Objectives - Organelle gene expression & signaling:
description
Transcript of 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
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