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RNA – A New Role

RNA – A New Role. Amy Anderson. On You!. Off You!. Introduction – How’d they do that?. 40+ years, still no complete answer…. Gene. Gene. Gene. Gene. How does gene expression get turned on and off?. Anabaena. Heterocysts. How do the cells differentiate?.

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RNA – A New Role

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  1. RNA – A New Role Amy Anderson

  2. On You! Off You! Introduction – How’d they do that? 40+ years, still no complete answer… Gene Gene Gene Gene How does gene expression get turned on and off?

  3. Anabaena Heterocysts How do the cells differentiate?

  4. Originally researchers looked for proteins… …GCCAATGTCAAAGATTTAGTAGAGACTTGCATATCGTTCACTCCGTGAGTAAGTTTTTGTAATTAACGT… Transcription …but nothing was found to account for everything. So we need something new.

  5. Where to look? E. coli!

  6. E. coli’s Regulatory Mechanism – small RNA • Small DNA sequence in between two genes • Transcribed RNA bound to mRNA of targeted genes • Binding blocked mRNA from being translated. Gene 1 Gene 2 …AATGCGCTA…GAATTATCGCG… 3. Blocks mRNA translation into protein This small RNA is called rhyB. It regulates the expression of proteins associated with the presence of iron. 1. Transcribed into RNA GCUA…GAAUUA 2. sRNA binds to mRNA containing targeted gene …GCUCUUACGUCGUCGAUUGCAGCGUA…

  7. Regulation of rhyB in E.coli RNA Polymerase Fe+2 Fe+2 Fe+2 Fe+2 Fe+2 Fe+2 Fe+2 Fe+2 Fe+2 Fe+2 ……..ACTAAAGTTGCCAATGTCAAAGATTTAGTAGAGACTTGCATATCGA…….TTCACTCCGTGAGTAAGTTTTTGTAATTAACGT…….. rhyB rhyB Promoter site Fe+2 fur Fe+2 Fe+2 Fe+2 Fe+2 Fe+2 Fe+2

  8. UUU ! UAUUACUU ! ! GGAGAAC Secondary Structure of rhyB …. GUUAACUAUUTUTTGAUGUGCCAA…. rhyB binding site mRNA of targeted gene

  9. DNA of multiple targeted genes mRNA of targeted genes with rhyB binding site

  10. Gene 1 Gene 2 …AATGCGCTA…GAATTATCGCG… …TTACGCGAT…CTTAATAGCGC… …TTACGCGAT…CTTAATAGCGC… …AATGCGCTA…GAATTATCGCG… …AATGCGCTA…GAATTATCGCG… Does Anabaena have a similar mechanism? What I was looking for: - Sequences that looked like an RNA (lollypops on a bubble) My Strategy: - BLAST the intergenic region surrounding each motif. - Look for partial matches that lie antisense (found on the opposite strand of) a gene (preferably one from my list). What I was given: - Set of motifs (sequences) that frequently showed up upstream from possible heterocyst related genes. - A list of possible heterocyst related genes Motifs: M10 - CACGTTATCTGTTGAGACCGGGTGTAAGGGTTT M11 - TACACCCTTTTCCAAACCCTTGATCTTTCGTTTTCATGCGTAAGT M12 - AAAACTCTACCCACAAGGGGATAGAGTTTTGTCAGTGGTCAGTGG ……

  11. ACGT…M10…AGGT ACGT…M10…AGGT ACGT…M10…AGGT ACGT…M10…AGGT ACGT…M10…AGGT ACGT…M10…AGGT ACGT…M10…AGGT ACGT…M10…AGGT ACGT…M10…AGGT ACGT…M10…AGGT ACGT…M10…AGGT ACGT…M10…AGGT ACGT…M10…AGGT ACGT…M10…AGGT ACGT…M10…AGGT alr4468 all2909 alr3693 asl1274 all4219 asl1111 all3558 all3239 all1124 all1683 asl2850 alr2744 all4668 all1782 alr4788 alr3240 all1781 all2908 all4218 alr2851 asr3694 all3557 all1682 alr4469 all4789 all1123 asr1275 alr2745 alr1112 asr4669 Intergenic regions Motif 10 was found in: D5 P13 D7 P16 D15 P19 D17 P20 D24 P21 P6 P22 P10 C26 P11

  12. BLAST hit alr0787 all0788 12-15 nucleotide overlap Most intergenic regions containing motif 10 hit this spot.

  13. Took an alignment of the portions that hit all0788. Seq 3 0 ---------- ---------- ---------- ---------- ---------- Seq 6 1 ---------- ---------- ---------- ---------- ---GGGTGTA Seq 10 0 ---------- ---------- ---------- ---------- ---------- Seq 7 0 ---------- ---------- ---------- ---------- ---------- Seq 4 0 ---------- ---------- ---------- ---------- ---------- Seq 5 0 ---------- ---------- ---------- ---------- ---------- Seq 1 1 ---------- ---------- ---------- ---------- ---GGGTGTA Seq 8 0 ---------- ---------- ---------- ---------- ---------- Seq 9 1 CAGGTTATCT GTTGAGACCG GGTGTAAGGG TTTAAGGGTA CAGGGGTGTA Seq 11 1 -AGGTTATCT GTTGAGACCG GGTGTAAGGG TTTAAGGGTA CAGGGGTGTA Seq 2 1 ---------- ---------- ---------- ---------- ---GGGTGTA Seq 12 1 ---------- ---------- ---------- ---------- ---GGGTGTA consensus 1 Seq 3 1 ---------- ---------- ------ACCC CTAC------ -ACCCTTCTC Seq 6 8 AGGGTTTCAA AAATTTATAC CCCTATACCC CTAC------ -ACCCTTGTC Seq 10 1 ---------- ---------- ----ATACCC CTAC------ -AACCTTTTC Seq 7 1 ---------- ---------- ----ATACCC CTAC------ -ACCCTTGTC Seq 4 1 ---------- ---------- -------CCC CTAT------ -ACCCTTGTC Seq 5 1 ---------- ---------- ---------- ---------- -ACCCTTGTC Seq 1 8 AGGGTTTCAA GCATTTATAC CCTTATACCC CTAC------ -ACCCTTGTC Seq 8 1 ---------- ---------- ----ATACCC CCAT------ -ACCCTTGTT Seq 9 51 AGGGTTTCAA GCATTTATAC TCCTACATCC CTAC------ -ATCCTTGTC Seq 11 50 AGGGTTTCAA GCATTTATAC CCTTATACCC CTAC------ -ACCCTTGTC Seq 2 8 AGGGTTTCAA GCATTTATAC CCTTGTATCC CTATCCCATA CACCCTTATC Seq 12 8 AGGGTTTCAA GCATTTATAC CCTCATACCC CTAT------ -ACCCTTGTC consensus 51 * **** * Seq 3 18 CAAACCCTTG ATTTCTC-GT TTTCATGCGT AAGTCCTA- Seq 6 51 TAAACCCTTG ATTTTTC-GT TTTCATGCGT AAGTCCTA- Seq 10 20 CAAAACCTTG ATCTTTC-GT TTTCATGTGT AAGTCCTA- Seq 7 20 CAAACCCTCG ATCTTTC-GT TTTCATGCGT AAGTCCTA- Seq 4 17 CAAACCCTTG ATGTTTT-AT TTTTCTGCGT AAGTCCTAT Seq 5 10 CAAACCCTTG ATCTTTC-GT TTTCATGCGT AAGTCCTA- Seq 1 51 CAAACCCTTG ATCTTTCCGT TTTCATGCGT AAGTCCTA- Seq 8 20 CAAACCCTTG ATCTTTC-GG TTTCATGCGT AAGTC---- Seq 9 94 CAAACCCTTG ATCTTTC-GT TTTCATGCCT AAGTCCTA- Seq 11 93 CAAACCCTCG ATCTTTC-GT TTTCATGCGT AAGT----- Seq 2 58 CAAAGCCTTG ATATTTC-TT TTTCATGGGT AAGTC---- Seq 12 51 CAAACCCTTT ATCTTTC-GT TTTCATGCGT AAGTC---- consensus 101 *** *** ** * * *** ** * ****

  14. Alignment of sub-sequences Seq 3 1 GGGTGTAAGG GTTTCAAGCA TTTATACCCT TGTATCCCTA TCCCATACAC Seq 7 1 GGGTGTAAGG GTTTCAAGCA TTTATACCCT CATACCCCTA T-------AC Seq 6 1 GGGTGTAAGG GTTTCAAGCA TTTATACCCT TATACCCCTA C-------AC Seq 1 1 GGGTGTAAGG GTTTCAAGCA TTTATACCCT TATACCCCTA C-------AC Seq 4 1 GGGTGTAAGG GTTTCAAAAA TTTATACCCC TATACCCCTA C-------AC Seq 2 1 GGGTGTAAGG GTTTCAAACA TTTATACCCC TAAACCCCTA T-------AT Seq 5 1 GGGTGTAAGG GTTTCAAGCA TTTATACTCC TACATCCCTA C-------AT consensus 1 ********** ******* * ******* * * ***** * Seq 3 51 CCTTATCCAA AGCCTTGATA TTTCT-TTTT CATGGGTAAG TC---- Seq 7 44 CCTTGTCCAA ACCCTTTATC TTTCG-TTTT CATGCGTAAG TC---- Seq 6 44 CCTTGTCCAA ACCCTCGATC TTTCG-TTTT CATGCGTAAG T----- Seq 1 44 CCTTGTCCAA ACCCTTGATC TTTCCGTTTT CATGCGTAAG TCCTA- Seq 4 44 CCTTGTCTAA ACCCTTGATT TTTCG-TTTT CATGCGTAAG TCCTA- Seq 2 44 CCTTCTCCAA ACCCTTAATC TTTAG-TTTT CATACGTAAG TCCTAT Seq 5 44 CCTTGTCCAA ACCCTTGATC TTTCG-TTTT CATGCCTAAG TCCTA- consensus 51 **** ** ** * *** ** *** **** *** **** *

  15. Secondary structures of aligned sequences

  16. BLASTed this bubble sequence

  17. Some BLAST Results About 80% landed in between parallel genes all2965 alr2966 alr2967 alr2968 all4019 all4020 all4021 all4022 all4023 alr4810 alr4811 alr4812 patN

  18. Summary and Conclusions • More work needs to be done. • It is possible that Anabaena employes small RNA as a regulatory mechanism (I found nothing that suggested otherwise). • When looking for any kind of pattern or sequence you must always consider the probability that it would happen by chance. • The data I showed was just a fraction of everything I had gathered. • I didn’t even scratch the surface of this new topic, but I learned a lot trying.

  19. Thanks to: Jeff Elhai Peter Wolk For being so patient with me :^)

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