Browsing by Author "Bhat, Vandita D."
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Item Engineering a Conserved RNA Regulatory Protein Repurposes its Biological Function in Vivo(eLife Sciences Publications Ltd, 2019-01-17) Bhat, Vandita D.; McCann, Kathleen L.; Wang, Yeming; Fonseca, Dallas R.; Shukla, Tarjani; Alexander, Jacqueline C.; Qiu, Chen; Wickens, Marv; Lo, Te-Wen; Hall, Traci M. Tanaka; Campbell, Zachary T.; 0000-0002-3768-6996 (Campbell, ZT); Bhat, Vandita D.; Shukla, Tarjani; Campbell, Zachary T.PUF (PUmilio/FBF) RNA-binding proteins recognize distinct elements. In C. elegans, PUF-8 binds to an 8-nt motif and restricts proliferation in the germline. Conversely, FBF-2 recognizes a 9-nt element and promotes mitosis. To understand how motif divergence relates to biological function, we first determined a crystal structure of PUF-8. Comparison of this structure to that of FBF-2 revealed a major difference in a central repeat. We devised a modified yeast 3-hybrid screen to identify mutations that confer recognition of an 8-nt element to FBF-2. We identified several such mutants and validated structurally and biochemically their binding to 8-nt RNA elements. Using genome engineering, we generated a mutant animal with a substitution in FBF-2 that confers preferential binding to the PUF-8 element. The mutant largely rescued overproliferation in animals that spontaneously generate tumors in the absence of puf-8. This work highlights the critical role of motif length in the specification of biological function.Item Global Pairwise RNA Interaction Landscapes Reveal Core Features of Protein Recognition(Nature Publishing Group) Zhou, Qin; Kunder, Nikesh; De La Paz, Jose Alberto; Lasley, Alexandra E.; Bhat, Vandita D.; Morcos, Faruck; Campbell, Zachary T.; 0000-0002-3768-6996 (Campbell, ZT); Zhou, Qin; Kunder, Nikesh; De La Paz, Jose Alberto; Lasley, Alexandra E.; Bhat, Vandita D.; Morcos, Faruck; Campbell, Zachary T.RNA-protein interactions permeate biology. Transcription, translation, and splicing all hinge on the recognition of structured RNA elements by RNA-binding proteins. Models of RNA-protein interactions are generally limited to short linear motifs and structures because of the vast sequence sampling required to access longer elements. Here, we develop an integrated approach that calculates global pairwise interaction scores from in vitro selection and high-throughput sequencing. We examine four RNA-binding proteins of phage, viral, and human origin. Our approach reveals regulatory motifs, discriminates between regulated and non-regulated RNAs within their native genomic context, and correctly predicts the consequence of mutational events on binding activity. We design binding elements that improve binding activity in cells and infer mutational pathways that reveal permissive versus disruptive evolutionary trajectories between regulated motifs. These coupling landscapes are broadly applicable for the discovery and characterization of protein-RNA recognition at single nucleotide resolution.Item Inhibition of Poly(A)-Binding Protein with a Synthetic RNA Mimic Reduces Pain Sensitization in Mice(Nature Publishing Group, 2018-10-22) Barragan-Iglesias, Paulino; Lou, Tzu-Fang; Bhat, Vandita D.; Megat, Salim; Burton, Michael D.; Price, Theodore J.; Campbell, Zachary T.; 0000-0002-6971-6221 (Price, TJ); 0000-0002-3768-6996 (Campbell, ZT); Barragan-Iglesias, Paulino; Lou, Tzu-Fang; Bhat, Vandita D.; Megat, Salim; Burton, Michael D.; Price, Theodore J.; Campbell, Zachary T.Nociceptors rely on cap-dependent translation to rapidly induce protein synthesis in response to pro-inflammatory signals. Comparatively little is known regarding the role of the regulatory factors bound to the 3' end of mRNA in nociceptor sensitization. Poly(A)-binding protein (PABP) stimulates translation initiation by bridging the Poly(A) tail to the eukaryotic initiation factor 4F complex associated with the mRNA cap. Here, we use unbiased assessment of PABP binding specificity to generate a chemically modified RNA-based competitive inhibitor of PABP. The resulting RNA mimic, which we designated as the Poly(A) SPOT-ON, is more stable than unmodified RNA and binds PABP with high affinity and selectivity in vitro. We show that injection of the Poly(A) SPOT-ON at the site of an injury can attenuate behavioral response to pain. Collectively, these results suggest that PABP is integral for nociceptive plasticity. The general strategy described here provides a broad new source of mechanism-based inhibitors for RNA-binding proteins and is applicable for in vivo studies.