New roles for RNA

New fields for bioinformatics?

Morten LindowCentre for Bioinformatic

University of Copenhagen

RNA modul• Torsdag

– Forelæsning• ncRNA, RNAi, miRNA

– Opgave gennemgang, Bioperl, næste hjemmeopgave

• Tirsdag– Morgen: Øvelse – lave færdig, snakke

snakke.– Eftermiddag: Opsamling

• Foredrag: mit projekt

Øvelser

• Formål:– Få basal øvelse i at håndtere sekvenser i perl– Værdsætte BioPerl– Prøve at finde miRNA i Arabidopsis

Plan for this lecture

• Abundance of ncRNA

• Types of ncRNA

• RNAi – discovery and mechanisms– miRNA

• RNAi as a technology

• RNAi – the genome’s immune system

• ncRNA and chromatin

What is the difference?

Source of variation

• What is the homology of protein coding genes between human and mice?– 99 % !!!!!!

• There are ~3 000 000 nucleotide differences between two humans.– 10 000 in protein coding genes (0.03%)

• Most are silent (third base)

• So where do the phenotypic differences between two individuals or two species come from?

Source of differences?

• Whole system properties:– Variations in gene expression (time/space).

• Control architecture is the primary source of complex traits variation

– Background for • Intercellular communication & integration• Interorgan communication & integration• Interindividual communication & integration

Systems biology

Control Architecture

Genome

Transcriptome

Proteome

Regulation by proteins Regulation by

RNA

Types of RNA

RNA

mRNAncRNA

Non-coding RNA. Transcribed RNA with a structural, functional or catalytic role

rRNARibosomal RNA

Participate in protein synthesis

tRNATransfer RNA

Interface betweenmRNA &

amino acids

snRNASmall nuclear RNA

-Incl. RNA thatform part of the

spliceosome

snoRNASmall nucleolar RNAFound in nucleolus,

involved in modificationof rRNA

miRNAMicro RNA

Small RNA involvedregulation of expression

OtherIncluding large RNA

with roles in chromotin structure and

imprinting

siRNASmall interfering RNAActive molecules in

RNA interference

stRNASmall temporal RNA.RNA with a role in

developmental timing

Discovery of RNAi

• Conventional antisense:– ~50% downregulation

• Fire et al. : Synergy when given together with sense RNA

Examples of RNAi

hairpin against pigment

GFP expressed in nuclei

Control dsRNA GFP specific dsRNA

Red = silencing of GFP

RNA interference

RNAi mechanismRNase III like enzyme

Endogenous vs exogenous

Inhibition of translation

Imperfect match Block translation

Near-perfect match Degrade mRNA

Defense against transposons?

• In C.elegans and Drosophila mutation of RNAi components activation of transposons

Is RNAi a genomic immune system?– The vertebrate adaptive immune system:

• Distinquish self from non-self• Amplify a response• Kill the intruder

Genomic immune system hypothesis 1

• Self/non-self discrimination (generation od dsRNA)– Multicopy transposons:

• read through from flanking promotors create complementary strands to form dsRNA

– Some transposons have terminal inverted repeats hairpin

Genomic immune system hypothesis 2

• Amplification of signal:– siRNA may work as

primers on the mRNA• Amplification by RNA

dependent RNA polymerase

• (only detected in plants & yeast)

Control Architecture

Genome

Transcriptome

Proteome

Regulation by proteins Regulation by

RNA

RNAi

Regulation of transcription

• What governs when a stretch of DNA is transcribed?– Transcription factors– Accessabilitity of DNA to transcription factors

• Structure of the chromatin– Proteins– RNAs........

DNA & chromosome structure

Movie

Transcription and chromatin structure

• Euchromatin – open structure – transcriptionally active

• Heterochromatin – closed structure – silenced

• Structure determined by– > methylation of cytosines histone binding

Spread of DNA methylation

Silent chromatin & RNAi

CREDIT: KATHARINE SUTLIFF/SCIENCE

• Centromers contain repeats and are often heterochromatic (silenced)

•Finding: Deletion of RNAi machinery causes desilencing centromeric regions

Control Architecture

Genome

Transcriptome

Proteome

Regulation by proteins Regulation by

RNA

Imprinting – methylationSplicing

RNAi

Ribozymes

Why use RNA in regulation?

• Less cost– Genomic space. Compare RNA vs protein– Metabolic space. Cost of aminoacids

• Speed– RNA produced and active very quickly

• Easier to evolve. Less costly to explore ’expression space’

So why proteins?

• More adaptable

• Better at sensing environment – responding to signal transduction etc