11/2/05

RNA Structure Prediction

11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction 1

 Announcements
Seminar
12:10 PM Fri BCB Faculty Seminar in E164 Lago
How to do sequence alignments on parallel computers
Srinivas Aluru, ECprE & Chair, BCB Program
http://www.bcb.iastate.edu/courses/BCB691F2005.html

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 Announcements
BCB 544 Projects - Important Dates:
Nov 2 Wed noon - Project proposals due to David/Drena

Nov 4 Fri 10A - Approvals/responses to students

Dec 2 Fri noon - Written project reports due

Dec 5,7,8,9 class/lab - Oral Presentations (20')

(Dec 15 Thurs = Final Exam)

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 RNA Structure & Function
Prediction
Mon Review - promoter prediction
RNA structure & function

Wed RNA structure prediction

2' & 3' structure prediction
miRNA & target prediction - perhaps..
RNA function prediction?
Won't have time to cover this…

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 Reading Assignment (for Mon/Wed)

Mount Bioinformatics
• Chp 8 Prediction of RNA Secondary Structure
• pp. 327-355
• Ck Errata: http://www.bioinformaticsonline.org/help/errata2.html

Cates (Online) RNA Secondary Structure Prediction Module

• http://cnx.rice.edu/content/m11065/latest/

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 Review last lecture:

RNA Structure & Function

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 RNA Structure & Function
• RNA structure
• Levels of organization
• Energetics (more about this on Wed)

• RNA types & functions

• Genomic information storage/transfer
• Structural
• Catalytic
• Regulatory

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 Covalent & non-covalent bonds in RNA

Primary:
Covalent bonds
Secondary/Tertiary
Non-covalent bonds
• H-bonds
(base-pairing)
• Base stacking

Fig 6.2
Baxevanis &
Ouellette 2005
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 Base-pairing in RNA
1) G-C, A-U, G-U ("wobble") & variants
U can form base-pairs with both A & G
2) Nucleotides in RNA are frequently modified
this is not very common in DNA
These features & flexible "single-stranded" RNA
backbone allow for many potential base-pairs

Modified bases are especially important) in tRNA:

e.g., pseudo-Uridine, rD, 5-CH3-C6-isopentenyl-A
7-CH3-G, many others…

See: IMB Image Library of Biological Molecules

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 Common structural motifs in RNA

Helices

Loops
• Hairpin
• Internal
• Bulge
• Multibranch

Pseudoknots

Fig 6.2
Baxevanis &
Ouellette 2005
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 RNA functions

Storage/transfer of genetic information

• Genomes
• many viruses have RNA genomes
single-stranded (ssRNA)
e.g., retroviruses (HIV)
double-stranded (dsRNA)

• Transfer of genetic information

• mRNA = "coding RNA" - encodes proteins

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 RNA functions
Structural
• e.g., rRNA, which is major structural component of
ribosomes (Gloria Culver, ISU)
BUT - its role is not just structural, also:

Catalytic
RNA in ribosome has peptidyltransferase activity
• Enzymatic activity responsible for peptide
bond formation between amino acids in growing
peptide chain
• Also, many small RNAs are enzymes
"ribozymes"
(W Allen Miller, ISU)

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 RNA functions
Regulatory
Recently discovered important new roles for RNAs
In normal cells:
• in "defense" - esp. in plants
• in normal development
e.g., siRNAs, miRNA

As tools:
• for gene therapy or to modify gene expression

• RNAi (used by many at ISU: Diane Bassham,

Thomas Baum, Jeff Essner, Kristen Johansen,
Jo Anne Powell-Coffman, Roger Wise, etc.)
• RNA aptamers (Marit Nilsen-Hamilton, ISU)

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 RNA types & functions
Types of RNAs Primary Function(s)
mRNA - messenger translation (protein synthesis)
regulatory

rRNA - ribosomal translation (protein synthesis) <catalytic>

t-RNA - transfer translation (protein synthesis)

hnRNA - heterogeneous nuclear precursors & intermediates of mature

mRNAs & other RNAs
scRNA - small cytoplasmic signal recognition particle (SRP)
tRNA processing <catalytic>

snRNA - small nuclear mRNA processing, poly A addition <catalytic>

snoRNA - small nucleolar rRNA processing/maturation/methylation

regulatory RNAs (siRNA, regulation of transcription and translation,

miRNA, etc.) other??

L Samaraweera 2005
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 Thanks to Chris Burge, MIT
for following slides
Slightly modified from:
Gene Regulation and MicroRNAs
Session introduction presented at
ISMB 2005, Detroit, MI

Chris Burge cburge@MIT.EDU

C Burge 2005 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction 15
 Expression of a Typical Eukaryotic Gene
Protein Coding Gene

…
DNA
Transcription Polyadenylation
exon intron

primary transcript / pre-mRNA

Splicing
For each of these
processes, there is
a ‘code’
AAAAAAAAA
mRNA Export (set of default
Translation Degradation recognition rules)

Protein
Folding, Modification,
Transport, Complex
Assembly
Protein Complex
Degradation

C Burge 2005 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction 16
 Gene Expression Challenges for
Computational Biology

• Understand the ‘code’ for each step in gene expression

(set of default recognition rules), e.g., the ‘splicing code’

• Understand the rules for sequence-specific recognition of

nucleic acids by protein and ribonucleoprotein (RNP) factors

• Understand the regulatory events that occur at each step and

the biological consequences of regulation

Lots of data
Genomes, structures, transcripts, microarrays, ChIP-Chip, etc.

C Burge 2005 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction 17
 Sequence-specific Transcription Factors

• have modular organization

» Understand DNA-binding specificity

Yan (ISU) A computational method to identify amino acid

residues involved in protein-DNA interactions

ATF-2/c-Jun/IRF-3 DNA complex

Panne et al. EMBO J. 2004

C Burge 2005 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction 18
 Early Steps in Pre-mRNA Splicing

• Formation of exon-spanning complex

hnRNP proteins
• Subsequent rearrangement to form
intron-spanning spliceosomes which
catalyze intron excision and exon ligation

Matlin, Clark & Smith Nature Mol Cell Biol 2005

C Burge 2005 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction 19
 Alternative Splicing

> 50% of human genes

undergo alternative splicing

Matlin, Clark & Smith Nature Mol Cell Biol 2005

Wang (ISU) Genome-wide Comparative Analysis of Alternative

Splicing in Plants

C Burge 2005 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction 20
 Splicing Regulation

ESE/ESS = Exonic Splicing Enhancers/Silencers

ISE/ISS = Intronic Splicing Enhancers/Silencers

Matlin, Clark & Smith Nature Mol Cell Biol 2005

C Burge 2005 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction 21
 C. elegans lin-4 Small Regulatory RNA

lin-4 precursor

lin-4 RNA
target mRNA

lin-4 RNA
V. Ambros lab “Translational
repression”

We now know that there are hundreds of microRNA genes

(Ambros, Bartel, Carrington, Ruvkun, Tuschl, others)

C Burge 2005 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction 22
 MicroRNA Biogenesis

N. Kim Nature Rev Mol Cell Biol 2005

C Burge 2005 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction 23
 miRNA and RNAi pathways
microRNA pathway RNAi pathway
MicroRNA primary transcript Exogenous dsRNA, transposon, etc.

Drosha

precursor Dicer Dicer

siRNAs
miRNA
target mRNA
RISC
RISC RISC

“translational repression”
and/or mRNA degradation
mRNA cleavage, degradation
C Burge 2005 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction 24
 miRNA Challenges for Computational Biology
• Find the genes encoding microRNAs
• Predict their regulatory targets
Computational Prediction of MicroRNA Genes & Targets

• Integrate miRNAs into gene regulatory pathways &

networks
Need to modify traditional paradigm of
"transcriptional control" primarily by protein-DNA
interactions to include miRNA regulatory mechanisms!

C Burge 2005 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction 25
 New Today:

RNA Structure Prediction

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 RNA structure prediction strategies
Secondary structure prediction

1) Energy minimization
(thermodynamics)

2) Comparative sequence analysis

(co-variation)

3) Combined experimental & computational

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 Secondary structure prediction strategies

1) Energy minimization (thermodynamics)

• Algorithm:
Dynamic programming to find
high probability pairs
(also, some Genetic algorithms)
• Software:
Mfold - Zuker
Vienna RNA Package - Hofacker
RNAstructure - Mathews
Sfold - Ding & Lawrence

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 Secondary structure prediction strategies

2) Comparative sequence analysis (co-variation)

• Algorithm:
Mutual information
Context-free grammars
• Software:
ConStruct
Alifold
Pfold
FOLDALIGN
Dynalign

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 Secondary structure prediction strategies

3) Combined experimental & computational

• Experiment:
Map single-stranded vs double-stranded
regions in folded RNA
• How?
Enzymes: S1 nuclease, T1 RNase
Chemicals: kethoxal, DMS

R Knight 2005 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction 30
Experimental RNA structure determination?

• X-ray crystallography

• NMR spectroscopy

• Enzymatic/chemical mapping

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 1) Energy minimization method

What are the assumptions?

Native tertiary structure or "fold" of an RNA
molecule is (one of) its "lowest" free energy
configuration(s)
Gibbs free energy = G in kcal/mol at 37C
= equilibrium stability of structure
lower values (negative) are more favorable
Is this assumption valid?
in vivo? - this may not hold, but we don't really know

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 Free energy minimization
What are the rules?

A U Basepair A=U
A U A=U What gives here?
G = -1.2 kcal/mole

A U Basepair
A=U
U A U=A
G = -1.6 kcal/mole

C Staben 2005 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction 33
 Energy minimization calculations:
Base-stacking is critical
AA -1.2 CG -3.0
UU GC

AU o r UA -1.6 GC -4.3
UA AU CG

AG, AC, CA, GA -2.1 GU -0.3

UC, UG, GU, CU UG

CC -4.8 XG, GX 0
GG YU, UY

- Tinocco et al.
C Staben 2005 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction 34
 Nearest-neighbor parameters
Most methods for free energy minimization
use nearest-neighbor parameters (derived from
experiment) for predicting stability of an RNA
secondary structure (in terms of G at 37C)

& most available software packages use

the same set of parameters:
Mathews, Sabina, Zuker & Turner, 1999

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 Energy minimization - calculations:
Total free energy of a specific
conformation for a specific RNA
molecule = sum of incremental
energy terms for:
• helical stacking
(sequence dependent)
• loop initiation
• unpaired stacking

(favorable "increments" are < 0)

Fig 6.3
Baxevanis &
Ouellette 2005
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 But how many possible conformations for a
single RNA molecule?
Huge number:
Zuker estimates (1.8)N possible secondary
structures for a sequence of N nucleotides
for 100 nts (small RNA…) =
3 X 1025 structures!
Solution? Not exhaustive enumeration…
 Dynamic programming
O(N3) in time
O(N2) in space/storage
iff pseudoknots excluded, otherwise:
O(N6 ), time
O(N4 ), space

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 2) Comparative sequence analysis
(co-variation)

Two basic approaches:

• Algorithms constrained by initial alignment
Much faster, but not as robust as unconstrained
Base-pairing probabilities determined by a
partition function
• Algorithms not constrained by initial alignment
Genetic algorithms often used for finding an
alignment & set of structures

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 RNA Secondary structure prediction:
Performance?
How evaluate?
• Not many experimentally determined structures
currently, ~ 50% are rRNA structures
so "Gold Standard" (in absence of tertiary structure):
compare with predicted RNA secondary
structure with that determined by comparative
sequence analysis (!!??) using Benchmark Datasets
NOTE: Base-pairs predicted by comparative sequence
analysis for large & small subunit rRNAs are 97% accurate
when compared with high resolution crystal structures!
- Gutell, Pace

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 RNA Secondary structure prediction:
Performance?
1) Energy minimization (via dynamic programming)
73% avg. prediction accuracy - single sequence
2) Comparative sequence analysis
97% avg. prediction accuracy - multiple sequences
(e.g., highly conserved rRNAs)
much lower if sequence conservation is lower &/or
fewer sequences are available for alignment
3) Combined - recent developments:
combine thermodynamics & co-variation
& experimental constraints? IMPROVED RESULTS

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 RNA structure prediction strategies
Tertiary structure prediction
Requires "craft" & significant user input & insight
1) Extensive comparative sequence analysis to predict
tertiary contacts (co-variation)
e.g., MANIP - Westhof
2) Use experimental data to constrain model building
e.g., MC-CYM - Major
3) Homology modeling using sequence alignment &
reference tertiary structure (not many of these!)
4) Low resolution molecular mechanics
e.g., yammp - Harvey

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