10/31/05

RNA Structure & Function

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 1

 Announcements
Seminar (Mon Oct 31)
12:10 PM IG Faculty Seminar in 101 Ind Ed II
Plant Steroid Hormone Signal Transduction
Yanhai Yin, GDCB

• BCB Link for Seminar Schedules (updated)

http://www.bcb.iastate.edu/seminars/index.html

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 2

 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)

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 3

 RNA Structure & Function
Prediction
Mon Review - promoter prediction
RNA structure & function
Wed RNA structure prediction
2' & 3' structure prediction
miRNA & target prediction
RNA function prediction?

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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/

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 5

 Review last lecture:

Promoter Prediction

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 6

 Promoter Prediction

• Overview of strategies
 What sequence signals can be used?
 What other types of information can be used?

• Algorithms  a bit more about these

in later lectures
• Promoter prediction software
• 3 major types
• many, many programs!

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Promoter prediction: Eukaryotes vs prokaryotes

Promoter prediction is easier in microbial genomes

Why? Highly conserved

Simpler gene structures
More sequenced genomes!
(for comparative approaches)

Methods? Previously, again mostly HMM-based

Now: similarity-based. comparative methods
because so many genomes available

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Promoter Prediction: Steps & Strategies

 Closely related to gene prediction!

• Obtain genomic sequence
• Use sequence-similarity based comparison
(BLAST, MSA) to find related genes
But: "regulatory" regions are much less well-
conserved than coding regions
• Locate ORFs
• Identify TSS (Transcription Start Site)
• Use promoter prediction programs
• Analyze motifs, etc. in sequence (TRANSFAC)

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 Promoter Prediction: Steps & Strategies
Identify TSS --if possible?
• One of biggest problems is determining exact TSS!
Not very many full-length cDNAs!
• Good starting point? (human & vertebrate genes)
Use FirstEF
found within UCSC Genome Browser
or submit to FirstEF web server

Fig 5.10
Baxevanis &
Ouellette 2005
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 Promoter prediction strategies
1) Pattern-driven algorithms

2) Sequence-driven algorithms

3) Combined "evidence-based"

BEST RESULTS? Combined, sequential

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 Promoter Prediction: Pattern-driven algorithms

• Success depends on availability of collections of

annotated binding sites (TRANSFAC & PROMO)
• Tend to produce huge numbers of FPs
• Why?
• Binding sites (BS) for specific TFs often variable
• Binding sites are short (typically 5-15 bp)
• Interactions between TFs (& other proteins) influence
affinity & specificity of TF binding
• One binding site often recognized by multiple BFs
• Biology is complex: promoters often specific to
organism/cell/stage/environmental condition

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Promoter Prediction: Pattern-driven algorithms

Solutions to problem of too many FP predictions?

• Take sequence context/biology into account
• Eukaryotes: clusters of TFBSs are common
• Prokaryotes: knowledge of  factors helps
• Probability of "real" binding site increases if
annotated transcription start site (TSS) nearby
• But: What about enhancers? (no TSS nearby!)
& Only a small fraction of TSSs have been
experimentally mapped
• Do the wet lab experiments!
• But: Promoter-bashing is tedious

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Promoter Prediction: Sequence-driven algorithms

• Assumption: common functionality can be deduced from

sequence conservation
• Alignments of co-regulated genes should highlight
elements involved in regulation
Careful: How determine co-regulation?
• Orthologous genes from difference species
• Genes experimentally determined to be
co-regulated (using microarrays??)
• Comparative promoter prediction:
"Phylogenetic footprinting" - more later….

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Promoter Prediction: Sequence-driven algorithms
Problems:
• Need sets of co-regulated genes
• For comparative (phylogenetic) methods
• Must choose appropriate species
• Different genomes evolve at different rates
• Classical alignment methods have trouble with
translocations, inversions in order of functional elements
• If background conservation of entire region is highly
conserved, comparison is useless
• Not enough data (Prokaryotes >>> Eukaryotes)
• Biology is complex: many (most?) regulatory elements
are not conserved across species!

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 Examples of promoter
prediction/characterization software

Lab: used MATCH, MatInspector

TRANSFAC
MEME & MAST
BLAST, etc.
Others?
FIRST EF
Dragon Promoter Finder (these are links in PPTs)
also see Dragon Genome Explorer (has specialized
promoter software for GC-rich DNA, finding CpG
islands, etc)
JASPAR

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 Global alignment of human & mouse obese
gene promoters (200 bp upstream from TSS)

Fig 5.14
Baxevanis &
Ouellette 2005
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 Check out optional review &
try associated tutorial:

Wasserman WW & Sandelin A (2004) Applied bioinformatics for

identification of regulatory elements. Nat Rev Genet 5:276-287
http://proxy.lib.iastate.edu:2103/nrg/journal/v5/n4/full/nrg1315_fs.html

Check this out: http://www.phylofoot.org/NRG_testcases/

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 Annotated lists of promoter databases &
promoter prediction software

• URLs from Mount Chp 9, available online

Table 9.12 http://www.bioinformaticsonline.org/links/ch_09_t_2.html
• Table in Wasserman & Sandelin Nat Rev Genet article
http://proxy.lib.iastate.edu:2103/nrg/journal/v5/n4/full/nrg1315_fs.htm

• URLs for Baxevanis & Ouellette, Chp 5:

http://www.wiley.com/legacy/products/subject/life/bioinformatics/ch05.htm#links

More lists:
• http://www.softberry.com/berry.phtml?topic=index&group=programs&subgroup=promo
ter
• http://bioinformatics.ubc.ca/resources/links_directory/?subcategory_id=104
• http://www3.oup.co.uk/nar/database/subcat/1/4/

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 New Today:

RNA Structure & Function

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

• RNA types & functions

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

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 RNA structure: 3 levels of organization

Rob Knight
Univ Colorado
10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 22
 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

G-C, A-U, G-U ("wobble") & variants

See: IMB Image Library of Biological Molecules
http://www.imbjena.de/ImgLibDoc/nana/IMAGE_NANA.html#sec_element

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 Common structural motifs in RNA

Helices

Loops
• Hairpin
• Interior
• Bulge
• Multibranch

Pseudoknots

Fig 6.2
Baxevanis &
Ouellette 2005
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 RNA functions
• Storage/transfer of genetic information

• Structural

• Catalytic

• Regulatory

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 26

 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??

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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 10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 31
 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 10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 32
 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 10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 33
 Steps in Transcription

Emerson Cell 2002

C Burge 2005 10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 34
 Sequence-specific Transcription Factors

• typically bind in clusters

» Regulatory modules

Kadonaga Cell 2004

C Burge 2005 10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 35
 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 10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 36
 Integration of
transcription &
RNA processing

Maniatis & Reed Nature 2002

C Burge 2005 10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 37
 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 10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 38
 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 10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 39
 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 10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 40
 Coupling of Splicing & Nonsense-mediated
mRNA Decay (NMD)

Maniatis & Reed Nature 2002

C Burge 2005 10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 41
 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 10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 42
 MicroRNA Biogenesis

N. Kim Nature Rev Mol Cell Biol 2005

C Burge 2005 10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 43
 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 10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 44
 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" by protein-DNA interactions
to include miRNA regulatory mechanisms

C Burge 2005 10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function 45