sb0c00119_si_001.pdf (4.47 MB)
Engineering REST-Specific Synthetic PUF Proteins to Control Neuronal Gene Expression: A Combined Experimental and Computational Study
journal contribution
posted on 2020-08-05, 23:29 authored by Stefania Criscuolo, Mahad Gatti Iou, Assunta Merolla, Luca Maragliano, Fabrizia Cesca, Fabio BenfenatiRegulation
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 16-repeat 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.