RNA Structure Prediction

RNA Structure Basics

The RNA ‘Rules’

Programs and Predictions

BIO520 Bioinformatics Jim 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) endCCA

Yeast Phenylalanine tRNA, 1.93A

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

Covalent Modifications-especially tRNAtRNA– rUrT, rrT, r, rD, rD, rS4U

– rC 3-CH3-C, 5-CH3-C

– rA I, 6-CH3-A, 6-isopentenyl-A

– rG 7-CH3-G, Q, Y

Nucleosides Nucleotides 1999 Jun-Jul;18(6-7):1579-81

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 G--C C--GA | U--A G--CA AA 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 UA U

A=UA=U

Basepair

G = -1.2 kcal/mole

A UU A

A=UU=A

Basepair

G = -1.6 kcal/mole

Base Pairing/Stacking

AAUU

-1.2 CGGC

-3.0

AU or UAUA AU

-1.6 GCCG

-4.3

AG, AC, CA, GAUC, UG, GU, CU

-2.1 GUUG

-0.3

CCGG

-4.8 XG, GXYU, UY

0

Bloomfield, Crothers, Tinoco, Physical Chemistry of Nucleic Acids

Hairpin Loops(GC closure)

N=3 +8

N=4,5 +5

N=6,7 +4

N=8,9 +5

N>=10 6+0.9(ln[N/10])

•Tertiary Interactions!

Internal Loops

G-X-CC-X-G

0

N=2-6 +2

N=7 +3

N>=8 3+0.9(ln[N/7])

5’ 3’ G--C G--C C--G A GG A A C T--A G--C T--A G--C

Single-Strand Bulges

5’ 3’ G--C G--C C--G A |G | A | T--A G--C T--A G--C

N=1 +3

N=2-3 +4

N=4-7 +5

N>=8 6+0.9ln(N/8)

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

1

2040

60

CCA-3’OH

DOMES, MOUNTAIN, CIRCLES

MFOLDStructure 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;28:57-73Proc Natl Acad Sci U S A 1998 Sep 29;95(20):11555-60

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, 1229 -1232 (2009)

Cross-replicating RNA enzymes