UC San Diego

.Decapping is a critical step in several mRNA decay pathways that are important for the regulation of gene expression. A major decapping complex consists of the decapping enzyme Dcp2 as well as several decapping enhancers. Although several interactions between decapping factors have been mapped, little is known about how Dcp2 activity is controlled to target the appropriate mRNAs for decapping or how these enhancers regulate this activity. Through a deletion analysis, I identified an important autoregulatory domain that is responsible for Dcp2 protein turnover. I demonstrated that the metazoan specific decapping enhancer, Hedls, modulates Dcp2 stability and requires the autoregulatory domain of Dcp2 for interaction. Furthermore, Hedls depletion reduces Dcp2 cellular activity independent of protein stability. Thus Hedls promotes Dcp2 mediated decapping by at least two mechanisms: controlling Dcp2 stability and enhancing Dcp2 catalytic activity. This could provide a way to modulate Dcp2 activity according to cellular demand, thereby preventing decapping of inappropriate targets. Hedls is required for Dcp2 concentration in mRNP granules called processing bodies (PBs), which contain mRNA substrates targeted for decay and the associated mRNA decay factors. Because Hedls also modulates Dcp2 stability, I hypothesized that Dcp2 might be stabilized when associated with mRNA substrates as a mechanism for controlling Dcp2 levels according to cellular substrate levels. To test this, PBs were used as a tool to determine whether Dcp2 stability increases when concentrated with mRNPs. Through a deletion analysis, I found that the C-terminus of Hedls is required for concentration in PBs. Nevertheless, its deletion did not affect Dcp2 association or stabilization. I also examined a catalytically inactive Dcp2 mutant predicted to trap Dcp2 with mRNPs, but I did not find an increase in stability. Finally, I co-transfected an mRNA reporter containing a stem loop from Rrp41 mRNA for which Dcp2 has a high affinity and did not find stabilization. Therefore, exactly what dictates Dcp2 stability remains unknown and is an important topic for further investigation. In sum, my studies revealed an unexpected mechanism of Dcp2 regulation that could serve to prevent promiscuous Dcp2 activity thereby ensuring decapping of only mRNAs targeted for decay