Molecular Mechanisms of mRNA Regulation and Decay

Division Of Basic Sciences - Nci

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Abstract

The Valkov laboratory focus is on investigating pathways that control and regulate gene expression in the cell after mature mRNAs are exported into the cytoplasm for translation. Specifically, we seek to understand molecular mechanisms and regulatory networks of mRNA decay. Previous work combined crystallization of individual proteins or isolated domains and interacting peptides to obtain in vitro insights with functional validation of structural hypotheses in cells through structure-guided mutagenesis of interacting interfaces. Our present approach is to biochemically reconstitute intact complexes with strictly defined multisubunit composition to address new challenges and outstanding questions in the field. To achieve this, we have developed the infrastructure and methodological expertise to enable routine production of large, intact complexes and to analyze their structure by high-resolution cryoelectron microscopy. One of the most urgent questions in mRNA decay field concerns the structural principles that govern the assembly and architecture of metazoan CCR4-NOT deadenylation complex. CCR4-NOT has critical functions in the shortening of poly(A) tails of bulk mRNA and translational repression of specific mRNA targets via interactions with diverse RNA-binding proteins. We reconstituted the multisubunit human CCR4-NOT complex using a step-wise approach. As was shown by others for the yeast CCR4-NOT complex, the intact human complex is significantly more active in shortening polyadenosine tails in vitro than its exonuclease module alone. Strict control over composition of the deadenylation machinery using the reconstitution approach allows us to discriminate between mechanistic hypotheses such as whether increased activity is due to allosteric effects or improved RNA binding. We are also utilizing the single-nucleotide precision of our biochemical assays to deriving quantitative models of poly(A) tail shortening in various contexts in order to understand how the CCR4-NOT achieves its exquisite selectivity for poly(A) and how specific mRNA targets may be recruited for decay by RNA-binding factors. One of our principal aims in this project is to determine the structure of the intact metazoan CCR4-NOT by cryoEM. An initial low-resolution view by negative stain in-house pointed to feasibility of this. We have now moved to high-resolution cryoEM work. With the repertoire of high-resolution structures of subcomplexes and individual domains of CCR4-NOT available, we are well placed to obtain an accurate high-resolution structural model of the metazoan CCR4-NOT complex. Removal of protective 7-methylguanosine cap from the 5' end of a transcript by decapping enzyme DCP2 is generally an irreversible step towards committing the bulk mRNA for degradation. Therefore, decapping is strictly regulated in the cell by a number of proteins termed activators or enhancers of decapping. Previously, we determined a high-resolution crystal structure of a ternary complex of fission yeast Dcp2 enzyme with an obligate coactivator Dcp1 and a peptide motif from disordered enhancer Edc1. We observed a substantial rotation of catalytic domain of Dcp2 stabilized by binding of Edc1 peptide motif, which allows residues from both Dcp2 and Dcp1 to cooperate in RNA binding. We proposed that this conformational change leads to decapping activation by increasing affinity for mRNA. Several recent studies from other groups have provided alternative mechanistic explanations for the precise action of Edc1 based on crystal structures in which different conformations of Dcp2 have been observed. The laboratory has now pivoted toward the study of the human decapping network in a defined in vitro system with purified factors. We produced all key full-length decapping factors with functionally relevant low-complexity regions intact. We discovered, that human full-length DCP2 enzyme is highly active in isolation in contrast to the yeast Dcp2, which contains a number of repressive motifs in the unstructured C-terminal region. We showed that the activating function of C-terminal region of human DCP2 can be attributed to its high affinity for RNA. Biochemical insights with purified proteins and complexes further yielded new insights into the regulation of DCP2 by known factors. We will continue to improve and extend our in vitro framework of mRNA decay and translational machinery and apply hybrid approaches to study their interactions. We are poised to biochemically rebuild the complete 5'-3' decay reaction in a test tube with purified proteins. This offers an exciting prospect of finally obtaining the long-sought molecular understanding of the mechanistic links between the up- and downstream events in this pathway. We will integrate in vitro work closely with functional studies in knockout cell lines generated by genome editing. We plan to study purified RNA-binding factors with intact low-complexity regions that recruit CCR4-NOT to specific mRNA targets to effect repression in strictly defined systems, which we can perturb and manipulate at will to answer questions about specificity and directionality of CCR4-NOT-mediated recruitment and repression. Finally, we will seek to understand whether artificially induced condensation of the diverse array of purified factors we have generated may influence RNA sequestration into phase-separated droplets and the effect of such separation on decay kinetics.