Kyle Gribbin University of Oregon Mentor: Margarita Rojas PI: Alice Barkan.
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Transcript of Kyle Gribbin University of Oregon Mentor: Margarita Rojas PI: Alice Barkan.
Cracking the PPR Code:Predicting and Manipulating PPR protein/RNA Interactions
Kyle GribbinUniversity of Oregon
Mentor: Margarita RojasPI: Alice Barkan
• All genes have RNA intermediates on their way to being expressed.
Argonaute
U2af65
How do these proteins know where to bind RNA?
RNA Binding Proteins Regulate Gene Expression
• Small area of contact with RNA• Idiosyncratic mechanisms of nucleotide recognition
PAZ
KH
Proteins bind Specific RNAs with RNA Binding Domains
Typical RNA binding domains:
•Proteins composed of repeating alpha helices that each recognize one nucleotide.
•The helices can be engineered to bind desired nucleic acids in sequence.
•My project concerns a new class of RNA binding repeat proteins: PentatricoPeptide Repeats
Puf domain TAL
domain
Repeat Proteins: A Novel Nucleic Acid Recognition Mechanism
Pentatricopeptide Repeat (PPR)
OrganelleTargeting Sequence
35 amino acid repeats (~4-30 RPTS)
PPRs: eucaryote-specific, RNA
binding module
?Small and Peeters, TIBS 2000
•Binds single stranded RNA
•Repeats have different amino acid sequences, allowing them to bind different nucleotides
Mito and ChloroplastRNA splicing, RNA editing, RNA
stabilization, RNA cleavage, translational activation and
repression.Act on SPECIFIC organellar
RNAs. Plant nuclear
genomes encode ~450 PPR proteins
PPR Proteins Affect Organellar Gene Expression
• Bound PPR proteins◦Stabilize RNA◦Regulate Splicing◦Regulate Translation
How do they know where to bind on the RNA?
GenePPR
PPR
PPR
ExposedSequence
SequesteredSequence
PPRBinding
site
How PPR Proteins Affect Gene Expression
Our lab has discovered a code by which PPR repeats bind nucleotides.
|||||||||||||GUAUCCUUAACCA
Combinatorial Amino Acid Code for Nucleotide Recognition by PPR Motifs•Modular Recognition
Repeats can be changed to bind specific nucleotides.•Two amino acid code defines nucleotide identity.
eg: N and position 6 and D at position 1 binds a Uracil.•Evidence that mismatches in the code can be tolerated.
To predict native binding sites for natural PPR proteins and to engineer new PPR proteins to bind desired RNA sequences.
To accomplish this, we will take a closer look at our model PPR protein: PPR10 19 PPR Motifs Binds 3 Chloroplast RNAs Stabilizes RNA Regulates Translation
|||||||||||||GUAUCCUUAACCA
Long Term Goal
Nucleotides outside of the box do not match with the code.
This suggests RNA loops out from the protein:
|||||||| |||GUAUCCUU CCA… /\
A A
“Linker” region
Loop out
PPR10’s 3 Native Binding Sites Suggests Interruption of a Contiguous RNA/Protein Interface
• Where can code mismatches be tolerated along the PPR10/RNA interface?
• Is the “linker” region a gap in binding, or are there interactions beyond the code?
Questions I Addressed
Wild type RNA 5’ GUAUCCUUAACCAUUUC 3’2 GAAUCCUUAACCAUUUC3 GUUUCCUUAACCAUUUC4 GUAACCUUAACCAUUUC5 GUAUGCUUAACCAUUUC6 GUAUCGUUAACCAUUUC7 GUAUCCAUAACCAUUUC
How will changing these nucleotides affect PPR10s affinity for the sequence?
Where Along a PPR/RNA Duplex can Code Mismatches be Tolerated?
32P RNAPPR10
Unbound RNA
Bound RNA
Protein concentration
Gel Mobility Shift Assay: Method for Determining PPR10/RNA Affinity
GAAUCCUUAACCAUUUCGUUUCCUUAACCAUUUC
GUAACCUUAACCAUUUC
GUAUCCUUAACCAUUUC
Unbound RNA
Bound RNA
Mutation at RNA Position 2,3, or 4 Cause Massive Loss of Binding Affinity
WT
Mutation at RNA position 5,6, or 7 Cause Small Loss of Binding Affinity
GUAUGCUUAACCAUUUCGUAUCGUUAACCAUUUC
GUAUCCAUAACCAUUUC
GUAUCCUUAACCAUUUC
Unbound RNA
Bound RNA
WT
|||||||||||||GUAUCCUUAACCA 234567
Wt
6
53
42
7
Fract
ion
RN
A
Bou
nd
PPR10 [nM]
5’ 3’
Loss of Binding Affinity Decreases as Mismatches move Towards the Center of PPR10/RNA Duplex
Wild Type RNA 5’ GUAUCCUUAACCAUUUC 3’ GUAUCCUUGGCCAUUUC GUAUCCUUUUCCAUUUC GUAUCCUUAAAACCAUUUC
|||||||| |||GUAUCCUU CCA /\
A A
Next Question: Is the “Linker” Region a Gap in Binding, or are there Interactions Beyond the Code?
GUAUCCUUAAAACCAUUUCGUAUCCUUUUCCAUUUC
GUAUCCUUGGCCAUUUCGUAUCCUUAACCAUUUC
Unbound RNA
Bound RNA
“Linker” Region Sequence Affects Binding Affinity
WT
Where can code mismatches be tolerated along the PPR10/RNA interface?• As mismatches move toward the center, the
loss of binding affinity decreases, OR• The cost of a mismatch could be affected by
how many stable interactions are surrounding it.
Is the “linker” region a gap in binding, or are there interactions beyond the Code? • The “linker” region of RNA must be interacting
with PPR10 in a way that does not use the 1 nt/1 repeat binding motif.
Conclusions
• Attempt to crystallize PPR10/RNA complex to visualize how the protein interacts with RNA.
• Investigate the affect of RNA point mutations on the 3’ side of the “linker” region
• Incorporate mismatch position data into prediction of native binding sites of the hundreds of unstudied PPR proteins.
Future Direction
Margarita RojasDr. Alice Barkan
NICHD Summer Research Program NIH-1R25HD070817and everyone in the Barkan Lab
Acknowledgements