10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function1 10/31/05 RNA Structure & Function

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function2 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)

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function3 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)

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function4 RNA Structure & Function Prediction Mon Review - promoter prediction RNA structure & function WedRNA structure prediction 2' & 3' structure prediction miRNA & target prediction RNA function prediction?

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function5 Reading Assignment (for Mon/Wed) Mount Bioinformatics Chp 8 Prediction of RNA Secondary Structure pp Ck Errata: Cates (Online) RNA Secondary Structure Prediction Module

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function6 Review last lecture: Promoter Prediction

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function7 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!

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function8 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 genomes available

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function9 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)

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function10 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 FirstEFFirstEF found within UCSC Genome BrowserUCSC Genome Browser or submit to FirstEF web serverFirstEF Fig 5.10 Baxevanis & Ouellette 2005

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function11 Promoter prediction strategies 1)Pattern-driven algorithms 2)Sequence-driven algorithms 3)Combined "evidence-based" BEST RESULTS? Combined, sequential

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function12 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

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function13 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

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function14 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….

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function15 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! Promoter Prediction: Sequence-driven algorithms

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function16 Examples of promoter prediction/characterization software Lab: used MATCH, MatInspectorMATCHMatInspector TRANSFAC MEME & MAST BLASTBLAST, etc. Others? FIRST EF Dragon Promoter FinderDragon Promoter Finder (these are links in PPTs) also see Dragon Genome Explorer (has specialized promoter software for GC-rich DNA, finding CpG islands, etc)Dragon Genome Explorer JASPAR

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function17 Global alignment of human & mouse obese gene promoters (200 bp upstream from TSS) Fig 5.14 Baxevanis & Ouellette 2005

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function18 Check out optional review & try associated tutorial: Wasserman WW & Sandelin A (2004) Applied bioinformatics for identification of regulatory elements. Nat Rev Genet 5: Check this out:

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function19 Annotated lists of promoter databases & promoter prediction software URLs from Mount Chp 9, available online Table Table in Wasserman & Sandelin Nat Rev Genet article URLs for Baxevanis & Ouellette, Chp 5: More lists: ter ter

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function20 New Today: RNA Structure & Function

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function21 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

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function22 Rob Knight Univ Colorado RNA structure: 3 levels of organization

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function23 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

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function24 G-C, A-U, G-U ("wobble") & variants Base-pairing in RNA See: IMB Image Library of Biological MoleculesIMB Image Library of Biological Molecules

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function25 Fig 6.2 Baxevanis & Ouellette 2005 Common structural motifs in RNA Helices Loops Hairpin Interior Bulge Multibranch Pseudoknots

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function26 RNA functions Storage/transfer of genetic information Structural Catalytic Regulatory

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function27 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

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function28 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, many small RNAs are enzymes "ribozymes" (W Allen Miller, ISU)

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function29 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)

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function30 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??

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function31 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 C Burge 2005

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function32 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

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function33 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

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function34 Steps in Transcription Emerson Cell 2002 C Burge 2005

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function35 Sequence-specific Transcription Factors typically bind in clusters » Regulatory modules Kadonaga Cell 2004 C Burge 2005

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function36 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 C Burge 2005

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function37 Maniatis & Reed Nature 2002 Integration of transcription & RNA processing C Burge 2005

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function38 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

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function39 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

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function40 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

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function41 Coupling of Splicing & Nonsense-mediated mRNA Decay (NMD) Maniatis & Reed Nature 2002 C Burge 2005

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function42 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

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function43 MicroRNA Biogenesis N. Kim Nature Rev Mol Cell Biol 2005 C Burge 2005

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function44 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

10/31/05 D Dobbs ISU - BCB 444/544X: RNA Structure & Function45 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" by protein-DNA interactions to include miRNA regulatory mechanisms