The Search for Small Regulatory RNA

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(No Transcript)
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The Search for Small Regulatory RNA
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Central Dogma DNA to RNA to Protein
hnRNA
mRNA
Replication
Processing /Translocation
rRNA
tRNA
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Central Dogma DNA to RNA to Protein
(unstable RNA)
mRNA
hnRNA
Replication
Processing /Translocation
rRNA
tRNA
(Stable RNA)
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Role of structure in stable RNA
tRNA
Tertiary structure
Secondary structure
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Other stable RNA
Telomerase
5'
3'
DNA Replication
RNA primer
DNA polymerase
DNA ligase
5'
3'
3'
5'
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Other stable RNA
Telomerase
DNA polymerase-independentrepeated sequence
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Other stable RNA
Telomerase
Telomere growth
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Other stable RNA
Spliceosome RNA
hnRNA
mRNA
introns
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Other stable RNA
RNAse P
tRNA maturation Protein RNA
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Other stable RNA
SRP RNA
Signal Recognition Particles
protein
RNA
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Other stable RNA

• Fundamental processes of life (left over from
  RNA world?)

• Nearly universal, highly conserved

• Not many

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Basis of RNA function
???
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Prediction of RNA secondary structure
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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
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.
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General Syntax of BioBIKE
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

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General Syntax of BioBIKE
and icons to help you work with functions
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Prediction of RNA secondary
Try it!
Go to www.people.vcu.edu/elhaij/bnfo650
Try Problem 2 with mystery sequence 1(a short
oligonucleotide)
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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)
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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

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The world of RNA has changed
Purple wild-type petunias
Chalcone synthase
More enzyme, more purple?
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The world of RNA has changed
Mutant petunias
Cosupression
Chalcone synthase
Overexpression of exogenous chalcone synthase
gene
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Basis of Cosuppression
RNA virus
Double-stranded RNA
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Basis of Cosuppression
RNA-InducedSilencingComplex
siRNADefense against foreign RNA
Hannon GJ (2002) Nature 418244-251
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Small Noncoding RNA
siRNA Inhibits expression of exogenous genes
RNAi Human exploitation of phenomenon
miRNA Inhibits expression of endogenous genes
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miRNA expression
Ambion Corp. http//www.ambion.com/techlib/resourc
es/miRNA/mirna_pro.html
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miRNA function
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Detection of transcribed RNA
Genome
Expect RNA found at regions of known genes
Tiling microarray
Shifts every 5 ntCovers 70 nt/spot
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Detection of transcribed RNA
Pooled results10 human chromosomes, 8 cell lines
Intergenicregions(31)
Introns(26)
Most RNA not attached to coding genes!
Many small ncRNA
Cheng et al (2005) Science 3081149-1154
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but Eubacteria?
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but Eubacteria?
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Role of RNA-binding protein Hfq
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How to identify sRNA?

• Label RNA with 32P Isolate, characterize

• Microarray detection

• Shotgun clone by size class, sequence

• Functional assay

• Computational prediction

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How to identify sRNA?
Sequence conservation
Lenz et al (2004) Cell 11869-82
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How to identify sRNA?
Structural conservation
Lenz et al (2004) Cell 11869-82
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How to identify sRNA?
Transcriptional features
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How to identify sRNA?
RNAz
Combines two measures
Thermodynamic stability (Z score)
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How to identify sRNA?
RNAz
Combines two measures
Thermodynamic stability (Z score)
Structural conservation Structural
Conservation Index (SCI)

• lt 1 if structure poorly conserved
• 1 if perfect conservation
• gt 1 if structure perfectly conserved, with
  compensatory mutations

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The world's most populous organism
ProchlorococcusA tiny marine cyanobacterium
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Strategy to find small RNAs
Prochlorococcus MED4
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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
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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
???