RNA Structure & Function Notes

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

10/31/05

RNA Structure & Function

10/31/05
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
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
Prediction
Mon Review - promoter prediction
RNA structure & function

Wed RNA structure prediction
2' & 3' structure prediction
miRNA & target prediction
RNA function prediction?

10/31/05
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
Review last lecture:

Promoter Prediction

10/31/05
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!
10/31/05
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

10/31/05
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)
10/31/05
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
10/31/05
Promoter prediction strategies

1) Pattern-driven algorithms

2) Sequence-driven algorithms

3) Combined "evidence-based"

BEST RESULTS? Combined, sequential
10/31/05
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
10/31/05
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
10/31/05
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.

10/31/05

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!
Promoter Prediction: Sequence-driven algorithms
10/31/05
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
10/31/05
Global alignment of human & mouse obese
gene promoters (200 bp upstream from TSS)
Fig 5.14
Baxevanis &
Ouellette 2005
10/31/05
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/
10/31/05
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/
10/31/05
New Today:


10/31/05

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

RNA types & functions
Genomic information storage/transfer
Structural
Catalytic
Regulatory

10/31/05

Rob Knight
Univ Colorado
RNA structure: 3 levels of organization
10/31/05

Fig 6.2
Baxevanis &
Ouellette 2005
Covalent & non-covalent bonds in RNA

Primary:
Covalent bonds

Secondary/Tertiary
Non-covalent bonds
H-bonds
(base-pairing)
Base stacking

10/31/05

G-C, A-U, G-U ("wobble") & variants
Base-pairing in RNA
http://www.imbjena.de/ImgLibDoc/nana/IMAGE_NANA.html#sec_element
See: IMB Image Library of Biological Molecules

10/31/05

Fig 6.2
Baxevanis &
Ouellette 2005
Common structural motifs in RNA

Helices

Loops
Hairpin
Interior
Bulge
Multibranch

Pseudoknots

10/31/05

RNA functions

Storage/transfer of genetic information

Structural

Catalytic

Regulatory

10/31/05

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

10/31/05

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)

10/31/05

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)
10/31/05

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
snoRNA - small nucleolar
mRNA processing, poly A addition <catalytic>
rRNA processing/maturation/methylation
regulatory RNAs (siRNA,
miRNA, etc.)
regulation of transcription and translation,
other??
10/31/05
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
Expression of a Typical Eukaryotic Gene
DNA

Transcription
Protein
Translation
mRNA
Splicing
exon intron
AAAAAAAAA
Polyadenylation
Protein Coding Gene
Folding, Modification,
Transport, Complex
Assembly
Protein Complex
Degradation
Degradation
primary transcript / pre-mRNA
Export
For each of these
processes, there is
a code
(set of default
recognition rules)
C Burge 2005
10/31/05
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
Steps in Transcription
Emerson Cell 2002
C Burge 2005
10/31/05
Sequence-specific Transcription Factors
typically bind in clusters
Regulatory modules
Kadonaga Cell 2004
C Burge 2005
10/31/05
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
Maniatis & Reed Nature 2002
Integration of
transcription &
RNA processing
C Burge 2005
10/31/05
Early Steps in Pre-mRNA Splicing
Matlin, Clark & Smith Nature Mol Cell Biol 2005
Formation of exon-spanning complex
Subsequent rearrangement to form
intron-spanning spliceosomes which
catalyze intron excision and exon ligation
hnRNP proteins
C Burge 2005
10/31/05
Alternative Splicing
Wang (ISU) Genome-wide Comparative Analysis of Alternative
Splicing in Plants
> 50% of human genes
undergo alternative splicing
C Burge 2005
10/31/05
Splicing Regulation
ESE/ESS = Exonic Splicing Enhancers/Silencers

ISE/ISS = Intronic Splicing Enhancers/Silencers
C Burge 2005
10/31/05
Coupling of Splicing & Nonsense-mediated
mRNA Decay (NMD)
Maniatis & Reed Nature 2002
C Burge 2005
10/31/05
C. elegans lin-4 Small Regulatory RNA
We now know that there are hundreds of microRNA genes
(Ambros, Bartel, Carrington, Ruvkun, Tuschl, others)
lin-4 precursor
lin-4 RNA
Translational
repression
V. Ambros lab
lin-4 RNA
target mRNA
C Burge 2005
10/31/05
MicroRNA Biogenesis
N. Kim Nature Rev Mol Cell Biol 2005
C Burge 2005
10/31/05
miRNA and RNAi pathways
RISC
Dicer
precursor
miRNA
siRNAs
Dicer
translational repression
and/or mRNA degradation
mRNA cleavage, degradation
RNAi pathway microRNA pathway
MicroRNA primary transcript
Exogenous dsRNA, transposon, etc.
target mRNA
Drosha
RISC
RISC
C Burge 2005
10/31/05
miRNA Challenges for Computational Biology
Find the genes encoding microRNAs
Predict their regulatory targets

Integrate miRNAs into gene regulatory pathways &
networks
Computational Prediction of MicroRNA Genes & Targets
C Burge 2005
Need to modify traditional paradigm of
"transcriptional control" by protein-DNA interactions
to include miRNA regulatory mechanisms

RNA Structure & Function Notes

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10/31/05

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

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