Title: The Search for Small Regulatory RNA
1(No Transcript)
2The Search for Small Regulatory RNA
3Central Dogma DNA to RNA to Protein
hnRNA
mRNA
Replication
Processing /Translocation
rRNA
tRNA
4Central Dogma DNA to RNA to Protein
(unstable RNA)
mRNA
hnRNA
Replication
Processing /Translocation
rRNA
tRNA
(Stable RNA)
5Role of structure in stable RNA
tRNA
Tertiary structure
Secondary structure
6Other stable RNA
Telomerase
5'
3'
DNA Replication
RNA primer
DNA polymerase
DNA ligase
5'
3'
3'
5'
7Other stable RNA
Telomerase
DNA polymerase-independentrepeated sequence
8Other stable RNA
Telomerase
Telomere growth
9Other stable RNA
Spliceosome RNA
hnRNA
mRNA
introns
10Other stable RNA
RNAse P
tRNA maturation Protein RNA
11Other stable RNA
SRP RNA
Signal Recognition Particles
protein
RNA
12Other stable RNA
- Fundamental processes of life (left over from
RNA world?)
- Nearly universal, highly conserved
13Basis of RNA function
???
14Prediction of RNA secondary structure
15Prediction of RNA secondary
Try it!
Go to www.people.vcu.edu/elhaij/bnfo650
Try Problem 1 with simple oligonucleotides
16Your name (no spaces)
Enter anything you like as a login name, but no
spaces or symbols.No password necessary.Click
New Login
17General 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.
18General 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
19General Syntax of BioBIKE
and icons to help you work with functions
20Prediction of RNA secondary
Try it!
Go to www.people.vcu.edu/elhaij/bnfo650
Try Problem 2 with mystery sequence 1(a short
oligonucleotide)
21Prediction of RNA secondary
Try it!
Go to www.people.vcu.edu/elhaij/bnfo650
Try Problem 3 with mystery sequence 2(a more
realistic oligonucleotide)
22Conclusions 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
- True structures can be predicted
- Surrounding sequence can obscure true structure
23The world of RNA has changed
Purple wild-type petunias
Chalcone synthase
More enzyme, more purple?
24The world of RNA has changed
Mutant petunias
Cosupression
Chalcone synthase
Overexpression of exogenous chalcone synthase
gene
25Basis of Cosuppression
RNA virus
Double-stranded RNA
26Basis of Cosuppression
RNA-InducedSilencingComplex
siRNADefense against foreign RNA
Hannon GJ (2002) Nature 418244-251
27Small Noncoding RNA
siRNA Inhibits expression of exogenous genes
RNAi Human exploitation of phenomenon
miRNA Inhibits expression of endogenous genes
28miRNA expression
Ambion Corp. http//www.ambion.com/techlib/resourc
es/miRNA/mirna_pro.html
29miRNA function
30Detection of transcribed RNA
Genome
Expect RNA found at regions of known genes
Tiling microarray
Shifts every 5 ntCovers 70 nt/spot
31Detection 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
32but Eubacteria?
33but Eubacteria?
34Role of RNA-binding protein Hfq
35How to identify sRNA?
- Label RNA with 32P Isolate, characterize
- Shotgun clone by size class, sequence
36How to identify sRNA?
Sequence conservation
Lenz et al (2004) Cell 11869-82
37How to identify sRNA?
Structural conservation
Lenz et al (2004) Cell 11869-82
38How to identify sRNA?
Transcriptional features
39How to identify sRNA?
RNAz
Combines two measures
Thermodynamic stability (Z score)
40How 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
41The world's most populous organism
ProchlorococcusA tiny marine cyanobacterium
42Strategy to find small RNAs
Prochlorococcus MED4
43Prediction 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
44Prediction 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
???