Kyle Gribbin University of Oregon Mentor: Margarita Rojas PI: 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) Organelle Targeting 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 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 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? Gene PPR Exposed Sequence Sequestered Sequence PPR Binding site How PPR Proteins Affect Gene Expression Our lab has discovered a code by which PPR repeats bind nucleotides.

||||||||||||| GUAUCCUUAAC CA 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 ||||||||||||| GUAUCCUUAAC CA 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 “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 G A AUCCUUAACCAUUUC 3 GU U UCCUUAACCAUUUC 4 GUA A CCUUAACCAUUUC 5 GUAU G CUUAACCAUUUC 6 GUAUC G UUAACCAUUUC 7 GUAUCC A UAACCAUUUC How will changing these nucleotides affect PPR10s affinity for the sequence? Where Along a PPR/RNA Duplex can Code Mismatches be Tolerated?

32 P RNA PPR10 Unbound RNA Bound RNA Protein concentration Gel Mobility Shift Assay: Method for Determining PPR10/RNA Affinity

G A AUCCUUAACCAUUUC GUUUCCUUAACCAUUUC 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 GUAUGCUUAACCAUUUC GUAUCGUUAACCAUUUC GUAUCCAUAACCAUUUC GUAUCCUUAACCAUUUC Unbound RNA Bound RNA WT

||||||||||||| GUAUCCUUAAC CA Wt Fraction RNA Bound 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 Next Question: Is the “Linker” Region a Gap in Binding, or are there Interactions Beyond the Code?

GUAUCCUUAAAACCAUUUC GUAUCCUUUUCCAUUUC GUAUCCUUGGCCAUUUC GUAUCCUUAACCAUUUC 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 Rojas Dr. Alice Barkan NICHD Summer Research Program NIH- 1R25HD and everyone in the Barkan Lab Acknowledgements