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Engineering RNA‐binding proteins with diverse activities

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With generations of efforts to understand RNA functions in diverse cellular processes, RNA‐binding proteins (RBPs) have emerged to be one of the central players in regulating RNA‐related pathways. RBPs control almost all aspects of RNA processing via recognizing their RNA target(s). Most of these proteins have a modular configuration, with one or more RNA‐binding domain for target recognition and various functional modules to affect the metabolism and biological functions of RNA. Thus, engineering RNA‐binding factors with customized specificity and function is extremely useful in biological and medical research. In this review, we discuss the current advances in engineering RBPs that specifically bind to diverse targets, with emphasis on the design strategies and their applications as new biological tools in various aspects of RNA metabolism and function. WIREs RNA 2015, 6:597–613. doi: 10.1002/wrna.1296 This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications
Synthetic regulatory circuits using engineered splicing factors. Red and green oval represent Gly‐rich and Arg/Ser (RS) domains. Pumilio/FBF repeats (PUF) and its targets are shown in blue. (a) Positive splicing feedback loop. The Gly‐PUF can specifically inhibit exon 2 inclusion by binding to the target, therefore increasing the splicing of its own messenger RNA (mRNA). A diagram of the design is shown at the right with the functional protein (Pa) controlling splicing to increase its own mRNA (Ra). (b) Negative splicing feedback loop. The design is similar to panel (a) except that RS‐PUF (Pb) is used to promote exon 2 inclusion, resulting in a decrease of its own mRNA (Rb). (c) Examples of splicing regulatory circuit containing two components. Two engineered splicing factors (ESFs) can regulate the splicing of each other through modified PUF domains (shown in light or dark blue) that recognize different targets (shown in same colors). A double inhibitory circuit (top) or a negative feedback circuit (bottom) can be constructed using standard parts shown in panel (a) or (b).
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Future application of engineered factors containing RNA‐binding proteins (RBPs). (a) Artificial RNA editing enzymes. (b) Engineered factors with RBDs to manipulate alternative polyadenylation. A protein or domain that affects RNA polyadenylation can be fused to designer RNA‐binding domain (RBD) to generate an artificial protein that enhances or suppresses the use of adjacent polyadenylation site. (c) Artificial factors with programmable RBDs can be engineered to release paused RNA polymerase. Designer RBD can be fused to a protein that mediates phosphorylation of P‐TEFb, thus regulating the transcription elongation. (d) RBDs fused with a protein translocation signal may transport RNA to different cellular compartments.
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Engineered Pumilio/FBF repeats (PUF) factors. (a) Combination of split GFP with PUF scaffold generated an RNA probe to image RNA in live cells. (b) Engineered PUF scaffold with a splicing regulatory domain (e.g., Arg/Ser‐rich domain or Gly‐rich domain) can control various alternative splicing events. (c) Combination of PUF domain with a nonspecific RNA endonuclease (PIN domain) can generate an artificial site‐specific RNA endonuclease that recognizes and cleaves RNA. (d) Fusion of GLD2 or CAF1 with PUF domain produced novel factors that can activate or inhibit mRNA translation. (e) Fusion protein of PUF with initiation factor eIF4E can active translation, whereas PUF can block translation by itself. (f) PUF domain can be linked with tristetraprolin to affect RNA stability.
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Structures of other RNA‐binding proteins (RBPs) in complex with cognate RNA targets. RNAs were colored with cyan. (a) Crystal structure of an AU‐rich RNA molecule recognized by two RNA recognition motifs (RRMs) of HuD protein (PDB entry 1G2E). (b) Two KH domains from the NusA protein bind RNA target in an extended conformation (PDB entry 2ASB). Other fragments of NusA are shown in gray. (c) Transcription factor TFIIIA contains zinc‐finger motifs. The crystal structure of a three‐finger polypeptide in complex with truncated 5sRNA (61 nt) of Xenopus laevis from the complete TFIIIA‐5S RNA complex is shown (PDB entry 1UN6). (d) Crystal structure of TRAP (PDB entry 1QAW) in complex with the RNA that contains 11 GAG or UAG triplets.
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RNA recognition by pentatricopeptide repeats (PPR) domain. (a) Left: structure of the PPR10PSAJ complex with a close‐up view of PPR‐RNA recognition (adapted from PDB entry 4M59). PPR10 forms a right‐handed two‐turn superhelical dimer, with 19 PPR motifs capped by three short α‐helices at the amino‐terminal domain (NTD, in yellow) and a single α‐helix at the C terminus. Each of the four RNA bases at the 5′ end is sandwiched by two residues at the end of adjacent motifs. Right: recognition of RNA bases by PPR as shown by the same PPR10PSAJ structure. (b) Schematic of PPR binding to cognate RNA. PPRs are shown as violet ellipses, with recognition code indicated below.
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RNA recognition by Pumilio/FBF repeats (PUF) domain. (a) Left: the crystal structure showing the PUF domain in complex with cognate RNA (PDB entry 2YJY). Right: a close‐up view of the RNA recognition code for each nucleotide in the structure. The hydrogen bonds between RNA and protein are indicated with dotted lines. (b) Schematic of the PUF binding to cognate RNA. PUF repeats are shown as red ellipses, with recognition code indicated below.
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Schematic diagram of RNA processing pathways during eukaryotic gene expression. Various RNA‐binding proteins (RBPs) are involved in multiple RNA processing steps, which can be manipulated by engineered RBPs with customized RNA‐binding specificity.
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RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications
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