RNAs and Proteins associated with P-body components

Post-transcriptional processes play a crucial role in the regulation of eukaryotic gene expression. miRNAs are small non-coding RNAs that have been well established as key regulators of gene expression at the translational level. More recently evidence accumulated that miRNAs can also accelerate mRNA degradation of some of their targets. Consistently, decapping activators of the general mRNA decay pathway (e.g. Ge-1, Me31B and HPat) have been shown to be important in miRNA-mediated degradation of mRNAs. However an important open question is how the miRNA effector complex can recruit the mRNA degradation machinery and thereby accelerate mRNA decay. To address this question we propose to TAP-tag proteins of the miRNA effector complex (AGO1 and GW182) and mRNA decapping activators (Ge-1, Me31B, and HPat) and start an extensive search for interacting factors with pull-down experiments in Drosophila cells. Moreover, we will use the TAP-tagged proteins to do a genome-wide analysis of mRNAs associated with above mentioned proteins. This will identify a large set of potential miRNA targets isolated by direct association with components of the miRNA effector complex that can be further divided in subsets dependend on their requirement for decapping activators. In combination with a miRNA microarray, which will identify the miRNAs on miRNA targets of pull-down experiments, this will enable us to do a comprehensive and comparative analysis of miRNA binding sites in natural 3`UTRs of differntly regulated miRNA targets. This set of data will not only be of great value for the field per se, but we can also start to search for possible differences in the 3`UTRs which could determine the extend of mRNA degradation upon miRNA targeting. This will be of great interest for the miRNA field since it is entirely unclear why a specific miRNA can induce mRNA degradation to various degrees, ranging from no degradation to complete degradation, on different miRNA targets. Overall, since Ge-1, Me31B and HPat are general decapping activator, we expect the proposed work to significantly contribute to our mechanistic understanding of the miRNA pathway but also to be important for the field of mRNA degradation and general translation repression.

Post-transcriptional processes play a crucial role in the regulation of eukaryotic gene expression. miRNAs are small non-coding RNAs that have been well established as key regulators of gene expression, which result in translation repression and/or mRNA destabilization upon binding to their target mRNAs. Key factors for miRNA-mediated mRNA degradation are the components of the miRNA effector complex (AGO1 and GW182) and the general mRNA degradation machinery (deadenylation and decapping enzymes). An important question we addressed in this project is how the mRNA decapping machinery gets recruited to mRNAs after targeting by miRNAs. We could identify the interaction of GW182, a component of the miRNA effector complex, and HPat, a decapping activator. In split-affinity purifications we could further demonstrate the binding of HPat to the miRNA effector complex consisting of both AGO1 and GW182. Furthermore, we identified the protein NOT1, a component of the CCR4-NOT deadenylation complex, as a critical factor for the interaction of HPat with the miRNA effector complex. Thus our results demonstrate that the decapping step is not only a consequence of deadenylation but decapping activators get recruited to the miRNA effector complex. The recruitment of HPat to the miRNA effector complex provides a mechanism to commit the mRNA for degradation.