1. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 1 Stanislav Luban 1,2 Daisuke Kihara 2,1 1. Department of Computer Sciences 2. Department of Biological Sciences Purdue University, West Lafayette, IN Comparative Study of Small RNA and Small Peptides in Complete Genome Sequences

2. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 2 Introduction: Structural Small RNA (sRNA) Genes which produce non-coding transcripts that function directly as structural, regulatory, or catalytic RNAs Include rRNAs, tRNAs, small nucleolar RNAs, spliceosomal RNAs, viral associated RNAs, microRNAs, ctRNAs, and others In Rfam (RNA families) database, 34496 sRNA entries distributed among 352 known families are stored In E. coli, about 50 sRNAs are known (figure from Rfam database: http://www.sanger.ac.uk/Software/Rfam/)

3. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 3 Methods: QRNA Model distinctive pattern of mutation: Conserved Structural RNA Pattern of compensatory mutations consistent with base-paired secondary structure Pair Stochastic Context-Free Grammar Model Conserved Coding Region Pattern of synonymous codon substitutions Pair Hidden Markov Model Other Types of Conserved Regions Approximated by “null hypothesis” that mutations occur position independently, without pattern Pair Hidden Markov Model Scores are log likelihoods used to calculate final log odds score for RNA model compared to other two models (Figure: Rivas et al, Current Biol. 2001)

4. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 4 Procedure for Extracting sRNAs Extract Intergenic Regions From 30 Sequenced Genomes Perform All Vs. All Nucleotide- Nucleotide BLAST Run QRNA, Extract Alignments Scoring as sRNAs vs. Coding and Null Hypothesis Regions Select Significant Alignments, Concatenate and Format into QRNA Program Input Verify Results Computationally And Experimentally (Yet To Be Done) Eliminate Alignment Regions Which Overlap >50% with E. coli Regulatory Regions Extend Regions Within 25 nt Of Other Regions Causing Them To Include Each Other Merge sRNA Regions Which Align or Exactly Overlap Into Families Eliminate Family Regions Not Found Using Both Query And Database Organism As Source

5. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 5 Genome Data Set 30 Microbial Genomes Used as Queries and Databases: Gammaproteobacteria Acinetobacter calcoaceticus Blochmannia floridanus Buchnera aphidicola Coxiella burnetii Erwinia carotovora Escherichia coli Haemophilus ducreyi Haemophilus influenzae Pasteurella multocida Photorhabdus luminescens Pseudomonas aeruginosa Pseudomonas putida Pseudomonas syringae Salmonella enterica Salmonella typhimurium Shewanella oneidensis Shigella flexneri Vibrio cholerae Vibrio parahaemolyticus Vibrio vulnificus Wigglesworthia brevipalpis Xanthomonas campestris Xanthomonas citri Xylella fastidiosa Yersinia pestis Alphaproteobacteria Agrobacterium tumefaciens Brucella melitensis Caulobacter crescentus Mesorhizobium loti Deinococci Deinococcus radiodurans

6. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 6 Result Statistics Total number of intergenic regions: 94464 Average number of intergenic regions per organism: 3148.8 Total combined length of intergenic regions: 16663732 nt Average length of intergenic region: 176.4 nt

7. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 7 sRNA Length vs. Score Plot Total: 29488 sRNAs

8. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 8 Total: 29488 sRNAs Number of sRNA Entries by Organism 1 - Pseudomonas putida 2 - Shigella flexneri 3 - Xanthomonas citri 4 - Shewanella oneidensis 5 - Wigglesworthia brevipalpis 6 - Haemophilus ducreyi 7 - Pseudomonas syringae 8 - Erwinia carotovora 9 - Escherichia coli 10 - Vibrio parahaemolyticus 11 - Mesorhizobium loti 12 - Buchnera aphidicola 13 - Brucella melitensis 14 - Yersinia pestis 15 - Xylella fastidiosa 16 - Pseudomonas aeruginosa 17 - Salmonella enterica 18 - Caulobacter crescentus 19 - Agrobacterium tumefaciens 20 - Blochmannia floridanus 21 - Pasteurella multocida 22 - Deinococcus radiodurans 23 - Vibrio cholerae 24 - Photorhabdus luminescens 25 - Coxiella burnetii 26 - Vibrio vulnificus 27 - Salmonella typhimurium 28 - Acinetobacter calcoaceticus 29 - Xanthomonas campestris 30 - Haemophilus influenzae

9. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 9 Conservation of sRNAs Total: 3768 families

10. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 10 Conservation of sRNAs Total: 3768 families E. Coli Total: 554 families Along with statistics for all entries, statistics for entries containing at least one entry from E. coli were added for comparison

11. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 11 Common Organism Combinations in Families Top 5 most frequent combinations of 4 and 7 organisms: Combination: Occurances: Ecoli, Senterica, Sflexneri, Styphimurium117 Ecarotovora, Ecoli, Senterica, Styphimurium26 Ecoli, Senterica, Styphimurium, Ypestis20 Ecarotovora, Ecoli, Sflexneri, Styphimurium18 Ecoli, Sflexneri, Styphimurium, Ypestis17 Ecarotovora, Ecoli, Pluminescens, Senterica, Sflexneri, Styphimurium, Ypestis 4 Acalcoaceticus, Ccrescentus, Mloti, Paeruginosa, Pputida, Psyringae, Xcampestris 2 Acalcoaceticus, Atumefaciens, Ccrescentus, Mloti, Pputida, Psyringae, Xcampestris 2 Acalcoaceticus, Atumefaciens, Ccrescentus, Mloti, Paeruginosa, Psyringae, Xcampestris 2 Acalcoaceticus, Atumefaciens, Ccrescentus, Mloti, Paeruginosa, Pputida, Xcampestris 2

12. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 12 Result Verification 71 total sRNAs related to E. coli already found to be annotated in Rfam database were used as benchmark Of those: 15 – found by computational method that were also listed in Rfam and not tRNAs 6 – not found due to shortcomings of method 29 – tRNAs already annotated as gene loci in E. coli genome sequence used 10 – E. coli plasmid loci not found in full E. coli genome sequence used 2 – 4.5S RNAs already annotated as gene loci in E. coli genome sequence used 2 – E. coli reverse transcriptase loci not found in full E. coli genome sequence used 1 – E. coli insertion sequence not found in full E. coli genome sequence used 1 – E. coli small RNA annotated separately, not found in full E. coli genome sequence used 1 – Antisense RNA already annotated as gene locus in E. coli genome sequence used 1 – Cloning vector with E. coli promoter not found in full E. coli genome sequence used 1 – E. coli transposable element not found in full E. coli genome sequence used 1 – Reporter vector not found in full E. coli genome sequence used 1 – E. coli retron not found in full E. coli genome sequence used

13. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 13 Candidates for Experimental Verification of Findings For the following 2 slides: Family designation expressed as [Organism name] [locus absolute start location] [locus absolute end location] and is synonymous with the first (header) entry of that family Entries refer to number of different organism (2 chromosomes counted separately) sRNA entries in the family Length (nt) and score only refer to the header entry of the family Scores calculated by QRNA program with log odds post for RNA likelihood as opposed to null hypothesis

14. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 14 Candidates for Experimental Verification of Findings Top 10 highest statistically scoring E. coli sRNA loci found by computational method: Family designation: Ecoli 3941194 3941327 Length: 133Score: 34.114 Family designation: Ecoli 2744345 2744445 Length: 100Score: 29.631 Family designation: Ecoli 780875 781068 Length: 193Score: 29.194 Family designation: Ecoli 2687537 2687689 Length: 152Score: 27.734 Family designation: Ecoli 2519348 2519548 Length: 200 Score: 23.876 Family designation: Ecoli 4169337 4169400 Length: 63Score: 21.625 Family designation: Ecoli 4038218 4038281 Length: 63Score: 21.596 Family designation: Ecoli 2751994 2752022 Length: 28Score: 20.893 Family designation: Ecoli 3420989 3421058Length: 69Score: 20.821 Family designation: Ecoli 3808832 3808858Length: 26Score: 16.995

15. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 15 Candidates for Experimental Verification of Findings Top 10 largest sRNA families found by computational method: Family designation: Styphimurium 3358766 3358804Entries: 18 Length: 38Score: 4.590 Family designation: Ecarotovora 3161909 3161946Entries: 15 Length: 37Score: 12.604 Family designation: Ecarotovora 1144121 1144141 Entries: 12 Length: 20Score: 5.265 Family designation: Styphimurium 3342804 3342899 Entries: 12 Length: 95Score: 4.328 Family designation: Ecarotovora 2597534 2597593 Entries: 10 Length: 59Score: 3.343 Family designation: Paeruginosa 2508264 2508282 Entries: 9 Length: 18Score: 7.068 Family designation: Styphimurium 975191 975219Entries: 8 Length: 28Score: 16.296 Family designation: Styphimuriu 3746886 3746903 Entries: 8 Length: 17Score: 1.146 Family designation: Ecarotovora 3477891 3477922 Entries: 8 Length: 31Score: 2.697 Family designation: Ecarotovora 4490537 4490683*Entries: 7 Length: 146 Score: 16.753 *This last entry was used a sample for detailed study and is discussed subsequently.

16. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 16 Detailed Study of Located Sample sRNA OrganismLocation (in genome)Length(nt)ScoreNeighboring Genes Ecarotovora 4490537-4490683 146 16.753 rpsM - rpmJ Pluminescens 5487752-5487866 114 10.791 rpsM - secY Ypestis 232330-232476 146 15.757 rpmJ - rpsM Styphimurium 3585744-3585879 135 41.980 rpsM - rpmJ Senterica 4243623-4243770 147 40.046 rpmJ - rpsM Ecoli 3440108-3440255 147 43.556 rpsM - rpmJ Sflexneri 3426855-3427002 147 41.980 rpsM - rpmJ Hit to Alpha_RBS RNA (Rfam: RF00140) (115 nt) Rfam Sequence: GUCCUUGAUAUUCUGUUUGAGUAUCCUGAAAACGGGCUUUUCAAGAUCAGAAUAUCAAAUUA AUUAAAAUAUAGGAGUGCAUAGUGGCCCGUAUUGCAGGCAUUAACAUUCCUGAU

17. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 17 Most Likely (Lowest Free Energy) Predicted Fold of 80 nt Segment of Sequence Mfold by Zuker et al, 2004 Used Detailed Study of Located Sample sRNA

18. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 18 Another Approach to Finding sRNAs in E. Coli: Paper Summary

19. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 19 Method Used in Paper to Find Putative sRNAs A database of all E. coli intergenic DNA sequences was created based on gene annotations in early release of the EcoGene database, and used as input to profile search program (pftools2.2, Swiss Bioinformatics Institute) set to find sigma-70 promoter Terminator motif was searched for in database using following search criteria: (1) An 11-nt A-rich region; (2) variable-length hairpin; (3) variable-length spacer; (4) 5-nt T-rich region nearest the hairpin; and (5) 7-nt distal extra T-rich region Predicted promoter and terminator pairs were combined to generate putative sRNAs if (1) pair was on same strand; and (2) pair was greater than 45 but less than 350 nt apart To verify, open reading frames and possible ribosome binding sites were searched for downstream of each promoter

20. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 20 Synopsis of Method Used in Paper Using the E. Coli MG1655 genome, DNA regions that contained a sigma-70 promoter within a short distance of a rho-independent terminator were searched for 227 putative sRNAs between 80 and 400 nt in length were predicted in E. coli by paper, 32 of which were already known to be sRNAs Transcripts of some of the candidate loci were verified using Northern hybridization Approach may possibly be used in annotating sRNA loci in other bacterial genomes

21. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 21 Verification of Paper Results with Results Using Our Method Along with other results, the paper gives a detailed listing of the 277 sRNAs predicted, including the designation, strand orientation (forward or reverse), left and right boundaries (nt from genome start position), and length (nt) of each sRNA Left and right boundary positions in genome given by paper were compared with left and right boundary positions of putative sRNAs found by our method If an sRNA candidate from the paper was within 100 nt of any sRNA predicted by our method, that sRNA was scored as ‘found’

22. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 22 Results of Verification 227 candidate sRNAs were predicted in E. coli by the paper Among them, 150 (66.1 %) were localized by our method, according to previously utilized criteria The test was re-run with a 50 nt threshold, yielding 140 hits (61.7 %), a 10 nt threshold, yielding 128 hits (56.4 %), and a 1000 nt threshold, yielding 187 hits (82.4 %)

23. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 23 Preliminary Procedure for Extracting Small Peptides Extract Intergenic Regions From 30 Sequenced Genomes Perform All Vs. All Nucleotide- Nucleotide BLAST Run QRNA, Extract Alignments Scoring as Coding vs. sRNA and Null Hypothesis Regions Select Significant Alignments, Concatenate and Format into QRNA Program Input Observe Results and Refine Extraction Method Extend Regions Within 25 nt Of Other Reions Causing Them To Include Each Other Merge sRNA Regions Which Align or Exactly Overlap Into Families Blast Resulting Family Entries Against SwissProt Database Score Regions Based on Quality of Fit Inside a Nearby Open Reading Frame

24. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 24 Preliminary Results of Small Peptide Search OrganismLocation (in genome)Length(nt)E-Value Erwinia Carotovora843815-8439481330.69 Aligns To gb|AAF36091.1| flagelliform silk protein [Nephila madagascariensis] Sequence aattccgtcgcatgttctctggtgagtacgacagcgcggattgctatctggatattcaggcgggatctggcggtacgg aagcgcaggactgggccagcatgctggtacgtatgtacctgcgttgggcggaagc Query: 133 LPPNAGTYVPACWPSPALPYRQIPPEYPDSNP 38 Subject: 1373 LPPLXTSXXPPPPPPPSXPLXSLPPSXPPSLP 1278 Tblastx Alignment Query Sequence Information

25. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 25 Preliminary Results of Small Peptide Search OrganismLocation (in genome) Length(nt)E-Value Pseudomonas syringae6171796-6172006 2100.23 Aligns To emb|CAD88221.2| C. elegans GRL-25 protein (corresponding sequence ZK 643.8) Sequence tgagttccggcagctcgtcatccagcttctgacgcaaccgcccggtcagaaacgcaaagccctcgagcaaccgct ccacatccggatcccgtccggcctgccccagaaacggcgccaacgccggactacgctcggcgaagcgacgac caagctggcgcagtgcagtgagttcgctctggtagtaatggttaaaggacacgggttacctgc Query: 62 PRATAPHPDPVRPAPETAPTP 124 Subject: 90 PPAPAPRPPPVAPAPRPLPPP 28 Tblastx Alignment Query Sequence Information

26. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 26 Conclusions Possible sRNAs are found from 20~39% of the intergenic regions in each organism Among them, ~31% of the sRNAs satisfy the log-odds score threshold of 5.0 or higher 137 “families” are conserved in equal to or more than 5 organisms Being well conserved, sRNAs may be responsible for fundamental functions of living organisms

27. 3.31.2005BIOL497 Undergraduate Presentation, Stanislav Luban, Member of Kihara Lab, Purdue Univ. 27 Future Direction Search for sRNAs will be expanded to a larger quantity of more diverse genomes Secondary structure prediction will be later employed in greater detail to verify well conserved sRNA regions among multiple evolutionarily distant organisms Experimental verification of the findings of this particular study under way (particularly for Shewanella oneidensis) Comparative genomics will be used to discover the function associated with each sRNA and possibly lead to learning its part in pathway