Abstract
Transcriptomes consist of several classes of RNA that have wide-ranging but often poorly described functions and the deregulation of which leads to numerous diseases. Engineering of functionalized RNA-binding proteins (RBPs) could therefore have many applications. Our previous studies suggested that the RanBP2-type Zinc Finger (ZF) domain is a suitable scaffold to investigate the design of single-stranded RBPs. In the present work, we have analyzed the natural sequence specificity of various members of the RanBP2-type ZF family and characterized the interaction with their target RNA. Surprisingly, our data showed that natural RanBP2-type ZFs with different RNA-binding residues exhibit a similar sequence specificity and therefore no simple recognition code can be established. Despite this finding, different discriminative abilities were observed within the family. In addition, in order to target a long RNA sequence and therefore gain in specificity, we generated a 6-ZF array by combining ZFs from the RanBP2-type family but also from different families, in an effort to achieve a wider target sequence repertoire. We showed that this chimeric protein recognizes its target sequence (20 nucleotides), both in vitro and in living cells. Altogether, our results indicate that the use of ZFs in RBP design remains attractive even though engineering of specificity changes is challenging.
Highlights
Recent advances in genome biology have revolutionized our notion of the transcriptome
Domains that bind to different RNA sequences, we selected five RanBP2-type ZFs for which the likely RNAbinding residues differed from ZRANB2 ZF2; (i) the Homo sapiens EWS ZF, (ii) the Arabidopsis thaliana ABI35Sup ZF, (iii) the Danio rerio ZRANB1B ZF, (iv) the Caenorhabditis elegans T0B2.5 ZF and (v) the Mus musculus RBM10 ZF (Fig. 1B)
All proteins were highly expressed, with purification yields reaching ~50–60 mg of GST-tagged (ZF)x2 per liter of culture. We examined their RNA-binding specificity using Systematic Evolution of Ligands by EXponential enrichment (SELEX)[29] experiments, in which high-affinity RNA sequences were selected from a random 25 nt-long single-stranded RNA (ssRNA) library (Supplementary Table S1) by the GST-fusion proteins pre-immobilized on glutathione-Sepharose beads (Fig. 2B)
Summary
Recent advances in genome biology have revolutionized our notion of the transcriptome. Since we wanted to target ssRNA, we have chosen a particular class of ZFs that naturally recognizes ssRNA with high affinity and specificity This family, named the RanBP2-type ZF family, was shown to bind to ssRNA24,25 and was initially discovered in the human splicing factor ZRANB2. The second main goal of this study was to show that we could engineer a functional tandem array of ZF domains in order to target a longer and more specific RNA in the context of a living cell This is why we generated a (ZF)x6 protein that combines RanBP2-type ZFs and ZFs that belong to other families. The statistical significance (E-value) is shown on the top of each LOGO diagram
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