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RNA Structure Prediction

RNA Structure Prediction. RNA Structure Basics The RNA ‘Rules’ Programs and Predictions. Assigned reading: Ch. 6 from Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, 3rd Ed. by Baxevanis and Ouellette. BIO520 Bioinformatics Jim Lund. mRNA - messenger RNA.

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RNA Structure Prediction

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  1. RNA Structure Prediction RNA Structure Basics The RNA ‘Rules’ Programs and Predictions Assigned reading: Ch. 6 from Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, 3rd Ed. by Baxevanis and Ouellette. BIO520 Bioinformatics Jim Lund

  2. mRNA - messenger RNA. tRNA - transfer RNA, small (~80 bases) sequences which bring amino acids to the ribosome. rRNA - ribosomal RNA, RNA + proteins = ribosome. viral RNA (ssRNA, dsRNA virii) miRNA: translational/transcriptional gene silencing. snoRNA, snRNA: splicing, RNA bp modification Transfer-Messenger RNAs (tmRNA), Small cytosolic RNAs (scRNA), Guide RNAs (gRNA) and more… RNA classes

  3. 1° Sequence (and modifications) 2° Base pairing 3° Overall Structure, non Watson-Crick pairs Experimental structures: tRNA, ribosome RNA structures

  4. RNA Tertiary Structure, tRNA 3’(aminoacyl) end CCA Anticodon Loop

  5. Yeast Phenylalanine tRNA, 1.93A

  6. Yeast Phenylalanine tRNA, 1.93A

  7. rRNA small subunit, X. laevis

  8. Watson-Crick pairing -> helices Loop regions Hairpin loops Internal loops Bulge loops Multibranch loops 2° RNA structures

  9. Covalent Modifications-especially tRNA rUrT, r, rD, rS4U rC 3-CH3-C, 5-CH3-C rA I, 6-CH3-A, 6-isopentenyl-A rG  7-CH3-G, Q, Y RNA Modifications Pairing Changes Critical to Wobble Anticodons Nucleosides Nucleotides 1999 Jun-Jul;18(6-7):1579-81

  10. G-C triple hydrogen bond A -U double hydrogen bond G-U single hydrogen bond RNA Base pairing

  11. The number of GC versus AU and GU base pairs. Higher energy bonds form more stable structures. Number of base pairs in a stem region. Longer stems result in more bonds. Number of base pairs in a hairpin loop region. Formation of loops with more than 10 or less than 5 bases requires more energy. Number of unpaired bases (interior loops or bulges). Unpaired bases decrease the stability of the structure. RNA structure energetics

  12. 2° Structure 5’3’ G--C G--C C--G A | U--A G--C A A A A A A Stem Bulge Hairpin Loop

  13. Base Pairs -- Good G:C better than A:T -- And local sequence matters! Bulges, Loops -- Bad Many small interactions---Stable Structure Only predict “Canonical Interactions” “The Rules”

  14. A U A U A=U A=U Basepair Basepair G = -1.2 kcal/mole A U U A A=U U=A G = -1.6 kcal/mole Base Pairs/Stacks Why 1.2 vs 1.6?

  15. Base Pairing/Stacking Bloomfield, Crothers, Tinoco, Physical Chemistry of Nucleic Acids

  16. Hairpin Loops(GC closure) • Tertiary Interactions!

  17. Internal Loops 5’3’ G--C G--C C--G A G G A A C T--A G--C T--A G--C

  18. Single-Strand Bulges 5’3’ G--C G--C C--G A | G | A | T--A G--C T--A G--C

  19. Mfold (M. Zuker) 2° structure RNAstructure/OligoWalk 2° structure, oligo/RNA target interactions alifold 2° structure constrained by muliple alignment. Pfold 2° structure guided by rules derived from known tRNA/rRNA structures Prediction Programs

  20. Mfold (GCG) M. Zuker Mfold input to Plotfold Non-graphic output -G option Graphics outputs SQUIGGLES mountains circles domes energy plots Prediction Programs Looks GREAT

  21. Squiggles CCA-3’OH 1 60 20 40

  22. DOMES, MOUNTAIN, CIRCLES

  23. Optimal & Suboptimal structures Can ask for multiple structures Energy increment and “window size” increment. View individually. How variable are the structures? Energy Plots MFOLDStructure Family

  24. ENERGY PLOT Variability in Pairing Optimal&Suboptimal

  25. P-Num Plot Most Variable

  26. Prediction Quality

  27. Complementary molecular surfaces Bridging cations Pseudoknotting “kinetic traps” in folding NOT always 2 first! Forces in RNA folds Annu Rev Biophys Biomol Struct 1999;28:57-73 Proc Natl Acad Sci U S A 1998 Sep 29;95(20):11555-60

  28. Physical methods X-ray diffraction, NMR Enzymatic methods S1, Rnases (find ss and ds regions). Chemical modification DMS… Mutagenesis G:C=>C:C=>C:G RNA Structure Probing

  29. Naturally occurring RNAaseP Group I introns Group II introns snRNA in the splicosome Artifical Engineered/evolved in the lab from natural ribozymes to have new substrate RNA. Cleave mRNA, drug-like action miRNA/siRNA Translational/transcriptional gene silencing Ribozymes

  30. Cross-replicating RNA enzymes Published by AAAS T. A. Lincoln et al., Science 323, 1229 -1232 (2009)

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