Chemical Synthesis of U1 snRNA Derivatives

Ohkubo, Akihiro; Kondo, Yasumasa; Suzuki, Makoto; Kobayashi, Haruki; Kanamori, Takashi; Yoshio, Masaki; Seio, Kohji; Nagai, Kiyoshi; Sekine, Mitsuo

doi:10.1021/ol401917r

Cited by 17 publications

(14 citation statements)

References 23 publications

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“…We have previously shown that AAV-mediated administration of ExSpeU1 in SMN2 transgenic mice recovers exon 7 inclusion in several target tissues including the brain, heart and skeletal muscle 40 . Additional approaches include delivery with lentiviral vectors for a cell-based therapy ex vivo 41 , or synthetic modified U1 snRNAs 63 . Through the development of the most appropriate delivery system it will be possible to translate the ExSpeU1 strategy into a reliable therapeutic opportunity.…”

Section: Discussionmentioning

confidence: 99%

Therapeutic activity of modified U1 core spliceosomal particles

et al. 2016

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Modified U1 snRNAs bound to intronic sequences downstream of the 5′ splice site correct exon skipping caused by different types of mutations. Here we evaluate the therapeutic activity and structural requirements of these exon-specific U1 snRNA (ExSpeU1) particles. In a severe spinal muscular atrophy, mouse model, ExSpeU1, introduced by germline transgenesis, increases SMN2 exon 7 inclusion, SMN protein production and extends life span. In vitro, RNA mutant analysis and silencing experiments show that while U1A protein is dispensable, the 70K and stem loop IV elements mediate most of the splicing rescue activity through improvement of exon and intron definition. Our findings indicate that precise engineering of the U1 core spliceosomal RNA particle has therapeutic potential in pathologies associated with exon-skipping mutations.

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Section: Discussionmentioning

confidence: 99%

Therapeutic activity of modified U1 core spliceosomal particles

et al. 2016

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“…These truncated RNAs were transcribed in the presence of 2 mM GMP to facilitate ligation of modified 5′ end oligonucleotides. The 5′ fragment of U1 snRNA with post-transcriptional modifications (5′-AmUmACΨΨACCU-3′ or 5′-AmUmACUUACCU-3′ where Ψ = pseudo-uridine, Am, Um = 2′-O-methyl nucleotides) were purchased from Dharmacon (GE Healthcare, Little Chalfont, UK) and ligated to the truncated U1 snRNAs by splint-assisted ligation with T4 DNA ligase ( Ohkubo et al, 2013 ). The plasmid template preparation, in vitro transcription and purification of RNA were carried out as described ( Price et al, 1995 ).…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5′ splice site recognition

Kondo

Oubridge

Roon

et al. 2015

eLife

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230

292

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U1 snRNP binds to the 5′ exon-intron junction of pre-mRNA and thus plays a crucial role at an early stage of pre-mRNA splicing. We present two crystal structures of engineered U1 sub-structures, which together reveal at atomic resolution an almost complete network of protein–protein and RNA-protein interactions within U1 snRNP, and show how the 5′ splice site of pre-mRNA is recognised by U1 snRNP. The zinc-finger of U1-C interacts with the duplex between pre-mRNA and the 5′-end of U1 snRNA. The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone around the splice junction but U1-C makes no base-specific contacts with pre-mRNA. The structure, together with RNA binding assays, shows that the selection of 5′-splice site nucleotides by U1 snRNP is achieved predominantly through basepairing with U1 snRNA whilst U1-C fine-tunes relative affinities of mismatched 5′-splice sites.DOI: http://dx.doi.org/10.7554/eLife.04986.001

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“…The short ExSpeU1 coding gene (˜650 bp) can be easily accommodated into Adeno Associated Viruses or Lentiviral Vectors forin vivo gene therapy (Rogalska et al , 2016;Donadonet al , 2018) or ex vivo cellular delivery (Dal Mas, Fortugno, et al, 2015;Dal Mas, Rogalska, et al, 2015;Nizzardo et al, 2015). Alternative delivery approaches that could be tested include the de novo chemical RNA synthesis (Ohkubo et al, 2013). The availability of patient-derived cellular models with the splicing mutations in form of nasal epithelial cells (I. M. Pranke et al, 2017) or organoids (Boj et al, 2017;Dekkers et al, 2013Dekkers et al, , 2016Sato et al, 2009Sato et al, , 2011 will allow a more direct assessment of the ExSpeU1s strategy on the CFTR functionality and the evaluation of the ExSpeU1 efficacy and safety profile in patient-derived target cells.…”

Section: Discussionmentioning

confidence: 99%

Rescue of common exon skipping mutations in Cystic Fibrosis with modified U1 snRNAs

Donegà¹,

Rogalska²,

Pianigiani³

et al. 2020

Preprint

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In Cystic Fibrosis (CF), correction of splicing defects represents an interesting therapeutic approach to restore normal CFTR function. In this study, we focused on ten common mutations/variants, 711+3A>G/C, 711+5G>A, 1863C>T, 1898+3A>G, 2789+5G>A, TG13T3, TG13T5, TG12T5 and 3120G>A that induce skipping of the corresponding CFTR exons 5, 9, 13, 16 and 18. To rescue the splicing defects we tested, in a minigene assay, a panel of modified U1 snRNAs, named Exon Specific U1s (ExSpeU1) that were engineered to bind to intronic sequences downstream of each defective exon. Using this approach, we show that all ten splicing mutations analysed are efficiently corrected by specific ExSpeU1s. Using cDNA-splicing competent minigenes, we also show that the ExspeU1-mediated splicing correction at the RNA level recovered the full-length CFTR protein for 1863C>T, 1898+3A>G, 2789+5G>A variants. In addition, detailed mutagenesis experiments performed on exon 13 led us to identify a novel intronic regulatory element involved in the ExSpeU1-mediated splicing rescue. These results provide a common strategy based on modified U1 snRNAs to correct exon skipping in a group of disease-causing CFTR mutations.

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Chemical Synthesis of U1 snRNA Derivatives

Cited by 17 publications

References 23 publications

Therapeutic activity of modified U1 core spliceosomal particles

Therapeutic activity of modified U1 core spliceosomal particles

Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5′ splice site recognition

Rescue of common exon skipping mutations in Cystic Fibrosis with modified U1 snRNAs

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