Heterotrimeric G proteins were originally discovered through efforts to understand the effects of hormones, such as glucagon and epinephrine, on glucose metabolism. On the other hand, many cellular metabolites, including glucose, serve as ligands for G protein-coupled receptors. Here we investigate the consequences of glucose-mediated receptor signaling, and in particular the role of a Gα subunit Gpa2 and a non-canonical Gβ subunit, known as Asc1 in yeast and RACK1 in animals. Asc1/RACK1 is of particular interest because it has multiple, seemingly unrelated, functions in the cell. The existence of such “moonlighting” operations has complicated the determination of phenotype from genotype. Through a comparative analysis of individual gene deletion mutants, and by integrating transcriptomics and metabolomics measurements, we have determined the relative contributions of the Gα and Gβ protein subunits to glucose-initiated processes in yeast. We determined that Gpa2 is primarily involved in regulating carbohydrate metabolism while Asc1 is primarily involved in amino acid metabolism. Both proteins are involved in regulating purine metabolism. Of the two subunits, Gpa2 regulates a greater number of gene transcripts and was particularly important in determining the amplitude of response to glucose addition. We conclude that the two G protein subunits regulate distinct but complementary processes downstream of the glucose-sensing receptor, as well as processes that lead ultimately to changes in cell growth and metabolism.

The yeast Saccharomyces cerevisiae has long been used to produce alcohol from glucose and other sugars. While much is known about glucose metabolism, relatively little is known about the receptors and signaling pathways that indicate glucose availability. Here, we compare the two glucose receptor systems in S. cerevisiae. The first is a heterodimer of transporter-like proteins (transceptors), while the second is a seven-transmembrane receptor coupled to a large G protein (Gpa2) that acts in coordination with two small G proteins (Ras1 and Ras2). Through comprehensive measurements of glucose-dependent transcription and metabolism, we demonstrate that the two receptor systems have distinct roles in glucose signaling: the G-protein-coupled receptor directs carbohydrate and energy metabolism, while the transceptors regulate ancillary processes such as ribosome, amino acids, cofactor and vitamin metabolism. The large G-protein transmits the signal from its cognate receptor, while the small G-protein Ras2 (but not Ras1) integrates responses from both receptor pathways. Collectively, our analysis reveals the molecular basis for glucose detection and the earliest events of glucose-dependent signal transduction in yeast.

While evolution is often considered from a DNA- and protein-centric view, RNA-based regulation can also impact gene expression and protein sequences. Here we examine interspecies differences in RNA-protein interactions using the conserved neuronal RNA-binding protein, Unkempt (UNK) as model. We find that roughly half of mRNAs bound in human are also bound in mouse. Unexpectedly, even when transcript-level binding was conserved across species differential motif usage was prevalent. To understand the biochemical basis of UNK-RNA interactions, we reconstitute the human and mouse UNK-RNA interactomes using a high-throughput biochemical assay. We uncover detailed features driving binding, show that in vivo patterns are captured in vitro, find that highly conserved sites are the strongest bound, and associate binding strength with downstream regulation. Furthermore, subtle sequence differences surrounding motifs are key determinants of species-specific binding. We highlight the complex features driving protein-RNA interactions and how these evolve to confer species-specific regulation.

RNA-protein interactions are pivotal to proper gene regulation. Many RNA-binding proteins possess multiple RNA-binding domains; however, how these domains interplay to select and regulate RNA targets remains poorly understood. Here, we investigate three multi-domain proteins, Musashi-1, Musashi-2, and unkempt, which share a high degree of RNA specificity, a common feature across RNA-binding proteins. We used massively parallel in vitro assays with unprecedented depth with random or naturally-derived RNA sequences and find that individual domains within a protein can have differing affinities, specificities, and motif spacing preferences. We conducted large scale competition assays between these proteins and determined how individual protein specificities and affinities influence competitive binding. Integration of binding and regulation in cells with in vitro specificities showed that target selection involves a combination of the protein intrinsic specificities described here, but cellular context is critical to drive these proteins to motifs in specific transcript regions. Finally, evolutionarily conserved RNA regions displayed evidence of binding multiple RBPs in cultured cells, and these RNA regions represent the highest affinity targets. This work emphasizes the importance of in vitro and in cultured cells studies to fully profile RNA-binding proteins and highlights the complex modes of RNA-protein interactions and the contributing factors in target selection.

Histone mRNA degradation is controlled by the unique 3’ stem-loop of histone mRNA and the stem-loop binding protein (SLBP). As part of this process, the 3’ stem-loop is trimmed by the histone-specific 3’ exonuclease (3’hExo) and uridylated by the terminal uridylyl transferase 7 (TUT7), creating partially degraded intermediates with short uridylations. The role of these uridylations in degradation is not fully understood. Our work examines changes in the stability of the ternary complex created by trimming and uridylation of the stem-loop to better understand the role of this process in the histone mRNA life cycle. In this study, we used fluorescence polarization and electrophoretic mobility shift assays to demonstrate that both SLBP and 3’hExo can bind to uridylated and partially degraded stem-loop intermediates, although with lower affinity. We further characterized this complex by performing 1-µs molecular dynamics simulations using the AMBER force field and Nanoscale Molecular Dynamics (NAMD). These simulations show that while uridylation helps maintain the overall shape of the stem-loop, the combination of uridylation and dephosphorylation of the TPNK motif in SLBP disrupts key RNA–protein interactions. They also demonstrate that uridylation allows 3’hExo to maintain contact with the stem-loop after partial degradation and plays a role in disrupting key base pairs in partially degraded histone mRNA intermediates. Together, these experiments and simulations suggest that trimming by 3’hExo, uridylation, and SLBP dephosphorylation weakens both RNA–protein interactions and the stem-loop itself. Our results further elucidate the role of uridylation and SLBP dephosphorylation in the early stages of histone mRNA degradation.

The yeast Saccharomyces cerevisiae has long been used to produce alcohol from glucose and other sugars. While much is known about glucose metabolism, relatively little is known about the receptors and signaling pathways that indicate glucose availability. Here we compare the two glucose receptor systems in S. cerevisiae . The first is a heterodimer of transporter-like proteins (transceptors), while the second is a seven-transmembrane receptor coupled to a large G protein (Gpa2) and two small G proteins (Ras1 and Ras2). Through comprehensive measurements of glucose-dependent transcription and metabolism, we demonstrate that the two receptor systems have distinct roles in glucose signaling: the G protein-coupled receptor directs carbohydrate and energy metabolism, while the transceptors regulate ancillary processes such as ribosome, amino acids, cofactor and vitamin metabolism. The large G protein transmits the signal from its cognate receptor, while the small G protein Ras2 (but not Ras1) integrates responses from both receptor pathways. Collectively, our analysis reveals the molecular basis for glucose detection and the earliest events of glucose-dependent signal transduction in yeast.