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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. (unstable RNA). mRNA. hnRNA. Replication. Processing / Translocation. rRNA. tRNA. (Stable RNA). - PowerPoint PPT Presentation
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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
???