rna structure prediction rna structure basics the rna ‘rules’ programs and predictions bio520...

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