Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

BACKGROUND: We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. RESULTS: We show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. CONCLUSION: Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology

Modulation artificielle de l'épissage alternatif

L'épissage alternatif est un mécanisme nucléaire complexe régulé par une multitude de facteurs naturels. En raison du nombre élevé (environ 15%) de maladies génétiques (fibrose kystique, (Ç-thalassémie, cancer, etc.) causées par un dérèglement de l'épissage, des stratégies ont été développées afin de moduler artificiellement l'épissage d'un ARN pré-messager. La stratégie actuellement la plus utilisée pour moduler l'épissage consiste à utiliser des oligonucléotides antisens ciblant directement un site d'épissage de façon à interférer avec l'utilisation de ce site au profit d'un site compétiteur. Cependant, étant donné que les sites d'épissage s'apparentent à une séquence consensus, cette stratégie soulève des problèmes de spécificité. Nous avons évalué la possibilité de moduler l'épissage par l'utilisation de protéines capables de lier les régions à proximité d'un site d'épissage. Cette étude constitue la première démonstration que le ciblage de la liaison d'une protéine en amont d'un site d'épissage 5 ' peut être utilisé pour moduler artificiellement la sélection de sites d'épissage."--Résumé abrégé par UMI

Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

Abstract Background We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. Results We show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. Conclusion Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.</p

Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals-1

<p><b>Copyright information:</b></p><p>Taken from "Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals"</p><p>BMC Biotechnology 2006;6():5-5.</p><p>Published online 13 Jan 2006</p><p>PMCID:PMC1379639.</p><p>Copyright © 2006 Gendron et al; licensee BioMed Central Ltd.</p>onucleotides (panel A, BS indicating the presence of a branch site sequence in the tail). Each mixture was tested in triplicate. (B) RT-PCR analysis of splicing mixtures. The ratio of the Bcl-xL and Bcl-xS products was plotted for different concentrations of oligomers. (C) Profiles obtained on the Agilent 2100 bioanalyzer are shown for a control mixture (lane 2) and mixtures incubated with the M4, M4B5 and M4B3+ oligonucleotides at concentrations of 18.75, 37.5 and 75 nM

Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals-0

<p><b>Copyright information:</b></p><p>Taken from "Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals"</p><p>BMC Biotechnology 2006;6():5-5.</p><p>Published online 13 Jan 2006</p><p>PMCID:PMC1379639.</p><p>Copyright © 2006 Gendron et al; licensee BioMed Central Ltd.</p>racts in the presence of increasing concentrations of oligonucleotides lacking a tail (M4) or carrying a 5' tail (M4A1, M4A1M, M4JV2, M4BC2 and M4B5). (A) Structure of the human Bcl-x pre-mRNA. Boxes and lines represent exons and introns, respectively. The dashed lines indicate plasmid sequences. The position of the 5' splice sites is shown as well as the position of the primers used in the RT-PCR assay. The portion of the pre-mRNA targeted by hybridization with various oligomers corresponds to nucleotide position -4 to -23, upstream of the Bcl-xL 5' splice junction. (B) Schematic structure of the oligonucleotides. The horizontal portion is complementary to position -4 to -23, upstream of the Bcl-xL 5' splice site. The presence of A1/A2 binding sites (A1BS), mutated A1/A2 binding sites (mutA1BS) or a branch site (BS) is indicated. (C) The RT-PCR assay was carried out on total RNA isolated from splicing reactions performed in triplicate. The RT-PCR products were fractionated by electrophoresis on an Agilent 2100 bioanalyser and the ratio of Bcl-xL/Bcl-xS products was calculated with standard deviations. The results were plotted in a graph that indicates the xL/xS ratio at various concentrations of oligonucleotides. (D) A computer generated image of fluorescent Bcl-x RT-PCR products is shown for the reactions performed in the absence of oligonucleotide (control, lane 2) and in the presence of oligonucleotides M4, M4A1 and M4A1M at concentrations of 1.5, 3.0 and 6.0 nM. The position and size of the Bcl-xL and Bcl-xS products and molecular weight markers are indicated

Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals-3

<p><b>Copyright information:</b></p><p>Taken from "Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals"</p><p>BMC Biotechnology 2006;6():5-5.</p><p>Published online 13 Jan 2006</p><p>PMCID:PMC1379639.</p><p>Copyright © 2006 Gendron et al; licensee BioMed Central Ltd.</p> μM of 2'-O-Me oligonucleotides complementary to the 5' end of U1 snRNA. A mock-treated extract was used as control. Increased amounts of the bifunctional oligonucleotide M4-25D were tested (7.4, 37 and 74 nM). Splicing products were fractionated in a 6% denaturing polyacrylamide gel. The position of the lariat splicing products is shown

Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals-4

<p><b>Copyright information:</b></p><p>Taken from "Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals"</p><p>BMC Biotechnology 2006;6():5-5.</p><p>Published online 13 Jan 2006</p><p>PMCID:PMC1379639.</p><p>Copyright © 2006 Gendron et al; licensee BioMed Central Ltd.</p>ntrol. Following incubation in a HeLa nuclear extract for 0, 30 and 60 minutes, the labeled oligos were fractionated in a 12% denaturing acrylamide gel. (B) The stability indices were tabulated. The apparent stability of each oligo was determined from the results presented in panel A by quantitating the amounts of oligonucleotides remaining after 60 minutes of incubation, and normalizing for the signal of the B3+ control oligonucleotide in each sample. A stability index of 1 is obtained when an oligonucleotide is as stable as B3+. A stability index greater or smaller than 1 indicates that the oligo is more or less stable than B3+, respectively