Iden%fying*the*interac%ons between*RNA7binding - - PDF document

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Iden%fying*the*interac%ons between*RNA7binding proteins*and*microRNAs

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Central Central Dogma Dogma

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Post-transcriptional regulation Post-transcriptional regulation

RBP miRNA

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Role of RBPs and miRNAs in human Role of RBPs and miRNAs in human diseases diseases

• RBPs

RBPs: :

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• miRNAs

miRNAs: :

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!Oostra!et!al.!Biochim!Biophys!Acta,!2009

Disease RNA)Binding)Protein Fragile!X!syndrome Fmr1 Spinal!muscular!atrophy SF2/ASF ! Disease RNA)Binding)Protein Breast!cancer

miR<125b,!miR<145,!miR<21,!miR< 155,!miR<210

Lung!cancer

miR<155,!let<7a

Alzheimer’s!disease

miR<29b<1,!miR<29a,!miR<9

Parkinson’s!disease

miR<30b,!miR<30c,!miR<26a,!miR< 133b,!miR<184∗,!let<7

Cartegni!et!al.!Nature!Gene=cs!2002 Li!et!al.!Genomics,!Proteomics!&!Bioinforma=cs!2012

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RNA-binding proteins (RBPs) RNA-binding proteins (RBPs)

• There are ~850 RBPs in human.
• They bind to short regions (4-12 nts) on

mRNAs.

• RBPs play important roles in:
• splicing
• polyadenylation
• mRNA stabilization
• mRNA localization
• translation

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microRNAs microRNAs

• There are >1000 miRNAs in the human

genome.

• A microRNA is a small non-coding RNA

molecule (~ 22 nts long) found in plants, animals, and some viruses

• They affect stability and translation
• f their target mRNAs.

!Vella,&M.C.&and&Slack,&F.J.&C.&elegans&microRNAs,&Wormbook.&ed!

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RNA secondary structure RNA secondary structure

• RNA does not

stay single stranded within the cell.

• Instead it folds

into complex structure where base pairs form between complementary nucleotides

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Fukunaga'et'al.'Genome'Biology'2014

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Our Goal Our Goal

• Most of the current methods or analysis consider a

single factor (RBP or miRNA) at a time.

• We are going to investigate cooperative and

competitive interactions between RBPs and miRNAs.

• Here’s an example for a

cooperative interaction:

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Kedde$et$al.$Nature$2010

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Mapping binding sites of RBPs and Mapping binding sites of RBPs and miRNAs on human 3’UTRs miRNAs on human 3’UTRs

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RBPs RBPs

• RNACompete motifs
• RNAcompete data is available for 200 RBPs

from several organisms

miRNAs miRNAs

• TargetScan predictions
• PicTar predictions

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Ray$et$al.$Nature$2013 Lewis$et$al.,$2005$and$$Grimson$et$al.,$2007 Anders$et$al,$Nucleic$Acids$Res.$2012

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Predicting the structure of 3’UTRs Predicting the structure of 3’UTRs

• Experimental determination of RNA

secondary structure is still difficult.

• An alternative way is to use computational

methods to predict RNA secondary structure.

• RNAfold
• RNAfold method predicts secondary structures of

single stranded RNA based on minimum free energy

• For each site we calculate the probability of being in a

paired region

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Not$a$binding$site$for any$factor Binding$site$of a$factor mRNA

Classifying sites according to secondary structure:

a) Inaccessible (Paired Inaccessible (Paired) )

b) Accessible (Unpaired) c) Complementary

%

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mRNA

Classifying sites according to secondary structure:

a) Inaccessible (Paired)

b) Accessible (Unpaired Accessible (Unpaired) )

c) Complementary

%

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RBP RBP Site microRNA Site

Classifying sites according to secondary structure:

a) Inaccessible (Paired) b) Accessible (Unpaired)

c) Complementary Complementary

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RBP

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Explaining the effects of PUM sites

• Zhao et al. have measured the stability of 3000

160nt long segments that are selected from conserved regions of human 3’UTRs.

• We analyzed the Supplementary Figure 7 of this

paper which shows mutation effects on mRNA stability.

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Zhao%et%al.%Nature%Biotechnology%2014

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SIPA1L2

Inaccessible ! ! Accessible " " Complementary ! ! #$%

This figure shows the mutation results of a segment of 3’UTR of SIPA1L2 gene .

• PUM binds to

positions 51:57

• HuR binds to

positions 134:140

Explaining the effects of PUM sites

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SIPA1L2

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PUM$Binding$site CPEB4$Binding$site

This figure shows the mutation results of a segment of 3’UTR of MYOD1 gene .

• PUM binds to

positions 51:57

• CPEB4 binds to

positions 34:40

Inaccessible ! ! Accessible " " Complementary ! ! #$%&'

Explaining the effects of PUM sites

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AKAP2:

PUM RBM41 TUT1

Inaccessible ! ! Accessible " " Complementary ! ! Compe22on RBM41, #$#% #$#%

This figure shows the mutation results of a segment of 3’UTR

• f AKAP2 gene.
• PUM binds to positions

123:129

• RBM41 binds to positions

128:134

• TUT1 binds to positions

126:132

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Explaining the effects of PUM sites

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Comparing sites with distinct structural contexts

• Zhao half life data for clones on Beas cell line are scanned

for RBPs and miRNAs binding site and the structure is predicted by RNAfold.

• For each factor (RBP or miRNA) we classify the clones into

5 distinct groups: A. mRNAs that have inaccessible sites for the factor, this site is not complementary to another factor's site and there is no accessible site of this factor. B. mRNAs that have at least one accessible sites for the factor C. mRNAs that have complementary sites for the factor and not having any accessible sites for the factor D. mRNAs that have no site for the factor E. mRNAs that have any site for the factor

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Results for IGFBP1-3

• (Ig%p''IS'A'KNOWN'STABILIZING'factor'and'we'validate'this'with'our'results.
• In'the'future'we'are'planning'to'analyse'the'factors'that'ig%'is'interacEng'with.)

mRNA'half'life'in'Beas'cell'line

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Conclusion

• We have developed a model that considers

sites of all known RBP and microRNA sites concurrently to explain post- transcriptional mechanisms.

• Using this model, we can explain the sites

that are functional / not functional.

• In the future, we will combine this model

with transcriptional effects to explain differential gene expression in cancer.

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Acknowledgement

• This study has been supported by Tubitak

(Scientific and Technological Research Council of Turkey) grant #113E159

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