a unique rna structure formed by a long-distance ... singh.pdf · natalia singh and ravindra singh...

Natalia Singh and Ravindra Singh Department of Biomedical Sciences

Iowa State University, USA [email protected]

A unique RNA structure formed by a long-distance interaction uncovers the therapeutic potential of a deep

intronic sequence

July 5, 2014, Salzburg, Austria

Organization of SMN protein

Howell et al., Future Med Chemistry, 6 (9), 1081-1099, 2014

Two SMN Genes as inverted repeats on 5q11-q13!

NAIP SMN2

AG-1 C212 NAIP SMN1

AG-1 C212

1 2a 2b 3 4 5 6 7 8

G C C

A A

1 2a 2b 3 4 5 6 7 8

A T T

C C

500 Kb 500 Kb

282 aa! 294 aa!

Spinal Muscular Atrophy (SMA)!!SMN1

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5% /% 6%5% 6%

$% &%5% 67%

Regulatory elements of SMN pre-mRNA

Master checkpoint

Howell et al., Future Med Chemistry, 6 (9), 1081-1099, 2014

8!!9#$%

:-5% :-/% :-6%8);<.)%/%

GUAAGUCUGCCAGCAUUAUGAAAGUGAAUCUUACUUUUGUAAAACUUUAUGGUUUGUGG!#$!$=9$>%?@()*%>A%#$!$B9$C%?@()*%BA%

#$!$=9$C%?@()*%CA%

#$!&=9&>%?@()*%5A%

#$!$B9&>%?@()*%$=A%

#$!&B9&C%?@()*%/A%#$!7=97>%?@()*%6A%

#$!&=9&C%?@()*%$$%#$!&B97>%?@()*%$&A%

#$!$B9&C%?@()*%$>A%#$!&=97>%?@()*%$BA%#$!&B97C%?@()*%$5A%

#$!$=97>%?@()*%$6A%#$!$=9&C%?@()*%$/A%

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ISS-N1 is a strong intronic splicing silencer

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5´SS !

Singh et al. Mol. Cell. Biol. 26, 1333-1346, 2006!

ISS-N1 is a target for an antisense oligonucleotide (ASO)-mediated correction of SMN2 exon 7 splicing

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GUAAGUCUGCCAGCAUUAUGAAAGUGAAUCUUACUUUUGUAAAACUUUAUGGUUUGUGG!

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5´SS !

Singh et al. Mol. Cell. Biol. 26, 1333-1346, 2006!US Patent # US 2007/0292408 !

Mutation!

3´SS !

Studies with ISS-N1-targeting ASOs

Sivanesan et al., Transl. Neurosci., 4, 1-7, 2013!

Relative efficacy of drug candidates in !7SMA mice!

Seo et al., Biochim. Biophys Acta, 1126, 271-83 2013!

ISS-N1 targeting ASO

ISS-N1 targeting ASO

Gene Therapy

days

Singh et al., RNA 16, 1167-1181, 2010

Antagonistic response of two ASOs that bind to targets differing in a single nucleotide

RNA 16, 1167-1181, 2010

ASO-based approach reveals a long-distance interaction (LDI)

Primary site of LDI

Nucleic Acids Research 41, 8144-8165, 2013!

Smallest motif for LDI


Effect of point mutations within the smallest motif for LDI


Effect of structure-associated mutations on LDI


Effect of compensatory mutations on LDI


Effect of negative regulators on LDI


Structure of SMN2 intron 7 (Probed by SHAPE)

ISS-N2!


Validation of the 5" strand of ISTL1

NAR 41, 8144-8165, 2013

Validation of the 3" strand of ISTL1 formation


Effect of L14 and F14 on RNA secondary structure of SMN2 intron 7


Structural basis of the effect of ASOs F14 pathway ! Exon 7 inclusion

L14 pathway ! Exon 7 exclusion


Is ISS-N2 a new antisense target ?

ISS-N2


Effect of ISS-N2-targeting ASOs on SMN2 exon 7 splicing

:-5% :-/% :-6%8);<.)%/%

:-B% :->% :-7% :-&I% :-&(% :-$%5´SS !

Mutation!


Mechanism of splicing correction by a deep intronic target


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