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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