Eukaryotic mRNAs can be controlled at many different steps. In the nucleus, transcription and mRNA processing are required to produce an mRNA that can be translated. In the cytoplasm, mature mRNAs can be regulated at the level of stability, transnational activity, and cellular location. The objective of the proposed research is to understand the biochemical mechanisms that control mature mRNAs in animal cells. We focus on the roles of poly (A) addition and removal in the transnational control of mRNAs during early development. These reactions are regulated by sequences that lie between the translation termination codon and the poly (A) tail--in the 3' untranslated region of the mRNA (3'UTR). Our ultimate goals are to understand, in molecular terms how poly (A) and elements in the 3'UTR control translation. We propose to study these reactions in frog oocytes and embryos. We focus on the period of oocyte maturation and use cycline and c-mos mRNAs, as models. We have shown that changes in poly (A) length can regulate translation during early development, and that sequences in the 3""""""""UTR govern the extent and timing of transnational activation and repression. We have developed straightforward in vivo and in vitro assays for the effects of poly (A) and sequences in the 3""""""""UTR. We propose to identify how poly (A) stimulates translation by combining in vivo and in vitro approaches. Using molecular genetics and biochemistry, we will identify the step(s) at which poly (A) exerts its effects and examine the role of the protein to which it binds. By constructing well-defined mutations in synthetic mRNAs, we will precisely identify elements in the 3""""""""UTR that control transnational activity in vivo and determine how they function. We will test our hypothesis that changes in the length of the c-mos mRNA's poly (A) tail, governed by signals in its 3""""""""UTR, are critical for control of the embryonic cell cycle. The work proposed will elucidate fundamental mechanisms of gene expression, and therefore has important practical applications. Regulated changes in poly (A) length occur in many, if not all, animal species, affect many mRNAs in the embryo, and continue throughout life. Our detailed investigations of the regulation of c-mos, a proto-oncogene normally expressed only in the germ line, bear on how cell division is controlled both in normal and abnormal growth.
| Lapointe, Christopher P; Stefely, Jonathan A; Jochem, Adam et al. (2018) Multi-omics Reveal Specific Targets of the RNA-Binding Protein Puf3p and Its Orchestration of Mitochondrial Biogenesis. Cell Syst 6:125-135.e6 |
| Aoki, Scott T; Porter, Douglas F; Prasad, Aman et al. (2018) An RNA-Binding Multimer Specifies Nematode Sperm Fate. Cell Rep 23:3769-3775 |
| Lapointe, Christopher P; Wickens, Marvin (2018) RNA Tagging: Preparation of High-Throughput Sequencing Libraries. Methods Mol Biol 1649:455-471 |
| Wilinski, Daniel; Buter, Natascha; Klocko, Andrew D et al. (2017) Recurrent rewiring and emergence of RNA regulatory networks. Proc Natl Acad Sci U S A 114:E2816-E2825 |
| Lapointe, Christopher P; Preston, Melanie A; Wilinski, Daniel et al. (2017) Architecture and dynamics of overlapped RNA regulatory networks. RNA 23:1636-1647 |
| Shin, Heaji; Haupt, Kimberly A; Kershner, Aaron M et al. (2017) SYGL-1 and LST-1 link niche signaling to PUF RNA repression for stem cell maintenance in Caenorhabditis elegans. PLoS Genet 13:e1007121 |
| Prasad, Aman; Porter, Douglas F; Kroll-Conner, Peggy L et al. (2016) The PUF binding landscape in metazoan germ cells. RNA 22:1026-43 |
| Aoki, Scott T; Kershner, Aaron M; Bingman, Craig A et al. (2016) PGL germ granule assembly protein is a base-specific, single-stranded RNase. Proc Natl Acad Sci U S A 113:1279-84 |
| Campbell, Zachary T; Wickens, Marvin (2015) Probing RNA-protein networks: biochemistry meets genomics. Trends Biochem Sci 40:157-64 |
| Porter, Douglas F; Koh, Yvonne Y; VanVeller, Brett et al. (2015) Target selection by natural and redesigned PUF proteins. Proc Natl Acad Sci U S A 112:15868-73 |
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