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

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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

???