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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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10/31/05RNA Structure & Function D Dobbs ISU - BCB 444/544X: 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
  • D Dobbs ISU - BCB 444/544X: RNA Structure & Function 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 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 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 Review last lecture:Promoter Prediction D Dobbs ISU - BCB 444/544X: RNA Structure & Function 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 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 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 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 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 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 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 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 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 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 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 Check out optional review & bp upstream from TSS)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 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 New Today: softwareRNA Structure & Function D Dobbs ISU - BCB 444/544X: RNA Structure & Function RNA Structure & Function software
  • 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 RNA structure: 3 levels of organization software Rob Knight Univ Colorado D Dobbs ISU - BCB 444/544X: RNA Structure & Function Covalent & non-covalent bonds in RNA software
  • 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 Base-pairing in RNA software 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 Common structural motifs in RNA software
  • Helices
  • Loops
  • Hairpin
  • Interior
  • Bulge
  • Multibranch
  • Pseudoknots
  • Fig 6.2 Baxevanis & Ouellette 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function RNA functions software
  • Storage/transfer of genetic information
  • Structural
  • Catalytic
  • Regulatory
  • D Dobbs ISU - BCB 444/544X: RNA Structure & Function RNA functions software
  • 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 RNA functions software
  • 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 RNA functions software
  • 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 RNA types & functions software D Dobbs ISU - BCB 444/544X: RNA Structure & Function Thanks to Chris Burge, MIT softwarefor following slidesSlightly modified from:Gene Regulation and MicroRNAsSession introduction presented at ISMB 2005, Detroit, MIChris [email protected]/* */ C Burge 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function Protein Coding Gene software … 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 Lots of data software 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 Steps in Transcription software Emerson Cell 2002 C Burge 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function • typically bind in clusters software » Regulatory modules Sequence-specific Transcription Factors Kadonaga Cell 2004 C Burge 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function • have modular organization software » 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 Integration of softwaretranscription & RNA processing Maniatis & Reed Nature 2002 C Burge 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function Early Steps in Pre-mRNA Splicing software • 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 Alternative Splicing software > 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 ESE/ESS = Exonic Splicing Enhancers/Silencers software 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 Coupling of Splicing & Nonsense-mediated mRNA Decay (NMD) software Maniatis & Reed Nature 2002 C Burge 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function lin-4 software 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 MicroRNA Biogenesis software N. Kim Nature Rev Mol Cell Biol 2005 C Burge 2005 D Dobbs ISU - BCB 444/544X: RNA Structure & Function microRNA pathway software 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 miRNA Challenges for Computational Biology software • 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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