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The Search for Small Regulatory RNA

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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 ... – PowerPoint PPT presentation

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Title: 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
4
Central Dogma DNA to RNA to Protein
(unstable RNA)
mRNA
hnRNA
Replication
Processing /Translocation
rRNA
tRNA
(Stable RNA)
5
Role of structure in stable RNA
tRNA
Tertiary structure
Secondary structure
6
Other stable RNA
Telomerase
5'
3'
DNA Replication
RNA primer
DNA polymerase
DNA ligase
5'
3'
3'
5'
7
Other stable RNA
Telomerase
DNA polymerase-independentrepeated sequence
8
Other stable RNA
Telomerase
Telomere growth
9
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
12
Other stable RNA
  • Fundamental processes of life (left over from
    RNA world?)
  • Nearly universal, highly conserved
  • Not many

13
Basis of RNA function
???
14
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
16
Your name (no spaces)
Enter anything you like as a login name, but no
spaces or symbols.No password necessary.Click
New Login
17
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
???
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