Abstract
RNAs play important roles in gene expression through translation and RNA splicing. Regulation of specific RNAs is useful to understand and manipulate specific transcripts. Pumilio and fem-3 mRNA-binding factor (PUF) proteins, programmable RNA-binding proteins, are promising tools for regulating specific RNAs by fusing them with various functional domains. The key question is: How can PUF-based molecular tools efficiently regulate RNA functions? Here, we show that the combination of multiple PUF proteins, compared to using a single PUF protein, targeting independent RNA sequences at the 3′ untranslated region (UTR) of a target transcript caused cooperative effects to regulate the function of the target RNA by luciferase reporter assays. It is worth noting that a higher efficacy was achieved with smaller amounts of each PUF expression vector introduced into the cells compared to using a single PUF protein. This strategy not only efficiently regulates target RNA functions but would also be effective in reducing off-target effects due to the low doses of each expression vector.
Highlights
Manipulation of specific RNAs is important for elucidating the roles of specificRNAs and for therapeutic purposes as represented by RNA interference and antisense strategies targeting specific mRNAs.Other than such complementary oligonucleotides, sequence-specific RNA-binding proteins, Pumilio and fem-3 mRNA-binding factor (PUF) proteins and pentatricopeptide repeat (PPR) proteins, and clustered regularly interspaced short palindromic repeats (CRISPR)-based systems, including dCas13 and dCas9-PAMer complexed with guide RNA, have been used for the customized sequence-specific regulation of mRNAs [1,2,3,4,5,6]
RNA sequence with high specificity both in vitro and in cellulo. By combining these four PUF proteins, we showed the use of multiple TTP-PUF proteins in the effective repression of target gene expression, even though the amount of each transfected expression vector was smaller than that of a single PUF
Further verification is needed to understand the off-target effects, we expect that this strategy will reduce off-target effects because of a lower dose of expression vectors
Summary
Manipulation of specific RNAs is important for elucidating the roles of specificRNAs and for therapeutic purposes as represented by RNA interference and antisense strategies targeting specific mRNAs.Other than such complementary oligonucleotides, sequence-specific RNA-binding proteins, Pumilio and fem-3 mRNA-binding factor (PUF) proteins and pentatricopeptide repeat (PPR) proteins, and clustered regularly interspaced short palindromic repeats (CRISPR)-based systems, including dCas and dCas9-PAMer complexed with guide RNA, have been used for the customized sequence-specific regulation of mRNAs [1,2,3,4,5,6]. Each repeat recognizes an RNA base through direct interaction between RNA bases and side chains of amino acids at the 12th and 16th positions [20] (Figure 1b). By changing the amino acid pairs of a PUF repeat, artificial RNA-binding domains corresponding to various 8-nucleotide (nt) RNA sequences can be designed [9,21,22]. As the length of RNA recognized by PUFs (8-nt) is not enough to specify the target RNAs within a huge transcriptome, efforts to increase the length of the RNA-binding sequences have been applied by increasing the number of PUF repeats [21,23,24,25,26]. Previous reports showed the assembly of two PUF proteins in adjacent regions on the RNA to image specific
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