Cellular function and homeostasis are regulated by careful choreography of RNA binding proteins, which play roles in messenger RNA (mRNA) transcription, turnover, subcellular localization, and ultimately translation. We propose to examine how the RNA-binding protein La-related protein 1 (Larp1) interacts with the untranslated regions (UTRs) of mRNAs to shunt them into pathways for translational repression or activation. Many of the RNAs with which Larp1 interacts encode proteins critical for the epithelial-mesenchymal transition (EMT), a series of changes that instigates invasive, metastatic behavior and drug resistance. Indeed, deregulation of Larp1 levels is linked to ovarian, lung, liver, and cervical cancer. Therefore, understanding the underlying mechanisms of Larp1 function will aid in designing diagnostic tools and treatment targets. Larp1 contains at least three putative RNA binding domains, the La- and RNA recognition motifs, which are predicted to form a structural module, and a highly conserved C-terminal region, named the DM15 motif, whose sequence only appears in Larp1 proteins. Larp1 has been proposed to interact with the UTRs of mRNAs and with poly-A binding protein (PABP), the protein that regulates the stability and translation of mRNAs. Recent data suggests that Larp1 relays information from the growth-responsive mTOR kinase to stimulate ribosome biogenesis through direct interactions with mTOR and with mRNAs encoding ribosomal proteins and translation-associated factors; the 5' terminal ends of these mRNAs contain oligopyrimidine tracts (5' TOPs). We hypothesize that the binding of specific RNA sequences in the 5' and 3' UTRs of mRNA by Larp1 modulates its conformation, and therefore its ability to directly interact with poly A-binding protein. We will: 1) Determine the molecular basis for the interaction between the DM15 motif of Larp1 and 5' TOP and poly-A mRNA. 2) Determine the contribution of the La-RRM module of Larp1 to its RNA binding specificity. 3) Determine how the RNA-binding domains of Larp1 cooperate to regulate mRNA binding and translation.
Aims 1 and 2 will be accomplished through in vitro biochemical and biophysical approaches and structure determination taking advantage of existing crystal forms.
Aim 3 will utilize an interdisciplinary approach combining in vitro biochemical characterization of Larp1-RNA recognition and high-throughput sequencing analysis of the RNAs that interact with Larp1 in cells. Not only will we reveal direct structural mechanisms, but our results will also demonstrate new modes of mRNA translation regulation. We will contribute the first structural data for Larp1. In addition, we will reveal the function of the Larp1-specific C-terminal region containing the DM15 motif. Finally, we will lend insight into the gene ontology and RNA sequence determinants underlying translational regulation by Larp1.
Careful regulation of protein synthesis is essential to cellular health; deregulation leads to several types of cancer. The goal of this research is to understand how a molecule named Larp1 regulates the many layers of protein synthesis; when deregulated, ovarian, cervical, liver, and breast cancer result. Our work will reveal the mechanisms of Larp1 that can be targeted for the future design of chemotherapeutics to treat these diseases.
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|Fonseca, Bruno D; Lahr, Roni M; Damgaard, Christian K et al. (2018) LARP1 on TOP of ribosome production. Wiley Interdiscip Rev RNA :e1480|
|Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E et al. (2017) La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs. Elife 6:|