There is a fundamental need to understand how RNA molecules that do not code for protein (non-coding RNA) are able to regulate gene expression. One method of gene regulation is executed in the cytosol, where non-coding RNAs interact with proteins to control the translation of messenger RNAs. The long-term goal of our laboratory is to characterize protein/RNA interactions that posttranscriptionally regulate gene expression. The overall objective of this R15 application is to characterize the molecular mechanism by which the RNA- binding protein LARP6 binds to its RNA targets. This work will determine the functional evolution of the LARP6 family of post-transcriptional regulators. The central hypothesis is that individual domains and motifs within LARP6 interact with one another to coordinate the specific RNA binding that dictates the physiological function of LARP6. The rationale that drives this proposal is that the evolutionary diversity within the LARP6 family of proteins will reveal how LARP6 binds to RNA. This work will expand the field's understanding of how RNA recognition motif domains are able to bind untranslated regions of mRNAs to control gene expression. This hypothesis will be tested through three specific aims: (1) Recombinantly express and characterize RNA binding by fish LARP6 proteins; (2) identify the role of non-canonical RRM sequences in RNA binding activity; and (3) determine the relationship between RNA binding and nuclear localization of LARP6. In the first aim, the LARP6 proteins from bony fish will be recombinantly expressed and purified for the analysis of RNA binding activity. These data will fill a gap in our understanding of the evolution of this regulatory protein.
The second aim will test what elements of the LARP6 RNA recognition motif are critical for specific recognition of the RNA ligand using a comparative genomics approach in concert with. Quantitative in vitro assays with recombinant LARP6 interspecies chimeric proteins and in vitro transcribed RNAs will report on changes in RNA binding activity.
The third aim will evaluate how RNA binding by LARP6 affects its location in the cell, which is a critical component of how LARP6 is able to control the translation of mRNAs in the cytosol. This approach is innovative because it uses a phylogenetic approach to understand protein function, taking advantage of bioinformatics, molecular biology, protein biochemistry, and cell culture. This work is significant because the results will define a posttranscriptional regulatory protein LARP6 binds to RNA sequences in order to control protein synthesis. This solid molecular biology foundation will also enable the use of fish genetic models to understand the global role of posttranscriptional regulation of gene expression in development and disease. As a result, it will provide novel insight into a fundamental cell biological problem with direct relevance to human health.
The proposed research is relevant to public health because it will expand our understanding of how gene expression is controlled at the level of protein translation. Such regulation is mediated by interactions between RNA and proteins and is a critical factor in pathways as diverse as development, carcinogenesis, and apoptosis. The proposed work will support the mission of NIGMS by identifying the molecular mechanism by which a novel RNA-binding protein is able to bind its targets.
| Castro, José M; Horn, Daniel A; Pu, Xinzhu et al. (2017) Recombinant expression and purification of the RNA-binding LARP6 proteins from fish genetic model organisms. Protein Expr Purif 134:147-153 |
| Villarreal, Michael A; Biediger, Nicole M; Bonner, Natalie A et al. (2017) Determining Zebrafish Epitope Reactivity to Commercially Available Antibodies. Zebrafish 14:387-389 |
| Ream, Jennifer A; Lewis, L Kevin; Lewis, Karen A (2016) Rapid agarose gel electrophoretic mobility shift assay for quantitating protein: RNA interactions. Anal Biochem 511:36-41 |
