Cameron Mackereth Affinity based definition of RNA motifs and strategic protein-RNA mutational design Cameron Mackereth Institut Européen de Chimie et Biologie / Inserm U1212 Bordeaux, France

Post-transcriptional maturation of mRNA - Splicing gene exon exon exon exon exon exon exon exon exon exon exon Proteins affect splicing patterns by recognizing RNA sequence elements within the pre-mRNA

Model system: Muscle-specific alternative splicing of the egl-15 gene from C. elegans Regulated by splicing protein SUP-12, together with one of ASD-1 or FOX-1 Molecular investigation mainly by NMR spectroscopy and isothermal titration calorimetry (ITC) In vivo assays by fluorescent reporters in live worms

egl-15 model system : pre-mRNA X mutations Mutually exclusive exons -> final egl-15 mRNA contains either exon 5B or 5A In the general case the first exon (5B) is used In muscle cells exon 5B is skipped and exon 5A is used The suppression of the first splice site acceptor is mediated by binding of the splicing factors ASD-1/FOX-1 and SUP-12

egl-15 model system : pre-mRNA mutations C. elegans EGL-15(B) other tissues EGL-15(A) muscle tissue EGL-15(B) other tissues EGL-15(A) muscle tissue

Fibroblast growth factor receptor EGL-15 (5A) binds EGL-17 EGL-17 muscle 5A EGL-15 is a fibroblast growth factor receptor Isoform 5B is critical for general C. elegans development (binds to LET-756) In muscle cells an alternate isoform 5A is produced EGL-15(5A) binds to the EGL-17 growth factor and causes migration of specific cells (sex myoblasts) LET-756 Fibroblast growth factor receptor 5B constitutive EGL-15 (5B) binds LET-756 Birnbaum et al, 2005, Dev Dyn 232:247

egl-15 model system : pre-mRNA X mutations Mutually exclusive exons -> final egl-15 mRNA contains either exon 5B or 5A In the general case the first exon (5B) is used In muscle cells exon 5B is skipped and exon 5A is used The suppression of the first splice site acceptor is mediated by binding of the splicing factors ASD-1/FOX-1 and SUP-12

egl-15 regulated by splicing factor SUP-12 SUP-12 protein from C. elegans SUP-12 (A) muscle-specific expression SUP-12 (B) RNA Recognition Motif (RRM) domain C.e. SUP-12 H.s. RBM24 H.s. RBM38

Structure of SUP-12 bound to GGUGUGC RNA

Using ITC to identify protein residues required to bind RNA

Isothermal Titration Calorimetry (ITC) SUP-12 binds equally well to RNA or DNA Isothermal Titration Calorimetry (ITC) KD 68 nM KD 290 nM KD 130 nM RNA GGUGUGC DNA GGUGUGC DNA GGTGTGC

SUP-12 mutants K36 A110 G113 E63 N108 N97 G40 N106 R76 Y78 H45 K104 RNA: GGUGUGC DNA: GGUGUGC DNA: GGTGTGC E63 N108 N97 G40 N106 R76 Y78 K104 H45 SUP-12 K74 Y44 R103 Affinity by isothermal titration calorimetry (ITC) RNA

affinity-weighted RNA motif Using ITC to create affinity-weighted RNA motif

SUP-12 motif by ITC affinity measurements - measure the relative affinity for 21 additional oligonucleotides in which each of the seven positions were changed to the other three bases

1 2 3 4 5 6 7 Nucleotide position Sequence KD /nM KA Relative affinity GGUGUGC 249  30 4.0 x 106 0.39 AGUGUGC 488 ± 24 2.0 x 106 0.20 UGUGUGC 427 ± 20 2.3 x 106 0.22 CGUGUGC 532 ± 28 1.9 x 106 0.19 2 0.52 GAUGUGC 2053 ± 239 4.9 x 105 0.06 GCUGUGC 2045 ± 175 GUUGUGC 368 ± 31 2.7 x 106 0.35 3 0.46 GGAGUGC 417 ± 31 2.4 x 106 0.28 GGCGUGC 787 ± 39 1.3 x 106 0.15 AGGGUGC 1068 ± 60 9.4 x 105 0.11 4 0.68 GGUAUGC 2688 ± 154 3.7 x 105 GGUCUGC 1468 ± 117 6.8 x 105 0.12 GGUUUGC 1178 ± 497 8.5 x 105 0.14 5 0.70 GGUGAGC 3861 ± 471 2.6 x 105 0.05 GGUGCGC 980 ± 99 1.0 x 106 0.18 GGUGGGC 2347 ± 201 4.3 x 105 0.07 6 0.76 GGUGUAC 4405 ± 456 2.3 x 105 0.04 GGUGUCC 2183 ± 106 4.6 x 105 0.09 GGUGUUC 1678 ± 106 6.0 x 105 7 0.24 GGUGUGA 410 ± 20 GGUGUGG 300 ± 21 3.3 x 106 GGUGUGU 141 ± 29 7.1 x 106 0.42

SUP-12 motif by ITC affinity measurements - measure the relative affinity for 21 additional oligonucleotides in which each of the seven positions were changed to the other three bases Position 3 U -> A 2-fold less binding Position 6 U -> A >20-fold less binding

Using ITC to confirm key contacts between an amino acid and RNA base

Testing key contacts Asn97 Asn106 Gua4 WT 2 3 4 5

Testing key contacts Asn97 Asn106 Gua4 WT N106A CH3 X Ala106 2 CH3 3 X Ala106 4 5 ~ 4 kJ/mol (one H-bond)

Testing key contacts Asn97 Asn106 Gua4 WT N106A CH3 X ~ 4 kJ/mol 2 CH3 3 X 4 5 ~ 4 kJ/mol (one H-bond)

Testing key contacts Asn97 Asn106 Gua4 WT N106A CH3 CH3 X Ala106 2 CH3 CH3 3 X Ala106 4 ΔΔG = 0 5

Testing key contacts Asn97 Asn106 Gua4 WT N106A CH3 CH3 X Ala106 2 CH3 CH3 3 X Ala106 4 ΔΔG = 0 5

Testing ITC results in live organisms

From: H. Kuroyanagi, Nature Methods, 2006 11:909-915 Modulation of egl-15 alternative splicing From: H. Kuroyanagi, Nature Methods, 2006 11:909-915 GFP RFP -GGTGTGC- (190 nM) -GGAGTGC- (420 nM) -GGTGCGC- (980 nM) -GGTGTAC- (4400 nM) Muscle cell expression WT - exon 5A Mutant - exon 5B

in vivo egl-15 alternative splicing Large object flow cytometer Scan thousands of worms Sort by size Convert data to a single line

WT egl-15 splicing in muscle cells -GGUGUGC- 290 nM -GGAGUGC- 420 nM Strain 1 Strain 2 head tail -GGUGCGC- 980 nM -GGUGUAC- 4400 nM RFP GFP

Slight but variable loss of exon 5B skipping -GGUGUGC- 190 nM -GGAGUGC- 420 nM Strain 1 Strain 2 Strain 1 Strain 2 head tail -GGUGCGC- 980 nM -GGUGUAC- 4400 nM RFP GFP

Moderate reduction in exon 5B skipping -GGUGUGC- 190 nM -GGAGUGC- 420 nM Strain 1 Strain 2 Strain 1 Strain 2 head tail -GGUGCGC- 980 nM -GGUGUAC- 4400 nM RFP GFP Strain 1 Strain 2

Strong loss of exon 5B skipping -GGUGUGC- 190 nM -GGAGUGC- 420 nM Strain 1 Strain 2 Strain 1 Strain 2 head tail -GGUGCGC- 980 nM -GGUGUAC- 4400 nM Strain 1 Strain 2 Strain 1 Strain 2

Using ITC to probe binding cooperativity

Interaction between SUP-12 and ASD-1 X ASD-1 (Fox-1) Investigate the interplay between ASD-1 and SUP-12 RRM domains bound to RNA U G C A U G G (Auweter et al. 2006) G G U G U G C SUP-12

Interaction between SUP-12 and ASD-1 X Investigate the interplay between ASD-1 and SUP-12 RRM domains bound to RNA

SUP-12 and ASD-1 bind independently + + GGUGUGC Isothermal titration calorimetry (ITC) UGCAUGGUGUGC KD 68 nM KD 69 nM No apparent binding co-operativity between SUP-12 and ASD-1 RRM domains

SUP-12 and ASD-1 bind independently GGUGUGC Binding affinity (ITC) SUP-12 ASD-1 UGCAUGGUGUGC

SUP-12 and ASD-1 bind independently GGUGUGC Mutant to disrupt RNA binding Binding affinity (ITC) SUP-12 ASD-1 UGCAUGGUGUGC

SUP-12 and ASD-1 bind independently GGUGUGC Disrupt protein interaction Binding affinity (ITC) SUP-12 ASD-1 UGCAUGGUGUGC

SUP-12 and ASD-1 bind independently Disrupt protein interaction UGCAUGG Binding affinity (ITC) ASD-1 SUP-12 UGCAUGGUGUGC Balance between favourable and unfavourable contacts

Acknowledgements Samir Amrane Denis Dupuy Karine Rebora Ilyass Zniber Axelle Grélard Estelle Morvan Hidehito Kuroyanagi (Tokyo Medical and Dental University)