In eukaryotic cells, components of the 5′ to 3′ mRNA degradation machinery can undergo a rapid phase transition. The resulting cytoplasmic foci are referred to as processing bodies (P-bodies). The molecular details of the self-aggregation process are, however, largely undetermined. Herein, we use a bottom-up approach that combines NMR spectroscopy, isothermal titration calorimetry, X-ray crystallography, and fluorescence microscopy to probe if mRNA degradation factors can undergo phase transitions in vitro. We show that the Schizosaccharomyces pombe Dcp2 mRNA decapping enzyme, its prime activator Dcp1, and the scaffolding proteins Edc3 and Pdc1 are sufficient to reconstitute a phase-separation process. Intermolecular interactions between the Edc3 LSm domain and at least 10 helical leucine-rich motifs in Dcp2 and Pdc1 build the core of the interaction network. We show that blocking of these interactions interferes with the clustering behavior, both in vitro and in vivo.

The eukaryotic mRNA 5 ′ cap structure is indispensible for pre-mRNA processing, mRNA export, translation initiation, and mRNA stability. Despite this importance, structural and biophysical studies that involve capped RNA are challenging and rare due to the lack of a general method to prepare mRNA in sufficient quantities. Here, we show that the vaccinia capping enzyme can be used to produce capped RNA in the amounts that are required for large-scale structural studies. We have therefore designed an efficient expression and purification protocol for the vaccinia capping enzyme. Using this approach, the reaction scale can be increased in a cost-efficient manner, where the yields of the capped RNA solely depend on the amount of available uncapped RNA target. Using a large number of RNA substrates, we show that the efficiency of the capping reaction is largely independent of the sequence, length, and secondary structure of the RNA, which makes our approach generally applicable. We demonstrate that the capped RNA can be directly used for quantitative biophysical studies, including fluorescence anisotropy and highresolution NMR spectroscopy. In combination with 13 C-methyl-labeled S-adenosyl methionine, the methyl groups in the RNA can be labeled for methyl TROSY NMR spectroscopy. Finally, we show that our approach can produce both cap-0 and cap-1 RNA in high amounts. In summary, we here introduce a general and straightforward method that opens new means for structural and functional studies of proteins and enzymes in complex with capped RNA.

In flowering plants, the asymmetrical division of the zygote is the first hallmark of apical-basal polarity of the embryo and is controlled by a MAP kinase pathway that includes the MAPKKK YODA (YDA). In Arabidopsis, YDA is activated by the membraneassociated pseudokinase SHORT SUSPENSOR (SSP) through an unusual parent-of-origin effect: SSP transcripts accumulate specifically in sperm cells but are translationally silent. Only after fertilization is SSP protein transiently produced in the zygote, presumably from paternally inherited transcripts. SSP is a recently diverged, Brassicaceae-specific member of the BRASSINOSTEROID SIGNALING KINASE (BSK) family. BSK proteins typically play broadly overlapping roles as receptorassociated signaling partners in various receptor kinase pathways involved in growth and innate immunity. This raises two questions: How did a protein with generic function involved in signal relay acquire the property of a signal-like patterning cue, and how is the early patterning process activated in plants outside the Brassicaceae family, where SSP orthologs are absent? Here, we show that Arabidopsis BSK1 and BSK2, two close paralogs of SSP that are conserved in flowering plants, are involved in several YDA-dependent signaling events, including embryogenesis. However, the contribution of SSP to YDA activation in the early embryo does not overlap with the contributions of BSK1 and BSK2. The loss of an intramolecular regulatory interaction enables SSP to constitutively activate the YDA signaling pathway, and thus initiates apical-basal patterning as soon as SSP protein is translated after fertilization and without the necessity of invoking canonical receptor activation.Arabidopsis thaliana | evolution | MAP kinase signaling | embryogenesis |

The scavenger decapping enzyme hydrolyses the protecting 5′ cap structure from short mRNAs that result from exosomal degradation. Based on static crystal structures and NMR data it is apparent that the dimeric enzyme has to undergo large structural changes to bind substrate in a catalytically competent conformation. Here, we study the yeast enzyme and show that the associated opening-closing motions can be orders of magnitude faster than the catalytic turnover rate. This excess of motion is induced by binding of a second ligand to the enzyme, which occurs under high substrate concentrations. We designed a mutant that disrupts the allosteric pathway that links the second binding event to the dynamics and show that this mutant enzyme is hyperactive. Our data reveals a unique mechanism of substrate inhibition, where motions that are required for catalytic activity also inhibit efficient turnover, when they are present in excess.

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Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase involved in development and human disease, including cancer. It is currently thought that the four-point one, ezrin, radixin, moesin (FERM)-kinase domain linker, which contains autophosphorylation site tyrosine (Y) 397, is not required for in vivo FAK function until late midgestation. Here, we directly tested this hypothesis by generating mice with FAK Y397-to-phenylalanine (F) mutations in the germline. We found that Y397F embryos exhibited reduced mesodermal fibronectin (FN) and osteopontin expression and died during mesoderm development akin to FAK kinase-dead mice. We identified myosin-1E (MYO1E), an actin-dependent molecular motor, to interact directly with the FAK FERM-kinase linker and induce FAK kinase activity and Y397 phosphorylation. Active FAK in turn accumulated in the nucleus where it led to the expression of osteopontin and other FN-type matrix in both mouse embryonic fibroblasts and human melanoma. Our data support a model in which FAK Y397 autophosphorylation is required for FAK function in vivo and is positively regulated by MYO1E.focal adhesion | myosin | fibronectin | melanoma | cancer F ocal adhesion kinase (FAK) is a nonreceptor tyrosine kinase involved in many biological processes, ranging from mesoderm development to cancer cell metastasis (1). FAK localizes to focal adhesions (2), where it becomes part of a multiprotein complex that links the extracellular matrix (ECM) to the intracellular actin cytoskeleton. FAK is also found in the nucleus, where it is believed to relay information from the cell cortex (3) and induce transcriptional changes (4). The domain architecture of FAK comprises a four-point one, ezrin, radixin, moesin (FERM) domain that is separated from a C-terminal catalytic kinase domain by the FERM-kinase linker. FAK kinase-dead (5) embryos die with mesodermal defects during late gastrulation. In contrast, mice with conditional FAK deletions in the epidermis (6) or breast epithelium (7) show resistance to carcinogenesis.Although FAK has important biological functions, the mechanisms regulating its activity are incompletely understood. For example, it is unclear whether the FERM-kinase linker that contains autophosphorylation site tyrosine (Y) 397 is required for FAK activity in vivo (8). In its closed, inactive conformation, the FAK kinase domain is autoinhibited through interaction with the N-terminal FERM domain. Y397 is nonphosphorylated (9). Upon activation by tethering (10) or other stimuli that induce conformational change (11), the linker region is exposed and Y397 becomes autophosphorylated, leading to the recruitment of the protooncogene SRC. FAK and SRC then form a transient complex, which stabilizes FAK in its active conformation and induces changes in cell shape and focal adhesion turnover in vitro (12). However, mice with a 19-aa deletion in the FAK linker that includes Y397 develop normally until midgestation (8).Here, we have mechanistically discerned the contributions of Y397 to FAK function in viv...

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The 5′ messenger RNA (mRNA) cap structure enhances translation and protects the transcript against exonucleolytic degradation. During mRNA turnover, this cap is removed from the mRNA. This decapping step is catalyzed by the Scavenger Decapping Enzyme (DcpS), in case the mRNA has been exonucleolyticly shortened from the 3′ end by the exosome complex. Here, we show that DcpS only processes mRNA fragments that are shorter than three nucleotides in length. Based on a combination of methyl transverse relaxation optimized (TROSY) NMR spectroscopy and X-ray crystallography, we established that the DcpS substrate length-sensing mechanism is based on steric clashes between the enzyme and the third nucleotide of a capped mRNA. For longer mRNA substrates, these clashes prevent conformational changes in DcpS that are required for the formation of a catalytically competent active site. Point mutations that enlarge the space for the third nucleotide in the mRNA body enhance the activity of DcpS on longer mRNA species. We find that this mechanism to ensure that the enzyme is not active on translating long mRNAs is conserved from yeast to humans. Finally, we show that the products that the exosome releases after 3′ to 5′ degradation of the mRNA body are indeed short enough to be decapped by DcpS. Our data thus directly confirms the notion that mRNA products of the exosome are direct substrates for DcpS. In summary, we demonstrate a direct relationship between conformational changes and enzyme activity that is exploited to achieve substrate selectivity.