Download Cracking the PPR code: predicting and manipulating protein/RNA

Kyle Gribbin
University of Oregon
Mentor: Margarita Rojas
PI: Alice Barkan
RNA Binding Proteins Regulate Gene
Expression
•
All genes have RNA intermediates on their way to being
expressed.
U2af65
Argonaute
How do these proteins know where to bind RNA?
Proteins bind Specific RNAs with RNA
Binding Domains
Typical RNA binding domains:
KH
PAZ
•
•
Small area of contact with RNA
Idiosyncratic mechanisms of nucleotide
recognition
Repeat Proteins: A Novel Nucleic Acid
Recognition Mechanism
Puf
domain
TAL domain
•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
Pentatricopeptide Repeat (PPR)
Organelle
Targeting Sequence
35 amino acid repeats
(~4-30 RPTS)
•Binds single stranded RNA
•Repeats have different amino acid
sequences, allowing them to bind
different nucleotides
PPRs: eucaryotespecific, RNA binding
module
?
Small and Peeters, TIBS 2000
PPR Proteins Affect Organellar Gene
Expression
Mito and Chloroplast
RNA splicing, RNA editing, RNA
stabilization, RNA cleavage, translational
activation and repression.
Act on SPECIFIC organellar RNAs.
Plant nuclear genomes
encode ~450 PPR proteins
How PPR Proteins Affect Gene Expression
•
Bound PPR proteins
◦ Stabilize RNA
◦ Regulate Splicing
◦ Regulate Translation
PPR
Binding
site
PPR
PPR
Exposed
Sequestered
Sequence
Sequence
PP
R
How do they know where to bind on the RNA?
Our lab has discovered a code by which
PPR repeats bind nucleotides.
Gene
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.
|||||||||||||
GUAUCCUUAACCA
Long Term Goal
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
PPR10’s 3 Native Binding Sites Suggests
Interruption of a Contiguous RNA/Protein
Interface
“Linker” region
Nucleotides outside of the box do not match with the code.
This suggests RNA loops
out from the protein:
|||||||| |||
GUAUCCUU/\CCA…
A A
Loop out
Questions I Addressed
•
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?
Where Along a PPR/RNA Duplex can Code
Mismatches be Tolerated?
Wild type RNA
2
3
4
5
6
7
5’ GUAUCCUUAACCAUUUC 3’
GAAUCCUUAACCAUUUC
GUUUCCUUAACCAUUUC
GUAACCUUAACCAUUUC
GUAUGCUUAACCAUUUC
GUAUCGUUAACCAUUUC
GUAUCCAUAACCAUUUC
How will changing these nucleotides
affect PPR10s affinity for the sequence?
Gel Mobility Shift Assay: Method for
Determining PPR10/RNA Affinity
32P
RNA
PPR10
Protein
concentration
Bound RNA
Unbound RNA
Mutation at RNA Position 2,3, or 4
Cause Massive Loss of Binding Affinity
GUAACCUUAACCAUUUC
WT
GUUUCCUUAACCAUUUC
GAAUCCUUAACCAUUUC
GUAUCCUUAACCAUUUC
Bound RNA
Unbound RNA
Mutation at RNA position 5,6, or 7
Cause Small Loss of Binding Affinity
GUAUCCAUAACCAUUUC
GUAUCGUUAACCAUUUC
WT
GUAUGCUUAACCAUUUC
GUAUCCUUAACCAUUUC
Bound RNA
Unbound RNA
Loss of Binding Affinity Decreases as
Mismatches move Towards the Center of
PPR10/RNA Duplex
Fraction RNA Bound
Wt
7
6
5
3
4
2
PPR10 [nM]
|||||||||||||
5’GUAUCCUUAACCA 3’
234567
Next Question: Is the “Linker” Region a Gap
in Binding, or are there Interactions Beyond
the Code?
|||||||| |||
GUAUCCUU/\CCA
A A
Wild Type RNA 5’ GUAUCCUUAACCAUUUC 3’
GUAUCCUUGGCCAUUUC
GUAUCCUUUUCCAUUUC
GUAUCCUUAAAACCAUUUC
“Linker” Region Sequence Affects
Binding Affinity
WT
GUAUCCUUAAAACCAUUUC
GUAUCCUUGGCCAUUUC
GUAUCCUUUUCCAUUUC
GUAUCCUUAACCAUUUC
Bound RNA
Unbound RNA
Conclusions
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.
Future Direction
•
•
•
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.
Acknowledgements
Dr. Alice Barkan
Margarita Rojas
NICHD Summer Research Program NIH1R25HD070817
and everyone in the Barkan Lab