Cell survival in changing environments requires appropriate regulation of gene expression, including posttranscriptional regulatory mechanisms. From reporter gene studies in glucose-starved yeast, it was proposed that translationally silenced eukaryotic mRNAs accumulate in P bodies and can return to active translation. We present evidence contradicting the notion that reversible storage of nontranslating mRNAs is a widespread and general phenomenon. First, genome-wide measurements of mRNA abundance, translation, and ribosome occupancy after glucose withdrawal show that most mRNAs are depleted from the cell coincident with their depletion from polysomes. Second, only a limited subpopulation of translationally repressed transcripts, comprising fewer than 400 genes, can be reactivated for translation upon glucose readdition in the absence of new transcription. This highly selective posttranscriptional regulation could be a mechanism for cells to minimize the energetic costs of reversing gene-regulatory decisions in rapidly changing environments by transiently preserving a pool of transcripts whose translation is rate-limiting for growth.

The receptor for activated C-kinase (RACK1), a conserved protein implicated in numerous signaling pathways, is a stoichiometric component of eukaryotic ribosomes located on the head of the 40S ribosomal subunit. To test the hypothesis that ribosome association is central to the function of RACK1 in vivo, we determined the 2.1-A crystal structure of RACK1 from Saccharomyces cerevisiae (Asc1p) and used it to design eight mutant versions of RACK1 to assess roles in ribosome binding and in vivo function. Conserved charged amino acids on one side of the beta-propeller structure were found to confer most of the 40S subunit binding affinity, whereas an adjacent conserved and structured loop had little effect on RACK1-ribosome association. Yeast mutations that confer moderate to strong defects in ribosome binding mimic some phenotypes of a RACK1 deletion strain, including increased sensitivity to drugs affecting cell wall biosynthesis and translation elongation. Furthermore, disruption of RACK1's position at the 40S ribosomal subunit results in the failure of the mRNA binding protein Scp160 to associate with actively translating ribosomes. These results provide the first direct evidence that RACK1 functions from the ribosome, implying a physical link between the eukaryotic ribosome and cell signaling pathways in vivo.

Initiation factor eIF4G is a key regulator of eukaryotic protein synthesis, recognizing proteins bound at both ends of an mRNA to help recruit messages to the small (40S) ribosomal subunit. Notably, the genomes of a wide variety of eukaryotes encode multiple distinct variants of eIF4G. We found that deletion of eIF4G1, but not eIF4G2, impairs growth and global translation initiation rates in budding yeast under standard laboratory conditions. Not all mRNAs are equally sensitive to loss of eIF4G1; genes that encode messages with longer poly(A) tails are preferentially affected. However, eIF4G1-deletion strains contain significantly lower levels of total eIF4G, relative to eIF4G2-delete or wild type strains. Homogenic strains, which encode two copies of either eIF4G1 or eIF4G2 under native promoter control, express a single isoform at levels similar to the total amount of eIF4G in a wild type cell and have a similar capacity to support normal translation initiation rates. Polysome microarray analysis of these strains and the wild type parent showed that translationally active mRNAs are similar. These results suggest that total eIF4G levels, but not isoform-specific functions, determine mRNA-specific translational efficiency.

m6A methylation is the most abundant and reversible chemical modification on mRNA with approximately one-fourth of eukaryotic mRNAs harboring at least one m6A-modified base. The recruitment of the mRNA m6A methyltransferase writer complex to phase-separated nuclear speckles is likely to be crucial in its regulation; however, control over the activity of the complex remains unclear. Supported by our observation that a core catalytic subunit of the methyltransferase complex, METTL3, is endogenously colocalized within nuclear speckles as well as in noncolocalized puncta, we tracked the components of the complex with a Cry2-METTL3 fusion construct to disentangle key domains and interactions necessary for the phase separation of METTL3. METTL3 is capable of self-interaction and likely provides the multivalency to drive condensation. Condensates in cells necessarily contain myriad components, each with partition coefficients that establish an entropic barrier that can regulate entry into the condensate. In this regard, we found that, in contrast to the constitutive binding of METTL14 to METTL3 in both the diffuse and the dense phase, WTAP only interacts with METTL3 in dense phase and thereby distinguishes METTL3/METTL14 single complexes in the dilute phase from METTL3/METTL14 multicomponent condensates. Finally, control over METTL3/METTL14 condensation is determined by its small molecule cofactor, S-adenosylmethionine (SAM), which regulates conformations of two gate loops, and some cancer-associated mutations near gate loops can impair METTL3 condensation. Therefore, the link between SAM binding and the control of writer complex phase state suggests that the regulation of its phase state is a potentially critical facet of its functional regulation.