RNA bioinformatics folding/interaction, stability/dynamics, thermodynamics/kinetics, … GT MASIM Fabrice Leclerc fabrice.leclerc@u-psud.fr November 16-17 2017 1
I2BC - Dept. M2GB Genomics, Molecular Genetics, Bioinformatics Team: RNA Sequence, Structure & function - D. Gautheret • RNA discovery: genomics, transcriptomics (cancer) - D. Gautheret • RNA structure & interactions: snoRNAs, ribozymes-viroids, RNA-RNA interactions, RNA-protein interactions - F. Leclerc • RNA function & processing: genetic code, RNA-based regulations - J. Lehmann • 4 senior researchers (C. Toffano: vice-director of the eBio platform), 2 Ph.D. students, 2 CDI IE, (1 invited professor)
Some recurrent problems & issues • 2D&3D modeling of RNA variants/mutants from 3D structures • sparse structural data • experimental 3D structures & biological-physiological conditions • 2D&3D modeling of RNA-RNA interactions • dense RNA-RNA interactome • predict RNA interactions; 2D to 3D transposition • 3D modeling of RNA/protein interactions • RNA binding ? - binding interface - binding mode - binding specificity (modeling/design) 3
Structural/biochemical data inconsistencies 41-nt 50-nt 56-nt 50-nt Wang et al. , Biochem., 1999 Khvorova et al. , Nat. Struct. Biol., 2003 Canny et al. , JACS, 2004 de la Peña et al. , EMBO J., 2003 4 4
2D&3D structures of H/ACA box s(no)RNAs (guide) A 30 Kloop C U G A U A G G A G A G C guide G C C G C G 20 U G 40 C G U C C G U G C G U A G U G A C 5' guide sequence 3' guide sequence C G G C G C G C G C 5’ A C Internal Loop G U C G target 10 G C 3’ G C 50 rRNA (tRNA) C G C G C G G C ANA Loop G C 5' 3' G U A C A 5
Structure/function relationships in H/ACA box s(no)RNAs 11 guide motifs (7 genes) 23 potential targets Pab_HACA ARN guide/ARN cible Pab/Pfu/Afu (PDB) 3-20 nt 17-18 nt K-turn/K-loop R 3-5 nt U A A G U G A A G guide L7Ae G C 0-6 nt G A ANA 5' U C C U C U G U GA stem 8-11 bp G C G G G G G G C 5' G C 10 bp Y R C U 0-3 nt C G Nop10 0-1 nt target C G 0-1 nt 0-1 nt LSU 15 productive guide-target U base pair annotations covarying mutations compatible mutations Gar1 no mutations observed 6 nt 5-6 nt 5-27 nt 5-8 nt nucleotide nucleotide 5´ guide sequence 3´ guide sequence present identity A 97% 75% 97% 0-1 nt N Cbf5 90% 50% 90% N 75% N 75% 5´ connector (zero length) connector (zero length) C G 9 bp lower stem Y R 7-9 bp variable-length region Y R C G variable-length loop G C G C variable-length stem G CA C A modular sub-structure 5´ 5´ A C A D A Toffano-Nioche et al., 2013; Toffano-Nioche et al., 2015 6 6
2D(3D) structural&energetic rules to classify “productive” guide RNAs 1. motif 10/5-6/9 tested rRNA H/ACA(-like) guide:targets 2. guide-target stability candidates targets 3. duplex 5’ stability 3-21 nt 7-9,11/j/k 10 10/5-6/9 R non-productive U guide fold G U A G productive fold 13 2 2 6 ANA 5´ A G C G A G G G C G A G A 5' U C G C U C C C G U U A U refolding 15 2 6 10 bp 5 guide:target target LSU energetics Y R 2588 unstable hybrid or 5' C G E = -30.2 kcal/mol E hybrid ≤ -27 0-1 nt duplex G & E 5’ = -6.2 kcal/mol 0-1 nt 0-1 nt E 5'duplex ≤ -6.0 9 R Y 5-6 nt stable productive duplex 15 8 5-23 nt guide G U 0-1 nt guide:target ANA 5' C C U C U G base-pairing G G A G A C 5' C G mispaired 9 bp C U RNA:RNA duplex C G target C G productive H/ACA E = -29.5 kcal/mol 15 8 guide:target E 5’ = -9.5 kcal/mol YA C A 5´ non-productive H/ACA guide:target Toffano-Nioche et al., 2015 7
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