1. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction1 11/2/05 RNA Structure Prediction

2. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction2 Announcements Seminar 12:10 PM Fri BCB Faculty Seminar in E164 Lago How to do sequence alignments on parallel computers Srinivas Aluru, ECprE & Chair, BCB Program http://www.bcb.iastate.edu/courses/BCB691F2005.html

3. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction3 Announcements BCB 544 Projects - Important Dates: Nov 2 Wed noon - Project proposals due to David/Drena Nov 4 Fri 10A - Approvals/responses to students Dec 2 Fri noon- Written project reports due Dec 5,7,8,9 class/lab- Oral Presentations (20') (Dec 15 Thurs = Final Exam)

4. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction4 RNA Structure & Function Prediction Mon Review - promoter prediction RNA structure & function Wed RNA structure prediction 2' & 3' structure prediction miRNA & target prediction - perhaps.. RNA function prediction? Won't have time to cover this…

5. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction5 Reading Assignment (for Mon/Wed) Mount Bioinformatics Chp 8 Prediction of RNA Secondary Structure pp. 327-355 Ck Errata: http://www.bioinformaticsonline.org/help/errata2.htmlhttp://www.bioinformaticsonline.org/help/errata2.html Cates (Online) RNA Secondary Structure Prediction Module http://cnx.rice.edu/content/m11065/latest/

6. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction6 Review last lecture: RNA Structure & Function

7. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction7 RNA Structure & Function RNA structure Levels of organization Energetics (more about this on Wed) RNA types & functions Genomic information storage/transfer Structural Catalytic Regulatory

8. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction8 Fig 6.2 Baxevanis & Ouellette 2005 Covalent & non-covalent bonds in RNA Primary: Covalent bonds Secondary/Tertiary Non-covalent bonds H-bonds (base-pairing) Base stacking

9. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction9 1)G-C, A-U, G-U ("wobble") & variants U can form base-pairs with both A & G 2)Nucleotides in RNA are frequently modified this is not very common in DNA These features & flexible "single-stranded" RNA backbone allow for many potential base-pairs Base-pairing in RNA See: IMB Image Library of Biological MoleculesIMB Image Library of Biological Molecules Modified bases are especially important) in tRNA: e.g., pseudo-Uridine, rD, 5-CH3-C6-isopentenyl-A 7-CH3-G, many others …

10. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction10 Fig 6.2 Baxevanis & Ouellette 2005 Common structural motifs in RNA Helices Loops Hairpin Internal Bulge Multibranch Pseudoknots

11. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction11 RNA functions Storage/transfer of genetic information Genomes many viruses have RNA genomes single-stranded (ssRNA) e.g., retroviruses (HIV) double-stranded (dsRNA) Transfer of genetic information mRNA = "coding RNA" - encodes proteins

12. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction12 RNA functions Structural e.g., rRNA, which is major structural component of ribosomes BUT - its role is not just structural, also: Catalytic RNA in ribosome has peptidyltransferase activity Enzymatic activity responsible for peptide bond formation between amino acids in growing peptide chain Also, many small RNAs are enzymes "ribozymes" (Gloria Culver, ISU) (W Allen Miller, ISU)

13. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction13 RNA functions Regulatory Recently discovered important new roles for RNAs In normal cells: in "defense" - esp. in plants in normal development e.g., siRNAs, miRNA As tools: for gene therapy or to modify gene expression RNAi (used by many at ISU: Diane Bassham, Thomas Baum, Jeff Essner, Kristen Johansen, Jo Anne Powell-Coffman, Roger Wise, etc.) RNA aptamers (Marit Nilsen-Hamilton, ISU)

14. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction14 RNA types & functions Types of RNAsPrimary Function(s) mRNA - messengertranslation (protein synthesis) regulatory rRNA - ribosomaltranslation (protein synthesis) t-RNA - transfertranslation (protein synthesis) hnRNA - heterogeneous nuclearprecursors & intermediates of mature mRNAs & other RNAs scRNA - small cytoplasmicsignal recognition particle (SRP) tRNA processing snRNA - small nuclear snoRNA - small nucleolar mRNA processing, poly A addition rRNA processing/maturation/methylation regulatory RNAs (siRNA, miRNA, etc.) regulation of transcription and translation, other?? L Samaraweera 2005

15. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction15 Thanks to Chris Burge, MIT for following slides Slightly modified from: Gene Regulation and MicroRNAs Session introduction presented at ISMB 2005, Detroit, MI Chris Burge cburge@MIT.EDUcburge@MIT.EDU C Burge 2005

16. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction16 Expression of a Typical Eukaryotic Gene DNA … Transcription Protein Translation mRNA Splicing exon intron AAAAAAAAA Polyadenylation Protein Coding Gene Folding, Modification, Transport, Complex Assembly Protein Complex Degradation primary transcript / pre-mRNA Export For each of these processes, there is a ‘code’ (set of default recognition rules) C Burge 2005

17. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction17 Gene Expression Challenges for Computational Biology Understand the ‘code’ for each step in gene expression (set of default recognition rules), e.g., the ‘splicing code’ Understand the rules for sequence-specific recognition of nucleic acids by protein and ribonucleoprotein (RNP) factors Understand the regulatory events that occur at each step and the biological consequences of regulation Lots of data Genomes, structures, transcripts, microarrays, ChIP-Chip, etc. C Burge 2005

18. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction18 Sequence-specific Transcription Factors have modular organization » Understand DNA-binding specificity Yan (ISU) A computational method to identify amino acid residues involved in protein-DNA interactions ATF-2/c-Jun/IRF-3 DNA complex Panne et al. EMBO J. 2004 C Burge 2005

19. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction19 Early Steps in Pre-mRNA Splicing Matlin, Clark & Smith Nature Mol Cell Biol 2005 Formation of exon-spanning complex Subsequent rearrangement to form intron-spanning spliceosomes which catalyze intron excision and exon ligation hnRNP proteins C Burge 2005

20. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction20 Alternative Splicing Matlin, Clark & Smith Nature Mol Cell Biol 2005 Wang (ISU) Genome-wide Comparative Analysis of Alternative Splicing in Plants > 50% of human genes undergo alternative splicing C Burge 2005

21. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction21 Splicing Regulation Matlin, Clark & Smith Nature Mol Cell Biol 2005 ESE/ESS = Exonic Splicing Enhancers/Silencers ISE/ISS = Intronic Splicing Enhancers/Silencers C Burge 2005

22. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction22 C. elegans lin-4 Small Regulatory RNA We now know that there are hundreds of microRNA genes (Ambros, Bartel, Carrington, Ruvkun, Tuschl, others) lin-4 precursor lin-4 RNA “Translational repression” V. Ambros lab lin-4 RNA target mRNA C Burge 2005

23. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction23 MicroRNA Biogenesis N. Kim Nature Rev Mol Cell Biol 2005 C Burge 2005

24. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction24 miRNA and RNAi pathways RISC Dicer precursor miRNA siRNAs Dicer “translational repression” and/or mRNA degradation mRNA cleavage, degradation RNAi pathway microRNA pathway MicroRNA primary transcript Exogenous dsRNA, transposon, etc. target mRNA Drosha RISC C Burge 2005

25. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction25 miRNA Challenges for Computational Biology Find the genes encoding microRNAs Predict their regulatory targets Integrate miRNAs into gene regulatory pathways & networks Computational Prediction of MicroRNA Genes & Targets C Burge 2005 Need to modify traditional paradigm of "transcriptional control" primarily by protein-DNA interactions to include miRNA regulatory mechanisms!

26. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction26 New Today: RNA Structure Prediction

27. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction27 RNA structure prediction strategies 1)Energy minimization (thermodynamics) 2) Comparative sequence analysis (co-variation) 3) Combined experimental & computational Secondary structure prediction

28. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction28 Secondary structure prediction strategies 1)Energy minimization (thermodynamics) Algorithm: Dynamic programming to find high probability pairs (also, some Genetic algorithms) Software: Mfold - Zuker Vienna RNA Package - Hofacker RNAstructure - Mathews Sfold - Ding & Lawrence R Knight 2005

29. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction29 Secondary structure prediction strategies 2) Comparative sequence analysis (co-variation) Algorithm: Mutual information Context-free grammars Software: ConStruct Alifold Pfold FOLDALIGN Dynalign R Knight 2005

30. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction30 Secondary structure prediction strategies 3) Combined experimental & computational Experiment: Map single-stranded vs double-stranded regions in folded RNA How? Enzymes: S1 nuclease, T1 RNase Chemicals: kethoxal, DMS R Knight 2005

31. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction31 Experimental RNA structure determination? X-ray crystallography NMR spectroscopy Enzymatic/chemical mapping

32. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction32 1) Energy minimization method What are the assumptions? Native tertiary structure or "fold" of an RNA molecule is (one of) its "lowest" free energy configuration(s) Gibbs free energy =  G in kcal/mol at 37  C = equilibrium stability of structure lower values (negative) are more favorable Is this assumption valid? in vivo? - this may not hold, but we don't really know

33. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction33 Free energy minimization What are the rules? A U A=U Basepair  G = -1.2 kcal/mole A U U A A=U U=A  G = -1.6 kcal/mole Basepair What gives here? C Staben 2005

34. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction34 Energy minimization calculations: Base-stacking is critical - Tinocco et al. C Staben 2005

35. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction35 Nearest-neighbor parameters Most methods for free energy minimization use nearest-neighbor parameters (derived from experiment) for predicting stability of an RNA secondary structure (in terms of  G at 37  C) & most available software packages use the same set of parameters : Mathews, Sabina, Zuker & Turner, 1999

36. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction36 Energy minimization - calculations: Total free energy of a specific conformation for a specific RNA molecule = sum of incremental energy terms for: helical stacking (sequence dependent) loop initiation unpaired stacking (favorable "increments" are < 0) Fig 6.3 Baxevanis & Ouellette 2005

37. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction37 But how many possible conformations for a single RNA molecule? Huge number: Zuker estimates (1.8) N possible secondary structures for a sequence of N nucleotides for 100 nts (small RNA…) = 3 X 10 25 structures! Solution? Not exhaustive enumeration…  Dynamic programming O(N 3 ) in time O(N 2 ) in space/storage iff pseudoknots excluded, otherwise: O(N 6 ), time O(N 4 ), space

38. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction38 2) Comparative sequence analysis (co-variation) Two basic approaches: Algorithms constrained by initial alignment Much faster, but not as robust as unconstrained Base-pairing probabilities determined by a partition function Algorithms not constrained by initial alignment Genetic algorithms often used for finding an alignment & set of structures

39. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction39 RNA Secondary structure prediction: Performance? How evaluate? Not many experimentally determined structures currently, ~ 50% are rRNA structures so "Gold Standard" (in absence of tertiary structure): compare with predicted RNA secondary structure with that determined by comparative sequence analysis (!!??) using Benchmark Datasets NOTE: Base-pairs predicted by comparative sequence analysis for large & small subunit rRNAs are 97% accurate when compared with high resolution crystal structures! - Gutell, Pace

40. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction40 RNA Secondary structure prediction: Performance? 1)Energy minimization (via dynamic programming) 73% avg. prediction accuracy - single sequence 2) Comparative sequence analysis 97% avg. prediction accuracy - multiple sequences (e.g., highly conserved rRNAs) much lower if sequence conservation is lower &/or fewer sequences are available for alignment 3) Combined - recent developments: combine thermodynamics & co-variation & experimental constraints? IMPROVED RESULTS

41. 11/02/05 D Dobbs ISU - BCB 444/544X: RNA Structure Prediction41 RNA structure prediction strategies Requires "craft" & significant user input & insight 1)Extensive comparative sequence analysis to predict tertiary contacts (co-variation) e.g., MANIP - Westhof 2)Use experimental data to constrain model building e.g., MC-CYM - Major 3)Homology modeling using sequence alignment & reference tertiary structure (not many of these!) 4)Low resolution molecular mechanics e.g., yammp - Harvey Tertiary structure prediction