RNA – A New RoleRNA – A New Role

Amy Anderson

Introduction – How’d they do that?Introduction – How’d they do that?

40+ years, still no complete answer…

Gene Gene

Off You!On You!

Gene Gene

How does gene expression get turned on and off?

Heterocysts

AnabaenaAnabaena

How do the cells differentiate?

Originally researchers looked for proteins…

…but nothing was found to account for everything.

So we need something new.So we need something new.

…GCCAATGTCAAAGATTTAGTAGAGACTTGCATATCGTTCACTCCGTGAGTAAGTTTTTGTAATTAACGT…

Transcription

Where to look?Where to look?

E. coli!E. coli!

E. coli’s Regulatory Mechanism – small RNAE. 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…

1. Transcribed into RNA

GCUA…GAAUUA

2. sRNA binds to mRNA containing targeted gene

…GCUCUUACGUCGUCGAUUGCAGCGUA…

3. Blocks mRNA translation into proteinThis small RNA is called This small RNA is called

rhyB.rhyB.

It regulates the expression It regulates the expression of proteins associated with of proteins associated with the presence of iron.the presence of iron.

……..ACTAAAGTTGCCAATGTCAAAGATTTAGTAGAGACTTGCATATCGA…….TTCACTCCGTGAGTAAGTTTTTGTAATTAACGT……..

rhyBfur

RNA Polymerase

Fe+2

Fe+2

Fe+2

Fe+2

Fe+2

Fe+2

Fe+2

Fe+2

Fe+2 Fe

+2

Fe+2

Fe+2

Fe+2

Fe+2

Fe+2Fe

+2

Fe+2

Regulation of Regulation of rhyB in rhyB in E.coliE.coli

rhyB Promoter site

GGAGAAC UAUUACUUUUU

Secondary Structure of Secondary Structure of rhyBrhyB

…. GUUAACUAUUTUTTGAUGUGCCAA….

mRNA of mRNA of targeted targeted genegene

rhyBrhyB binding site binding site

!!!

DNA of multiple targeted genes

mRNA of targeted genes with rhyB binding site

Does Anabaena have a similar mechanism?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

……

Gene 1 Gene 2…AATGCGCTA…GAATTATCGCG…

…AATGCGCTA…GAATTATCGCG……TTACGCGAT…CTTAATAGCGC…

…AATGCGCTA…GAATTATCGCG……TTACGCGAT…CTTAATAGCGC…

Intergenic regions Motif 10 was found Intergenic regions Motif 10 was found in:in:

asl1111 alr1112ACGT…M10…AGGT

asl1274 asr1275ACGT…M10…AGGT all2909 all2908ACGT…M10…AGGT

all3558 all3557ACGT…M10…AGGT

alr3693 asr3694ACGT…M10…AGGT

all4219 all4218ACGT…M10…AGGT

alr4468 alr4469ACGT…M10…AGGT

alr4788 all4789ACGT…M10…AGGT

asl2850 alr2851ACGT…M10…AGGT

all3239 alr3240ACGT…M10…AGGT

all4668 asr4669ACGT…M10…AGGT

all1124 all1123ACGT…M10…AGGT

alr2744 alr2745ACGT…M10…AGGT

all1683 all1682ACGT…M10…AGGT

all1782 all1781ACGT…M10…AGGT

D5

D7

D15

D17

D24

P6

P10

P11

P13

P16

P19

P20

P21

P22

C26

all0788

alr0787

BLAST hitBLAST hit

12-15 nucleotide overlap

Most intergenic regions containing motif 10 hit this spot.

Took an alignment of the portions that hit Took an alignment of the portions that hit all0788.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 *** *** ** * * *** ** * ****

Alignment of sub-Alignment of sub-sequencessequences

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 **** ** ** * *** ** *** **** *** **** *

Secondary structures of aligned sequencesSecondary structures of aligned sequences

BLASTed this bubble sequence

Some BLAST ResultsSome BLAST Results

About 80% landed in between parallel genes

all2965 alr2968alr2967alr2966

all4019 all4020 all4021 all4022 all4023

alr4810 alr4811 alr4812

patN

Summary and ConclusionsSummary 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.

Thanks to:Thanks to:

Peter WolkPeter Wolk

Jeff ElhaiJeff Elhai

For being so patient with For being so patient with me :^)me :^)