Our major goal is to understand how levels of oncoproteins are controlled, since their sustained synthesis promotes a genetic program the end result of which is tumor formation. As such, oncoprotein production is subject to tight regulatory controls. While transcriptional control has been appreciated for decades, the last several years have presented explosive growth in our appreciation and understanding of posttranscriptional ontrol mechanisms. Two key mechanisms that limit oncoprotein production are mRNA degradation and translational suppression. For example, A+U-rich elements (AREs) within 3'UTRs flag a mRNA for rapid degradation via a sequential pathway involving segmental removal of the poly(A) tail followed by decapping and combined 5'-3' and 3'-5' degradation. Association of proteins such as AUF1 with the ARE initiates this enzymatic cascade of mRNA deconstruction. Association of microRNAs, -22 nt in length, with microRNA response elements (MREs) in target transcripts suppresses their translation and in some cases promotes their degradation by removal of the poly(A) tail. The mRNA encoded by the MYCN oncogene, a neural transcription factor often overexpressed in neuroblastoma, contains AREs, at least one MRE, and several additional unidentified mRNA decay elements.
In Aim 1 of this proposal, we will determine if the MRE contributes to degradation of MYCN mRNA, and we will seek to identify the other mRNA decay elements. We will also examine whether the numerous MYCN decay elements act in an independent, additive, or coordinated fashion. During the prior funding period, we found unexpectedly that AUF1 association with the ARE of MYC mRNA promotes its translation. Using RNA interference, we can reduce AUF1 levels in cells by.95%. In^Aim 2,.we plan to utilize-these eells-to perform genetic add-back experiments of~At)F1 isoforms to determine which ones are necessary and sufficient for MYC translation. The 3'UTR of MYC mRNA also contains three 10-12 nt elements perfectly conserved between six species. Two of these elements lay within predicted stem-loop structure. We thus plan to determine if secondary structure exists and if so, examine its roles in AUF1 binding and translation/decay of MYC mRNA. Finally, we present biochemical and genetic evidence for a novel multi-subunit enzyme that degrades the poly(A) tail of mRNAs. We plan to purify this activity and examine its roles in degradation of MYC, MYCN, and perhaps other ARE-bearing mRNAs.
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