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