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

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

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

Yeast Phenylalanine tRNA, 1.93A

rRNA small subunit, X. laevis

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

RNA Modifications tRNA Covalent Modifications-especially tRNA rT, r , rD –rU  rT, r , rD, rS 4 U –rC  3-CH 3 -C, 5-CH 3 -C –rA  I, 6-CH 3 -A, 6-isopentenyl-A –rG  7-CH 3 -G, Q, Y Nucleosides Nucleotides 1999 Jun-Jul;18(6-7):

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

RNA structure energetics 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.

2° Structure 5’ 3’ G--C C--G A | U--A G--C A A A

“The Rules” 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”

Base Pairs/Stacks A U A=U Basepair  G = -1.2 kcal/mole A U U A A=U U=A Basepair  G = -1.6 kcal/mole

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

Hairpin Loops (GC closure) Tertiary Interactions!

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

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

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

Squiggles CCA-3’OH

DOMES, MOUNTAIN, CIRCLES

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

ENERGY PLOT

P-Num Plot

Prediction Quality

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

RNA Structure Probing 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

Ribozymes 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

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