1. New roles for RNA New fields for bioinformatics? Morten Lindow Centre for Bioinformatic University of Copenhagen

2. RNA modul Torsdag –Forelæsning ncRNA, RNAi, miRNA –Opgave gennemgang, Bioperl, næste hjemmeopgave Tirsdag –Morgen: Øvelse – lave færdig, snakke snakke. –Eftermiddag: Opsamling Foredrag: mit projekt

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

4. 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

5. What is the difference?

6. 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?

7. 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

8. Control Architecture Genome Transcriptome Proteome Regulation by proteins Regulation by RNA

9. Types of RNA RNA mRNA ncRNA Non-coding RNA. Transcribed RNA with a structural, functional or catalytic role rRNA Ribosomal RNA Participate in protein synthesis tRNA Transfer RNA Interface between mRNA & amino acids snRNA Small nuclear RNA Incl. RNA that form part of the spliceosome snoRNA Small nucleolar RNA Found in nucleolus, involved in modification of rRNA miRNA Micro RNA Small RNA involved regulation of expression siRNA Small interfering RNA Active molecules in RNA interference stRNA Small temporal RNA. RNA with a role in developmental timing Other Including large RNA with roles in chromotin structure and imprinting

10. Discovery of RNAi Conventional antisense: –~50% downregulation Fire et al. : Synergy when given together with sense RNA

11. Examples of RNAi hairpin against pigment GFP expressed in nuclei Control dsRNAGFP specific dsRNA Red = silencing of GFP

12. RNA interference

13. RNAi mechanism RNase III like enzyme

14. Endogenous vs exogenous

15. Inhibition of translation Imperfect match  Block translation Near-perfect match  Degrade mRNA

16. 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

17. 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

18. 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)

19. Control Architecture Genome Transcriptome Proteome Regulation by proteins Regulation by RNA RNAi

20. 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........

21. DNA & chromosome structure Movie

22. Transcription and chromatin structure Euchromatin – open structure – transcriptionally active Heterochromatin – closed structure – silenced Structure determined by –> methylation of cytosines  histone binding

23. Spread of DNA methylation

24. Silent chromatin & RNAi CREDIT: KATHARINE SUTLIFF/SCIENCE Centromers contain repeats and are often heterochromatic (silenced) Finding: Deletion of RNAi machinery causes desilencing centromeric regions

25. Control Architecture Genome Transcriptome Proteome Regulation by proteins Regulation by RNA Imprinting – methylation Splicing RNAi Ribozymes

26. 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’

27. So why proteins? More adaptable Better at sensing environment – responding to signal transduction etc