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10/31/05 RNA Structure & Function

10/31/05 RNA Structure & Function. Announcements. Seminar (Mon Oct 31) 12:10 PM IG Faculty Seminar in 101 Ind Ed II Plant Steroid Hormone Signal Transduction Yanhai Yin, GDCB BCB Link for Seminar Schedules (updated) http://www.bcb.iastate.edu/seminars/index.html. Announcements.

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10/31/05 RNA Structure & Function

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  1. 10/31/05RNA Structure & Function D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  2. Announcements • Seminar (Mon Oct 31) • 12:10 PM IG Faculty Seminar in 101 Ind Ed II • Plant Steroid Hormone Signal Transduction • Yanhai Yin, GDCB • BCB Link for Seminar Schedules (updated) • http://www.bcb.iastate.edu/seminars/index.html D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  3. 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) D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  4. RNA Structure & Function Prediction Mon Review - promoter prediction RNA structure & function Wed RNA structure prediction 2' & 3' structure prediction miRNA & target prediction RNA function prediction? D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  5. 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.html • Cates (Online) RNA Secondary Structure Prediction Module • http://cnx.rice.edu/content/m11065/latest/ D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  6. Review last lecture:Promoter Prediction D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  7. Promoter Prediction • Overview of strategies •  What sequence signals can be used? •  What other types of information can be used? • Algorithms  a bit more about these • in later lectures • Promoter prediction software • 3 major types • many, many programs! D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  8. Promoter prediction: Eukaryotes vs prokaryotes Promoter prediction is easier in microbial genomes Why? Highly conserved Simpler gene structures More sequenced genomes! (for comparative approaches) Methods? Previously, again mostly HMM-based Now: similarity-based. comparative methods because so many genomesavailable D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  9. Promoter Prediction: Steps & Strategies • Closely related to gene prediction! • Obtain genomic sequence • Use sequence-similarity based comparison • (BLAST, MSA) to find related genes • But: "regulatory" regions are much less well-conserved than coding regions • Locate ORFs • Identify TSS (Transcription Start Site) • Use promoter prediction programs • Analyze motifs, etc. in sequence(TRANSFAC) D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  10. Promoter Prediction: Steps & Strategies Identify TSS --if possible? • One of biggest problems is determining exact TSS! Not very many full-length cDNAs! • Good starting point? (human & vertebrate genes) Use FirstEF found within UCSC Genome Browser or submit to FirstEF web server Fig 5.10 Baxevanis & Ouellette 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  11. Promoter prediction strategies • Pattern-driven algorithms • Sequence-driven algorithms • Combined "evidence-based" • BEST RESULTS? Combined, sequential D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  12. Promoter Prediction: Pattern-driven algorithms • Success depends on availability of collections of annotated binding sites (TRANSFAC & PROMO) • Tend to produce huge numbers of FPs • Why? • Binding sites (BS) for specific TFs often variable • Binding sites are short (typically 5-15 bp) • Interactions between TFs (& other proteins) influence affinity & specificity of TF binding • One binding site often recognized by multiple BFs • Biology is complex: promoters often specific to organism/cell/stage/environmental condition D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  13. Promoter Prediction: Pattern-driven algorithms Solutions to problem of too many FP predictions? • Take sequence context/biology into account • Eukaryotes: clusters of TFBSs are common • Prokaryotes: knowledge of  factors helps • Probability of "real" binding site increases if annotated transcription start site (TSS) nearby • But: What about enhancers? (no TSS nearby!) & Only a small fraction of TSSs have been experimentally mapped • Do the wet lab experiments! • But: Promoter-bashing is tedious D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  14. Promoter Prediction: Sequence-driven algorithms • Assumption: common functionality can be deduced from sequence conservation • Alignments of co-regulated genes should highlight elements involved in regulation Careful: How determine co-regulation? • Orthologous genes from difference species • Genes experimentally determined to be co-regulated (using microarrays??) • Comparative promoter prediction: "Phylogenetic footprinting" - more later…. D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  15. Promoter Prediction: Sequence-driven algorithms Problems: • Need sets of co-regulated genes • For comparative (phylogenetic) methods • Must choose appropriate species • Different genomes evolve at different rates • Classical alignment methods have trouble with translocations, inversions in order of functional elements • If background conservation of entire region is highly conserved, comparison is useless • Not enough data (Prokaryotes >>> Eukaryotes) • Biology is complex: many (most?) regulatory elements are not conserved across species! D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  16. Examples of promoter prediction/characterization software Lab: used MATCH, MatInspector TRANSFAC MEME & MAST BLAST, etc. Others? FIRST EF Dragon Promoter Finder(these are links in PPTs) also see Dragon Genome Explorer (has specialized promoter software for GC-rich DNA, finding CpG islands, etc) JASPAR D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  17. Global alignment of human & mouse obese gene promoters (200 bp upstream from TSS) Fig 5.14 Baxevanis & Ouellette 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  18. Check out optional review & try associated tutorial: Wasserman WW & Sandelin A (2004) Applied bioinformatics for identification of regulatory elements. Nat Rev Genet 5:276-287 http://proxy.lib.iastate.edu:2103/nrg/journal/v5/n4/full/nrg1315_fs.html Check this out: http://www.phylofoot.org/NRG_testcases/ D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  19. Annotated lists of promoter databases & promoter prediction software • URLs from Mount Chp 9, available online Table 9.12http://www.bioinformaticsonline.org/links/ch_09_t_2.html • Table in Wasserman & Sandelin Nat Rev Genet article http://proxy.lib.iastate.edu:2103/nrg/journal/v5/n4/full/nrg1315_fs.htm • URLs for Baxevanis & Ouellette, Chp 5: http://www.wiley.com/legacy/products/subject/life/bioinformatics/ch05.htm#links More lists: • http://www.softberry.com/berry.phtml?topic=index&group=programs&subgroup=promoter • http://bioinformatics.ubc.ca/resources/links_directory/?subcategory_id=104 • http://www3.oup.co.uk/nar/database/subcat/1/4/ D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  20. New Today: RNA Structure & Function D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  21. RNA Structure & Function • RNA structure • Levels of organization • Bonds & energetics • (more about this on Wed) • RNA types & functions • Genomic information storage/transfer • Structural • Catalytic • Regulatory D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  22. RNA structure: 3 levels of organization Rob Knight Univ Colorado D Dobbs ISU - BCB 444/544X: RNA Structure & Function

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

  24. Base-pairing in RNA G-C, A-U, G-U ("wobble") & variants See: IMB Image Library of Biological Molecules http://www.imbjena.de/ImgLibDoc/nana/IMAGE_NANA.html#sec_element D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  25. Common structural motifs in RNA • Helices • Loops • Hairpin • Interior • Bulge • Multibranch • Pseudoknots Fig 6.2 Baxevanis & Ouellette 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  26. RNA functions • Storage/transfer of genetic information • Structural • Catalytic • Regulatory D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  27. 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 D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  28. RNA functions • Structural • e.g., rRNA, which is major structural component of ribosomes (Gloria Culver, ISU) • 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, manysmall RNAs are enzymes "ribozymes" (W Allen Miller, ISU) D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  29. 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) D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  30. RNA types & functions D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  31. Thanks to Chris Burge, MITfor following slidesSlightly modified from:Gene Regulation and MicroRNAsSession introduction presented at ISMB 2005, Detroit, MIChris Burgecburge@MIT.EDU C Burge 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function

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

  33. Lots of data Genomes, structures, transcripts, microarrays, ChIP-Chip, etc. 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 C Burge 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  34. Steps in Transcription Emerson Cell 2002 C Burge 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  35. • typically bind in clusters » Regulatory modules Sequence-specific Transcription Factors Kadonaga Cell 2004 C Burge 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  36. • have modular organization » Understand DNA-binding specificity Sequence-specific Transcription Factors 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 D Dobbs ISU - BCB 444/544X: RNA Structure & Function

  37. Integration of transcription & RNA processing Maniatis & Reed Nature 2002 C Burge 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function

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

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

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

  41. Coupling of Splicing & Nonsense-mediated mRNA Decay (NMD) Maniatis & Reed Nature 2002 C Burge 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function

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

  43. MicroRNA Biogenesis N. Kim Nature Rev Mol Cell Biol 2005 C Burge 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function

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

  45. 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 Need to modify traditional paradigm of "transcriptional control" by protein-DNA interactions to include miRNA regulatory mechanisms C Burge 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function

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