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.
Highlights
The protein coding information in eukaryotic organisms is split by intronic sequences containing regulatory elements and microRNAs
We describe the identification of a splicing inhibitory RNA aptamer, termed AK48 (Aptamer K homology (KH)-binding 48), which was selected against the KH domains of Blom7a by Systematic Evolution of Ligands by Exponential Enrichment (SELEX)
Blom7a consists of two KH domains which co-localize with RNA
Summary
The protein coding information in eukaryotic organisms is split by intronic sequences containing regulatory elements and microRNAs. One distinct subcomplex associated with the spliceosome is the CDC5L/ SNEVPrp19/Pso complex, which consists of SNEVPrp19/Pso, CDC5L, PLRG1, SPF27 (BCAS2) and Hsp forming the core complex, while additional proteins are associated [2,3]. This complex is necessary for the catalytic steps of pre-mRNA splicing since its immunodepletion results in blocking of pre-mRNA splicing in vitro [4,5]. Inhibition of the interaction between different subunit members like CDC5L and PLRG1 [4] block splicing, whereby disruption of the multimerisation of SNEVPrp19/Pso even blocks spliceosome assembly [5]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have