions (as seen in bacteria)

Organelle protein complex assemblyand protein turn-over

Protease Location and Functionin plastid

ClpP/ClpCATP-dependent serine protease

stromadegrades mis-targeted proteins and cytb6/f subunits

FtsHmembrane-bound, ATP-dependent metallo- protease

stromal face of thylakoid membranesdegrades photo-damaged PSI protein D1 from stromal side

DegPserine heat-shock protease

lumenal side of thylakoid membranesdegrades photo-damaged PSI protein D1 from lumen side

Bacterial – type proteases in plastids