Rna Activation PDF Download

Bulk RNA degradation irreversibly removes an mRNA from the translating pool to regulate gene expression. Transcripts must be degraded in a coordinated manner during cell cycle, development and in response to stimuli. A dense network of proteins assembles on a transcript to ensure timely and specific destruction of the RNA. This results in trimming of the 3' poly(A) tail followed by removal of the 5' methyl-7 guanine (m7G) cap structure, leading to rapid exonucleolysis of the message. A key question has been understanding how protein factors at the 3' end trigger decapping at the 5' end. Pat1 is a central scaffolding protein that interacts with multiple decay factors to control distinct steps during RNA turnover. Previous work has demonstrated that Pat1 enhances binding of the heteroheptameric Lsm1-7 complex to the 3' end and promotes decapping by the Dcp1/Dcp2 enzyme complex at the 5' end. Due to the multifunctional nature of Pat1, however, we lack a mechanistic understanding of how it regulates RNA turnover. In this work, I have used a reconstituted system to understand how Pat1 interacts with and activates distinct factors during 5'-3' degradation. First, I show how Pat1 interacts with and enhance the RNA binding of Lsm1-7. This increased affinity is selective for adenine-rich oligoRNAs, which in turn broadens the specificity of the Lsm1-7 complex. Second, I show how Pat1 interacts with short linear motifs in the disordered C-terminal tail of Dcp2 to activate decapping by either recruiting the enzyme complex to substrate or alleviating autoinhibition to promote catalysis. Both activation of Lsm1-7 and decapping require a bipartite interaction between two domains of Pat1 and involve distinct surfaces and motifs. Last, I uncover how different decay factors tune both the size and assembly of Pat1, which may be leveraged to organize an active decapping complex. This biochemically reconstituted system provides a framework for how Pat1 can regulate multiple protein cofactors and steps during bulk RNA turnover.