A system for imaging the regulatory noncoding Xist RNA in living ...
The Search for Small Regulatory RNA
description
Transcript of The Search for Small Regulatory RNA
The Search for Small Regulatory RNA
Central Dogma: DNA to RNA to Protein
Replication
Processing /Translocation
hnRNA
rRNA tRNA
mRNA
Central Dogma: DNA to RNA to Protein
Replication
Processing /Translocation
hnRNA
rRNA tRNA
mRNA
(Stable RNA)
(unstable RNA)
Role of structure in stable RNAtRNA
Secondary structure
Tertiary structure
G U A
G U A
GU A
G UAG U A
GU A
G UA
Other stable RNATelomerase
5'
5' 5'3' 3'
3'
RNA primer
DNA polymerase
DNA ligase
DNA Replication
Other stable RNATelomerase
DNA polymerase-independentrepeated sequence
Other stable RNA
Telomere growth
Telomerase
Other stable RNASpliceosome RNA
hnRNA mRNAintrons
Other stable RNARNAse P
tRNA maturation
Protein + RNA
Other stable RNA
Signal Recognition Particles
protein RNA
SRP RNA
Other stable RNA
• Fundamental processes of life (left over from RNA world?)
• Nearly universal, highly conserved
• Not many
Basis of RNA function
Structure FunctionSequence ???
Prediction of RNA secondary structure
• Energy minimization
• Base-pairing energy
• Stacking energy
Prediction of RNA secondary
Try it!
Try Problem 1 with simple oligonucleotides
Go to www.people.vcu.edu/~elhaij/bnfo650
Enter anything you like as a login name, but no spaces or symbols.No password necessary.Click New Login
Your name (no spaces)
General Syntax of BioBIKE
Function-name Argument(object)
Keyword object Flag
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.
• Function-name (e.g. SEQUENCE-OF or LENGTH-OF)
• Argument: Required, acted on by function
• Keyword clause: Optional, more information
General Syntax of BioBIKE
• Flag: Optional, more (yes/no) information
Function-name Argument(object)
Keyword object Flag
Function boxes contain the following elements:
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
• Clear/Delete icon: Removes information you entered or removes box entirely
• Action icon: Brings up a menu enabling you to execute a function, copy and paste, information, get help, etc
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
• Surrounding sequence can obscure true structure
• True structures can be predicted
• Limited to 2-dimensional structure
• Ignorant of pseudoknots
The world of RNA has changed
tryptophan anthocyaninChalcone synthase
More enzyme, more purple?
Purple wild-type petunias
The world of RNA has changed
Chalcone synthase
Overexpression of exogenous chalcone
synthase gene
Overall expression
of gene
Mutant petunias
Cosupression
Basis of Cosuppression
RNA virus
Double-stranded RNA
Basis of Cosuppression
siRNADefense against
foreign RNA
RNA-InducedSilencingComplex
Hannon GJ (2002) Nature 418:244-251
Small Noncoding RNA
siRNA – Inhibits expression of exogenous genes
miRNA – Inhibits expression of endogenous genes
RNAi – Human exploitation of phenomenon
miRNA expression
Ambion Corp. http://www.ambion.com/techlib/resources/miRNA/mirna_pro.html
miRNA function
Detection of transcribed RNAGenome
mRNA mRNA
Tiling microarray
Shifts every 5 ntCovers 70 nt/spot
Expect
RNA found at regions of
known genes
Cheng et al (2005) Science 308:1149-1154
Detection of transcribed RNAPooled results
10 human chromosomes, 8 cell lines
Most RNA not attached to
coding genes!
Many small
ncRNA
Knownregions
(differentsources)
Introns(26%)
Intergenicregions(31%)
Cheng et al (2005) Science 308:1149-1154
…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 TerminatorShort sequence
How to identify sRNA?RNAz
Combines two measures:
Thermodynamic stability (Z score)
MFE – MFErandom
σrandom
Z = MFEMinimum 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
ProchlorococcusA tiny marine
cyanobacterium
Prochlorococcus MED4
Strategy to find small RNAs
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
dmtB trpD
Try Problem 5, testing whether RNAz predicts a small RNA between
dmtB and trpD
???