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:

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

Plastid gene expression overview

Translation

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 regulategene expression

RNA Polymerases and promoters

Polymerase

Subunits Consensus promoter

Bacterial αββ’ 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 ATACGACTCACTATAGGG

AGANuclear -encoded

plastid (NEP) T7-like core &+/- specificity

factoroverlaps 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

initiated 5’ end

Organelle transcripts - initiated vs. processed 5’ ends

PPP

PPP

* processed 5’ end

*

Processed transcripts have 5’ mono-phosphate

Substrate for ligatione.g. RNA oligo nucleotide for 5’ RACEe.g. Self-ligation -> Circularization

Polymerase-initiated transcripts have 5’ PP or 5’PPP terminiSubstrate only after de-phosphorylation w/tobacco acid pyrophosphatese (TAP)

Compare 5’ RACE products +/—TAP

Organelle transcript initiated vs. processed 5’ ends

5’PPPinitiated transcript –not a ligation substrate

PLigate

RNAAdaptor

naturally processed or TAP-treated transcript

Geneprim

erAdaptorprimer

cDNA

3’

3’

Products containing initiated 5’ ends appear only after TAP treatment

Processed transcripts have 5’ mono-phosphate

Substrate for ligatione.g. RNA oligo nucleotide for 5’ RACEe.g. Circularization

Initiated transcripts have 5’ PP or 5’PPP terminiSubstrate 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’PPPInitiated transcript –not a ligation substrate

3’

cDNA

Gene primer

2

5’P

Geneprim

er1

3’

Amplify and sequence across ligation junctionto identify 5’ and 3’ end sequences

5’P

Geneprimer 1

3’

Gene primer

2

Identification of promoters in Arabidopsis plastids

[Swiatecka-Hagenbruch Mol Genet Genomics 277:725]

+ T = + tobacco acid pyrophosphatase treatment- T = without pyrophosphatase treatmentg = 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]

[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 ]

I (↓) II(↑)−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 in vivo

[Lysenko, Plant Cell Rep. 26:845]

SIG2 and SIG6 are essential in Arabidopsis– 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 PETE nuclear gene promoters • Fused to GUS reporter gene• GUS activity measured in response to light

changes

Possible 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

psbB operon processing in maize

[Barkan et al. EMBOJ 13:3170]

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

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)

psbB operon processing in maize

[Barkan et al. EMBOJ13:3170]

missing in crp1/crp1 mutant

seedlings

B

A

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)

PET A,B, C& D protein translation in wild-type and crp1 mutant maize

[Barkan et al. EMBOJ 13:3170]

35S-labeled leaf proteins

35S-labeled in organello

synthesized proteins

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

petD startcodon

petB stopcodon

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

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?

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

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

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]

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 I and Group II have distinct splicing mechansimsGroup 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

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

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]

Trans-splicing Chlamydomonas psaA transcripts

[Gillham 1994 Organelle Genes and Genomes]

i1 3’ end

i1 5’ end

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

Model for plastid mRNA turn-over

[from Monde et al. Biochimie 82:573]

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

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 S 12 (RPS12) of plant mitochondria

RNA editing occurs 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]

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

RNA editing – genetic analysis defines a trans-acting factor

[from Kotera et al. Nature 433:326]

[from Kotera et al. Nature 433:326]

RNA editing – genetic analysis defines a trans-acting factor

[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?

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) 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 identifiedo Binding increased 10X in the lighto PSI reduced thioredoxin required for

bindingo Binding abolished by oxidationo 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

Photosynthetic redox chemistry & plastid gene expression

[Pfannschmit Trends Plant Sci 8:33]

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

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

Redox regulation of PSBA protein synthesis in Chlamydomonas

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

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

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 membranes

degrades 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