1. www..uni-rostock.de Bioinformatics Computational methods to discover ncRNA in bacteria Ulf Schmitz ulf.schmitz@informatik.uni-rostock.de Bioinformatics and Systems Biology Group www.sbi.informatik.uni-rostock.de

2. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA2 Outline 1. Problem description 2. Streptoccocus pyogenes 3. The RNome, transcriptome 4. Characteristics of bacterial ncRNA 5. Approaches to find fRNA 6. Conclusion / Outlook

3. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA3 Streptococcus pyogenes important human pathogen (group A streptococcus or GAS) causes following diseases: –pyoderma (111 million cases/year) –pharyngitis (616 million cases/year and 517,000 deaths/year) pyoderma (source: DermNet NZ) pharyngitis (source: UCSD) completely adapted to humans as it’s only natural host causes purulent infections of the skin and mucous membranes and rarely life-threatening systemic diseases

4. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA4 Streptococcus pyogenes  varies in multiplication rate -> associated with type of infection  to understand the regulation, one studied the growth-phase regulatory factors and gene expression in response to specific environmental differences within the host  a novel growth phase assosiated two-component-type regulator was identified  fasBCA operon, present in all 12 tested M serotypes  contained two potential HPK genes (FasB, FasC) and one RR (FasA)  shows its maximum expression and activity at the transition phase  and to potentially support the aggressive spreading of the bacteria in its host HPK = Histidine protein kinase RR = response regulator

5. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA5 Streptococcus pyogenes downstream of the fas operon they identified a ~300 nucleotide transcript (fasX) not encoding for a peptide/protein –but also growth phase related –main effector molecule of fas regulon ncRNA or fRNA

6. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA6 ncRNA gltX-L fasB fasCfasA p fas p fasX tt p rnpA rnpA-L fasX 1kb

7. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA7 RNome or transcriptome RNA mRNA ncRNA / fRNA snmRNA / sRNA Structural RNA tRNA rRNA miRNAsiRNAsnRNAsnoRNAstRNA putative gene expression regulators (also protein interaction – and housekeeping ncRNAs where found)

8. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA8 RNome or transcriptome fRNAFunctional RNAessentially synonymous with non-coding RNA miRNAMicroRNA21-24 nucleotide RNAs probably acting as translational regulators mRNA siRNASmall interfering RNAactive molecules in RNA Interference snRNASmall nuclear RNAincludes spliceosomal RNAs snmRNASmall non-mRNAessentially synonymous with small ncRNAs snoRNASmall nucleolar RNAmost known snoRNAs are involved in rRNA modification stRNASmall temporal RNAfor example, lin-4 and let-7 in Caenorhabditis elegans Non-coding RNA (ncRNA) genes produce functional RNA molecules rather than encoding proteins and here are the nominees: mRNAmessenger RNA - transcript of a protein coding gene rRNAribosomal RNA - form large parts of the ribosome, the protein producing machinary tRNA transfer RNA - also involved in protein production, carrying single amino acids to the growing amino acid chain of a protein ncRNAnon coding RNA - found in intergenic regions, playing miscellaneous roles types of RNA:

9. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA9 Functions of ncRNA …target mRNAs via imperfect sequence complementarity binding may result in: blockage of ribosome entry (translation repression) melting of inhibitory secondary structures (translation activation) loop-loop kissing complex dissolving fold the fold back structure

10. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA10 Streptococcus pyogenes genomes SerotypeLengthDate M1 GAS1852441 bpSep 19 2001 MGAS102701928252 bpMay 4 2006 MGAS103941899877 bpAug 3 2004 MGAS107501937111 bpMay 4 2006 MGAS20961860355 bpMay 4 2006 MGAS3151900521 bpJul 18 2002 MGAS50051838554 bpAug 8 2005 MGAS61801897573 bpAug 8 2005 MGAS82321895017 bpJan 31 2002 MGAS94291836467 bpMay 4 2006 SSI-11836467 bpMay 4 2006 Genome Info & Features: Genes:1805 Protein coding1697 Length1,852,441 nt Structural RNAs:73 GC Content:38% Pseudo genes:35 Coding:83% Topology:circular MoleculedsDNA

11. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA11 Intergenic sequence inspector (ISI) IGR extractor Annotated genome IGR filteringBLASTBLAST AnalyserGenview Final results IGR databank Filtered IGR databank BLAST results Aligned features Sequence features Bacterial genomes database

12. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA12 Characteristics of bacterial ncRNA intergenic sequence/structure conservation between related genomes encoded by free-standing genes, oriented in opposite fashion to both flanking genes 50 to 400 nt long (avrg. >200nt) higher G+C content than average intergenic space σ 70 promoter ρ – independent terminator imperfect sequence complementary with target mRNA

13. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA13 Characteristics of bacterial ncRNA CA 90 T Promotor T 82 T 84 G 78 A 65 C 54 A 45 T 80 A 95 T 45 A 60 A 50 T 96 -35-10 16-19bp 5-9bp Startpoint intrinsic terminator

14. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA14 The structure approach with RNAz 1.multiple sequence alignment 2.measure of thermodynamic stability (z score) 3.measure for RNA secondary structure conservation Function of many ncRNAs depend on a defined secondary structure

15. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA15 calculation of the MFE (minimum free energy) as a measure of thermodynamic stability MFE depends on the length and the base composition of the sequence –and is therefor difficult to interpret in absolute terms RNAz calculates a normalized measure of thermodynamic stability by –compares the MFE m of a given (native) sequence –with the MFEs of a large number of random sequences with similar length and base composition. A z-score is calculated as, where µ and σ are the mean and standard deviations, resp., of the MFEs of the random samples negative z score indicates the a sequence is more stable than expected by chance The structure approach Thermodynamic stability

16. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA16 RNAz predicts a consensus secondary structure for an alignment –results in a consensus MFE E A RNAz compares this consensus MFE to the average MFE of the individual sequences Ē and calculates a structure conservation index: SCI will be low if no consensus fold can be found. The structure approach Structural conservation

17. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA17 The structure approach z-score and SCI, are used to classify an alignment as “structural RNA” or “other”. RNAz uses a support vector machine (SVM) learning algorithm which is trained on a set of known ncRNAs.

18. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA18 Analysis pipeline of Freiburg group extraction of intergenic regions ≥50nt BLASTN E-value ≤10 -8 discard no reverse complement Unify overlapping Clustering Scoring local alignment of IGRs with BLASTN of candidate sequences to reduce redundancy using ClustalW using RNAz

19. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA19 Summary / Conclusion there are ‘reliable’ computational methods to find ncRNA coding genes in bacteria key methods involve: –IGR extraction and filtering –observing sequence conservation in related genomes (BLAST search, ClustalW alignment) –checking for structure conservation and thermodynamic stability next step is to proof their existance experimentally via microArrays or Northern Blots

20. www..uni-rostock.de Ulf Schmitz, Computational methods to discover ncRNA20 Outlook might it be possible to predict target mRNA? Thanks for your attention!