Inhibition of Pre-mRNA Splicing by a Synthetic Blom7α-Interacting Small RNA

Abstract

Originally the novel protein Blom7α was identified as novel pre-mRNA splicing factor that interacts with SNEVPrp19/Pso4, an essential protein involved in extension of human endothelial cell life span, DNA damage repair, the ubiquitin-proteasome system, and pre-mRNA splicing. Blom7α belongs to the heteronuclear ribonucleoprotein K homology (KH) protein family, displaying 2 KH domains, a well conserved and widespread RNA-binding motif. In order to identify specific sequence binding motifs, we here used Systematic Evolution of Ligands by Exponential Enrichment (SELEX) with a synthetic RNA library. Besides sequence motifs like (U/A)1–4 C2–6 (U/A)1–5, we identified an AC-rich RNA-aptamer that we termed AK48 (Aptamer KH-binding 48), binding to Blom7α with high affinity. Addition of AK48 to pre-mRNA splicing reactions in vitro inhibited the formation of mature spliced mRNA and led to a slight accumulation of the H complex of the spliceosome. These results suggest that the RNA binding activity of Blom7α might be required for pre-mRNA splicing catalysis. The inhibition of in-vitro splicing by the small RNA AK48 indicates the potential use of small RNA molecules in targeting the spliceosome complex as a novel target for drug development.

Biology of the mRNA Splicing Machinery and Its Dysregulation in Cancer Providing Therapeutic Opportunities

“…The RNA-binding protein KHDC4 that affects both 5′ and 3′ splice site selection is also part of the NTC, where it associates with PRPF19 [ 348 ]. Using a Systematic Evolution of Ligands by EXponential enrichment (SELEX) approach with recombinant KHDC4 or its conserved RNA binding domain as bait, an RNA-aptamer was identified that specifically binds to KHDC4 and inhibits splicing in vitro, as demonstrated by the accumulation of synthetic pre-mRNA and a slight inhibition of spliceosome assembly [ 349 ]. Presumably, the aptamer inhibits binding of KHDC4 to its target sequence on the pre-mRNA, thereby preventing proper positioning of the NTC and activation of the catalytic site.…”