2. The Search for Small Regulatory RNA

3. Central Dogma: DNA to RNA to Protein Replication Processing / Translocation hnRNA rRNAtRNA mRNA

4. Central Dogma: DNA to RNA to Protein Replication Processing / Translocation hnRNA rRNAtRNA mRNA (Stable RNA) (unstable RNA)

5. Role of structure in stable RNA tRNA Secondary structure Tertiary structure G U A G U A G U A G U A G U A G U A G U A

6. Other stable RNA Telomerase 5' 3' RNA primer DNA polymerase DNA ligase DNA Replication

7. Other stable RNA Telomerase DNA polymerase-independent repeated sequence

8. Other stable RNA Telomere growth Telomerase

9. Other stable RNA Spliceosome RNA hnRNAmRNA introns

10. Other stable RNA RNAse P tRNA maturation Protein + RNA

11. Other stable RNA Signal Recognition Particles proteinRNA SRP 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 Structure FunctionSequence ???

14. Prediction of RNA secondary structure Energy minimization Base-pairing energy Stacking energy

15. Prediction of RNA secondary Try it! Try Problem 1 with simple oligonucleotides Go to www.people.vcu.edu/~elhaij/bnfo650

16. Enter anything you like as a login name, but no spaces or symbols. No password necessary. Click New Login Your name (no spaces)

17. 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.

18. 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:

19. 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

20. Prediction of RNA secondary Try it! Go to www.people.vcu.edu/~elhaij/bnfo650 Try Problem 2 with mystery sequence 1 (a short oligonucleotide)

21. 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)

22. 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

23. The world of RNA has changed tryptophan anthocyanin Chalcone synthase More enzyme, more purple? Purple wild-type petunias

24. The world of RNA has changed Chalcone synthase Overexpression of exogenous chalcone synthase gene Overall expression of gene Mutant petunias Cosupression

25. Basis of Cosuppression RNA virus Double-stranded RNA

26. Basis of Cosuppression siRNA Defense against foreign RNA RNA- Induced Silencing Complex Hannon GJ (2002) Nature 418:244-251

27. Small Noncoding RNA siRNA – Inhibits expression of exogenous genes miRNA – Inhibits expression of endogenous genes RNAi – Human exploitation of phenomenon

28. miRNA expression Ambion Corp. http://www.ambion.com/techlib/resources/miRNA/mirna_pro.html

29. miRNA function

30. Detection of transcribed RNA Genome mRNA Tiling microarray Shifts every 5 nt Covers 70 nt/spot Expect RNA found at regions of known genes Cheng et al (2005) Science 308:1149-1154

31. Detection of transcribed RNA Pooled results 10 human chromosomes, 8 cell lines Most RNA not attached to coding genes! Many small ncRNA Known regions (different sources) Introns (26%) Intergenic regions (31%) Cheng et al (2005) Science 308:1149-1154

32. …but Eubacteria?

34. Role of RNA-binding protein Hfq

35. How to identify sRNA? Label RNA with 32 P; Isolate, characterize Microarray detection Shotgun clone by size class, sequence Functional assay Computational prediction

36. How to identify sRNA? Sequence conservation Lenz et al (2004) Cell 118:69-82

37. How to identify sRNA? Structural conservation Lenz et al (2004) Cell 118:69-82

38. How to identify sRNA? Transcriptional features Promoter Terminator Short sequence

39. How to identify sRNA? RNAz Combines two measures: Thermodynamic stability (Z score) MFE – MFE random σ random Z = MFE Minimum Free Energy

40. 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

41. The world's most populous organism Prochlorococcus A tiny marine cyanobacterium

42. Prochlorococcus MED4 Strategy to find small RNAs Prochlorococcus #2 Prochlorococcus #3 Prochlorococcus #4 Conserved?

43. 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

44. 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 ???