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RNA structures limit the scope of oligonucleotide-facilitated splicing modulation

Posted by pbio on 07 May 2009 at 22:16 GMT

Author: Ravindra Singh
Position: Assistant Professor
Institution: University of Massachusetts Medical School
Additional Authors: Natalia Singh,
Submitted Date: April 04, 2007
Published Date: April 5, 2007
This comment was originally posted as a “Reader Response” on the publication date indicated above. All Reader Responses are now available as comments.

The recent paper by Hua et al (1) used Antisense Oligonucleotides (ASOs) against sequences of SMN2 exon 7. Such approach has potential to identify targets for the ASO-mediated correction of SMN2 exon 7 splicing in Spinal Muscular Atrophy (SMA), a debilitating disease of children and infants. This approach may also identify novel cis-elements, as well as validate the nature of cis-elements described earlier. Effect of an ASO is entirely dependent upon the accessibility of the target site, with loop being the best target (2). Regulatory elements located within the strong RNA structures cannot be targeted by ASOs. We have recently reported the probed structure of SMN2 exon 7 that contains two terminal loop structures, TSL1 and TSL2 (3). Fifteen ASOs used by Hua et al did not show any effect on SMN2 exon 7 splicing. Since all these ASOs targeted TSL1 or TSL2, we believe these ASOs were not able to anneal to their respective targets within exon 7.

We have previously proposed that exon 7 contains three regulatory elements i.e. Exinct (Extended inhibitory context), Conserved tract and 3’-Cluster (4). In this study Hua et al confirm the existence of our three elements (1). For example, six ASOs that targeted Exinct (Microwalk-A) promoted exon 7 inclusion in SMN2. Similarly, seven ASOs that targeted 3’-Cluster (Microwalk-B) promoted exon 7 inclusion in SMN2. As expected, all ASOs that targeted conserved tract promoted exon 7 skipping. However, unexpectedly, a number of ASOs that targeted either Exinct or 3’-Cluster also produced inhibitory effects. There are no clear interpretations for these contradictions. Because of these contradictions, overall results do not justify alteration of boundaries of the exonic regulatory elements previously proposed. Noticeably, there are discrepancies between experiments performed in vitro and in vivo. In addition, since most ASO effects were measured at high concentrations (in micro-molar range), we believe some of the in vivo effects could be indirect and non-specific. This could be particularly true for experiments in which effects were measured 48 hours after transfection with the high concentrations of ASOs. Our group is also interested in ASO-mediated correction of SMN2 exon 7 splicing. Towards this goal, we have located an efficient ASO target within SMN2 intron 7 (5). In contrast to Hua et al (1), we have shown low nano-molar concentrations of ASOs promote SMN2 exon 7 inclusion (5). Most importantly, we observed a robust ASO response within 24 hours (5). In case of Hua et al, we believe structurally inaccessible targets may have required high concentrations of short ASOs.
As our work on RNA structure is quite recent and was not referenced by Hua et al (1), we feel that may be an advantage for the PLos readers to be aware of this study.

Acknowledgements: RNS acknowledges support from MDA, NIH and FSMA.

1. Hua, Y., Vickers, T.A., Baker, B.F., Bennett, C.F. and Krainer, A.R. (2007) Enhancement of SMN2 Exon 7 Inclusion by Antisense Oligonucleotides Targeting the Exon. PLoS Biol, 5, e73
2. Joli, F., Bouchemal, N., Laigle, A., Hartmann, B. and Hantz, E. (2006) Solution structure of a purine rich hexaloop hairpin belonging to PGY/MDR1 mRNA and targeted by antisense oligonucleotides. Nucleic Acids Res 34, 5740-51.
3. Singh, N.N., Singh, R.N. and Androphy, E.J. (2007) Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes.
Nucleic Acids Res, 35, 371-89.
4. Singh, N.N., Androphy, E.J. and Singh, R.N. (2004) In vivo selection reveals combinatorial controls that define a critical exon in the spinal muscular atrophy genes. RNA, 10, 1291-305.
5. Singh, N.K., Singh, N.N., Androphy, E.J. and Singh, R.N. (2006) Splicing of a critical exon of human Survival Motor Neuron is regulated by a unique silencer element located in the last intron. Mol Cell Biol, 26, 1333-46.

Competing interests declared: Authors have invented a novel method of in vivo selection of the entire exon. Using this method, authors have discoverered several cis-elements against which antisense oligonucleotides (ASOs) were used in this study. University of Massachusetts Medical School has a US patent pending against those cis-elements. Authors work on ASO-mediated correction of SMN2 splicing and have made seminal contributions to the field.