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Book Synopsis Specificity and Manipulation of PUF-RNA Interactions by :
Download or read book Specificity and Manipulation of PUF-RNA Interactions written by and published by . This book was released on 2015 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Post-transcriptional control is essential for biological processes. RNA-binding factors are critical for eliciting mRNA-specific control. The Pumilio and Fem-3 binding factor (PUF) family of proteins is a single-stranded RNA-binding family conserved throughout eukaryotes. PUF proteins bind mRNAs with high specificity in order to control their translation, localization and stability. PUF regulation is dependent upon specific RNA recognition through protein-RNA contacts within the PUF RNA-binding domain (RBD). In these studies, we have elucidated mechanisms governing PUF-RNA interactions and determined binding properties of manipulated PUF scaffolds. We performed structure-based mutational analyses of PUF binding to RNA across several PUF scaffolds. This work led to the identification of critical PUF-RNA interactions and uncovered a binding pattern applicable across PUF proteins. We discovered a mechanism that determines alternate RNA base-flipping patterns observed in Saccharomyces cerevisiae Puf4p and Caenorhabditis elegans FBF-2 crystal structures, and we determined overlapping in vivo regulatory targets of S. cerevisiae Puf4p and Puf5p. Modularity of PUF RBDs allows for their engineered specificity. Three important amino acids, termed the tripartite recognition motif (TRM), impart RNA base specificity for each PUF repeat. We showed the generality of a TRM recognition code across PUF RBD scaffolds and PUF repeats, demonstrating the use of specific TRM combinations to target desired RNA sequences. We also found that additional PUF-RNA contacts do not substantially increase binding affinity or specificity by testing RNA-binding of extended PUF RBDs. Together, these data have furthered our knowledge of how PUF proteins recognize target mRNAs, uncovered details of in vivo PUF regulation, and further developed the PUF RBD as a scaffold for engineered RNA-targeting.