Download The search for small regulatory RNA

The Search for Small
Regulatory RNA
Central Dogma:
DNA to RNA to Protein
hnRNA
mRNA
Replication
Processing /
Translocation
rRNA
tRNA
Central Dogma:
DNA to RNA to Protein
hnRNA
mRNA (unstable RNA)
Replication
Processing /
Translocation
rRNA
tRNA
(Stable RNA)
Role of structure in stable RNA
tRNA
Tertiary
structure
Secondary
structure
G U A
G U A
Other stable RNA
Telomerase
5' 3'
DNA Replication
RNA primer
DNA polymerase
DNA ligase
3' 5'
3' 5'
Other stable RNA
Telomerase
DNA polymerase-independent
repeated sequence
Other stable RNA
Telomerase
Telomere growth
Other stable RNA
Spliceosome RNA
hnRNA
mRNA
introns
Other stable RNA
RNAse P
tRNA maturation
Protein + RNA
Other stable RNA
SRP RNA
Signal Recognition
Particles
protein
RNA
Other stable RNA
• Fundamental processes of life
(left over from RNA world?)
• Nearly universal, highly conserved
• Not many
Basis of RNA function
Sequence ???
Structure
Function
Prediction of RNA secondary structure
• Energy minimization
• Base-pairing energy
• Stacking energy
Prediction of RNA secondary
Try it!
Go to
www.people.vcu.edu/~elhaij/bnfo650
Try Problem 1 with simple oligonucleotides
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General Syntax of BioBIKE
Function-name
Argument
(object)
Keyword
object
The basic unit of BioBIKE is the
function box. It consists of the
name of a function, perhaps one or
more required arguments, and
optional keywords and flags.
A function may be thought of as a
black box: you feed it information,
it produces a product.
Flag
General Syntax of BioBIKE
Function-name
Argument
(object)
Keyword
object
Flag
Function boxes contain the
following elements:
• Function-name (e.g. SEQUENCE-OF or LENGTH-OF)
• Argument: Required, acted on by function
• Keyword clause: Optional, more information
• Flag: Optional, more (yes/no) information
General Syntax of BioBIKE
Function-name
Argument
(object)
Keyword
object
Flag
… and icons to help you work with
functions:
•
Option icon: Brings up a menu of keywords and flags
•
Action icon: Brings up a menu enabling you to execute
a function, copy and paste, information, get help, etc
Clear/Delete icon: Removes information you entered
or removes box entirely
•
Prediction of RNA secondary
Try it!
Go to
www.people.vcu.edu/~elhaij/bnfo650
Try Problem 2 with mystery sequence 1
(a short oligonucleotide)
Prediction of RNA secondary
Try it!
Go to
www.people.vcu.edu/~elhaij/bnfo650
Try Problem 3 with mystery sequence 2
(a more realistic oligonucleotide)
Conclusions re RNA structure prediction
• A visually beautiful structure means nothing
• A negative free energy means little
• Function cannot be predicted from ability
of sequence to form structure
• Limited to 2-dimensional structure
• Ignorant of pseudoknots
• True structures can be predicted
• Surrounding sequence can obscure true structure
The world of RNA has changed
Purple
wild-type
petunias
Chalcone synthase
tryptophan
anthocyanin
More enzyme, more purple?
The world of RNA has changed
Mutant
petunias
Cosupression
Chalcone synthase
Overexpression of
exogenous chalcone
synthase gene
Overall
expression
of gene
Basis of Cosuppression
RNA virus
Double-stranded RNA
Basis of Cosuppression
RNAInduced
Silencing
Complex
siRNA
Defense against
foreign RNA
Hannon GJ (2002) Nature 418:244-251
Small Noncoding RNA
siRNA – Inhibits expression of exogenous genes
RNAi – Human exploitation of phenomenon
miRNA – Inhibits expression of endogenous genes
miRNA expression
Ambion Corp. http://www.ambion.com/techlib/resources/miRNA/mirna_pro.html
miRNA function
Detection of transcribed RNA
Genome
mRNA
Tiling microarray
Shifts every 5 nt
Covers 70 nt/spot
mRNA
Expect
RNA found at
regions of
known genes
Cheng et al (2005)
Science 308:1149-1154
Detection of transcribed RNA
Pooled results
10 human chromosomes, 8 cell lines
Intergenic
regions
(31%)
Many
small
ncRNA
Known
regions
(different
sources)
Introns
(26%)
Cheng et al (2005)
Science 308:1149-1154
Most RNA not
attached to
coding genes!
…but Eubacteria?
…but Eubacteria?
Role of RNA-binding protein Hfq
How to identify sRNA?
• Label RNA with 32P; Isolate, characterize
• Microarray detection
• Shotgun clone by size class, sequence
• Functional assay
• Computational prediction
How to identify sRNA?
Sequence conservation
Lenz et al (2004) Cell 118:69-82
How to identify sRNA?
Structural conservation
Lenz et al (2004) Cell 118:69-82
How to identify sRNA?
Transcriptional features
Promoter
Short sequence
Terminator
How to identify sRNA?
RNAz
Combines two measures:
Thermodynamic stability
(Z score)
Z=
MFE – MFErandom
σrandom
MFE
Minimum Free Energy
How to identify sRNA?
RNAz
Combines two measures:
Thermodynamic stability
(Z score)
Structural conservation
Structural Conservation Index (SCI)
• < 1 if structure poorly conserved
• = 1 if perfect conservation
• > 1 if structure perfectly conserved, with
compensatory mutations
The world's most populous organism
Prochlorococcus
A tiny marine
cyanobacterium
Strategy to find small RNAs
Prochlorococcus MED4
Prochlorococcus #2
Prochlorococcus #3
Prochlorococcus #4
Conserved?
Prediction of RNA secondary
Try it!
Go to
www.people.vcu.edu/~elhaij/bnfo650
Try Problem 4, testing whether RNAz
can find a known small RNA
Prediction of RNA secondary
Try it!
Go to
www.people.vcu.edu/~elhaij/bnfo650
Try Problem 5, testing whether RNAz
predicts a small RNA between
dmtB and trpD
dmtB
???
trpD