DEEP MUTATIONAL SCANNING IN VIVO OF AN RNA RECOGNITION MOTIF

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. RNA-binding proteins (RBPs) mediate virtually all aspects of RNA metabolism, including, transcription, processing, subcellular localization, and regulation of translation and decay. Since these functions underlie multiple cellular processes, genetic changes that disrupt RBP function can lead to human pathologies, such as neurological disorders and cancer. The RNA Recognition Motif (RRM) is the most common RNA-binding domain in eukaryotes. We have undertaken an approach, deep mutational scanning, to study the in vivo function of a single RRM. This approach queries the relative fitness effect of mutations in every position in the domain of interest. In this case we exploit the necessity of a functional Poly(A) binding protein (Pab1) for yeast viability. We created a library of yeast expression plasmids with an average of three mutations in the RRM2 domain of Pab1, and transformed the plasmids into yeast expressing a wild-type Pab1 under a tetracycline-repressible promoter. After addition of tetracycline to the culture, transformants that have a functional Pab1 RRM2 domain survive while nonfunctional mutants are lost. The RRM2 domain in the input and the selected library pools are ready to be sequenced using the Illumina sequencing platform. We anticipate that the results of this experiment will help to characterize the RNA-binding activity of this domain as well as yet unidentified protein regions that are important for function. Furthermore, the data extracted from these experiments might be used to predict how RRM mutations associated with several human genetic diseases affect the function of this domain.