The Search for Small Regulatory RNA Central Dogma

The Search for Small Regulatory RNA

Central Dogma: DNA to RNA to Protein hn. RNA m. RNA Replication Processing / Translocation r. RNA t. RNA

Central Dogma: DNA to RNA to Protein hn. RNA m. RNA (unstable RNA) Replication Processing / Translocation r. RNA (Stable RNA) t. RNA

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

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

Other stable RNA Telomerase DNA polymerase-independent repeated sequence

Other stable RNA Telomerase Telomere growth

Other stable RNA Spliceosome RNA hn. RNA m. RNA introns

Other stable RNAse P t. RNA maturation Protein + RNA

Other stable RNA SRP RNA Signal Recognition Particles protein RNA

Other stable RNA • Fundamental processes of life (left over from RNA world? ) • Nearly universal, highly conserved • Not many

Basis of RNA function Sequence ? ? ? Structure Function

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

Prediction of RNA secondary Try it! Go to www. people. vcu. edu/~elhaij/bnfo 650 Try Problem 1 with simple oligonucleotides

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General Syntax of Bio. BIKE Function-name Argument (object) Keyword object The basic unit of Bio. BIKE 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. Flag

General Syntax of Bio. BIKE Function-name Argument (object) Keyword object Flag Function boxes contain the following elements: • Function-name (e. g. SEQUENCE-OF or LENGTH-OF) • Argument: Required, acted on by function • Keyword clause: Optional, more information • Flag: Optional, more (yes/no) information

General Syntax of Bio. BIKE 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 • Action icon: Brings up a menu enabling you to execute a function, copy and paste, information, get help, etc Clear/Delete icon: Removes information you entered or removes box entirely •

Prediction of RNA secondary Try it! Go to www. people. vcu. edu/~elhaij/bnfo 650 Try Problem 2 with mystery sequence 1 (a short oligonucleotide)

Prediction of RNA secondary Try it! Go to www. people. vcu. edu/~elhaij/bnfo 650 Try Problem 3 with mystery sequence 2 (a more realistic oligonucleotide)

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 • Limited to 2 -dimensional structure • Ignorant of pseudoknots • True structures can be predicted • Surrounding sequence can obscure true structure

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

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

Basis of Cosuppression RNA virus Double-stranded RNA

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

Small Noncoding RNA si. RNA – Inhibits expression of exogenous genes RNAi – Human exploitation of phenomenon mi. RNA – Inhibits expression of endogenous genes

mi. RNA expression Ambion Corp. http: //www. ambion. com/techlib/resources/mi. RNA/mirna_pro. html

mi. RNA function

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

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

…but Eubacteria?

…but Eubacteria?

Role of RNA-binding protein Hfq

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

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

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

How to identify s. RNA? Transcriptional features Promoter Short sequence Terminator

How to identify s. RNA? RNAz Combines two measures: Thermodynamic stability (Z score) Z= MFE – MFErandom σrandom MFE Minimum Free Energy

How to identify s. RNA? 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

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

Strategy to find small RNAs Prochlorococcus MED 4 Prochlorococcus #2 Prochlorococcus #3 Prochlorococcus #4 Conserved?

Prediction of RNA secondary Try it! Go to www. people. vcu. edu/~elhaij/bnfo 650 Try Problem 4, testing whether RNAz can find a known small RNA

Prediction of RNA secondary Try it! Go to www. people. vcu. edu/~elhaij/bnfo 650 Try Problem 5, testing whether RNAz predicts a small RNA between dmt. B and trp. D dmt. B ? ? ? trp. D