Engineering REST-Specific Synthetic PUF Proteins to Control Neuronal Gene Expression: A Combined Experimental and Computational Study

Abstract

Regulation of gene transcription is an essential mechanism for differentiation and adaptation of organisms. A key actor in this regulation process is the repressor element 1 (RE1)-silencing transcription factor (REST), a transcriptional repressor that controls more than 2000 putative target genes, most of which are neuron-specific. With the purpose of modulating REST expression, we exploited synthetic, ad hoc designed, RNA binding proteins (RBPs) able to specifically target and dock to REST mRNA. Among the various families of RBPs, we focused on the Pumilio and FBF (PUF) proteins, present in all eukaryotic organisms and controlling a variety of cellular functions. Here, a combined experimental and computational approach was used to design and test 8-and 16-repeat PUF proteins specific for REST mRNA. We explored the conformational properties and atomic features of the PUF-RNA recognition code by Molecular Dynamics simulations. Biochemical assays revealed that the 8-and 16repeat PUF-based variants specifically bind the endogenous REST mRNA without affecting its translational regulation. The data also indicate a key role of stacking residues in determining the binding specificity. The newly characterized REST-specific PUF-based constructs act as excellent RNA-binding modules and represent a versatile and functional platform to specifically target REST mRNA and modulate its endogenous expression.

Mechanisms and Strategies for Determining m6A RNA Modification Sites by Natural and Engineered m6A Effector Proteins

“…To achieve selectivity for specific endogenous mRNAs, it is desirable to use modified PUFs that can recognize longer sequences. [64,[67][68][69] Notably, PUFs can target RNAs in mitochondria, which cannot be achieved using CRISPR-Cas. [83] Although the presence of m 6 A has been reported in plant mitochondrial transcripts, [88,89] its presence in mammalian cells has not been observed, [90] but PUF has the potential to control mitochondrial RNA modifications.…”

“…PUFs have not been used to control RNA modification in cells. To achieve selectivity for specific endogenous mRNAs, it is desirable to use modified PUFs that can recognize longer sequences [64,67–69] . Notably, PUFs can target RNAs in mitochondria, which cannot be achieved using CRISPR‐Cas [83] .…”

“…Because each repeat consisting of approximately 36 amino acid residues recognizes a single nucleotide at specific amino acid residues, artificial RNA‐binding proteins targeting various 8‐nt sequences can be designed by simply substituting the amino acid residues [61–66] . Furthermore, by connecting multiple RNA‐binding repeats, engineered PUF proteins can be designed to bind to more than 8 nt, which would satisfy the requirement for specificity to the target locus within the transcriptome [64,67–69] . This programmable RNA‐binding mode has been used for sequence‐specific control of RNA metabolism (Figure 4a) [70] .…”

“…[61][62][63][64][65][66] Furthermore, by connecting multiple RNA-binding repeats, engineered PUF proteins can be designed to bind to more than 8 nt, which would satisfy the requirement for specificity to the target locus within the transcriptome. [64,[67][68][69] This programmable RNA-binding mode has been used for sequence-specific control of RNA metabolism (Figure 4a). [70] Fusion proteins of PUFs with the RNA decay factor, tristetraprolin, significantly repressed protein production from mRNA containing PUF-binding sites at the 3'UTR.…”

Effective RNA Regulation by Combination of Multiple Programmable RNA-Binding Proteins

“…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 also showed the assembly of two PUF proteins in adjacent regions on the RNA to image specific transcripts [27][28][29][30].…”