Yeast poly(A)-binding protein (Pab1) controls translation initiation in vivo primarily by blocking mRNA decapping and decay.

Poly(A)-binding protein (Pab1 in yeast) is involved in mRNA decay and translation initiation, but its molecular functions are incompletely understood. We found that auxin-induced degradation of Pab1 reduced bulk mRNA and polysome abundance in WT but not in a mutant lacking the catalytic subunit of decapping enzyme (Dcp2), suggesting that enhanced decapping/degradation is a major driver of reduced translation at limiting Pab1. An increased median poly(A) tail length conferred by Pab1 depletion was likewise not observed in the dcp2Δ mutant, suggesting that mRNA isoforms with shorter tails are preferentially decapped/degraded at limiting Pab1.

Purification and Analysis of eIF2α Phosphorylation by Stress-Activated Protein Kinase Gcn2 from S. cerevisiae.

Gcn2 is the sole eIF2α kinase in budding yeast, responsible for inhibiting general translation while inducing translation of transcriptional activator Gcn4, a master regulator of amino acid biosynthesis, in nutrient-starved cells. Gcn2 is activated by interactions between multiple regulatory domains that overcome the inherent latency of its protein kinase domain, including a pseudokinase domain, one related to histidyl-tRNA synthetase, a ribosome-binding and dimerization domain, and a region that binds the trans-acting activators Gcn1/Gcn20, which respond to deacylated tRNAs engendered by amino acid starvation or other impediments to translation elongation that lead to ribosome stalling and collisions. Here, we describe methods for purifying Gcn2 from yeast cells and assaying its protein kinase activity against a recombinant segment of eIF2α.

Decapping activators Edc3 and Scd6 act redundantly with Dhh1 in post-transcriptional repression of starvation-induced pathways.

Degradation of many yeast mRNAs involves decapping by the Dcp1:Dcp2 complex. Previous studies on decapping activators Edc3 and Scd6 suggested their limited roles in mRNA decay. RNA-seq analysis of mutants lacking one or both proteins revealed that Scd6 and Edc3 have largely redundant activities in targeting numerous mRNAs for degradation that are masked in the single mutants.

Differential effects of 40S ribosome recycling factors on reinitiation at regulatory uORFs in GCN4 mRNA are not dictated by their roles in bulk 40S recycling.

Recycling of 40S ribosomal subunits following translation termination, entailing release of deacylated tRNA and dissociation of the empty 40S from mRNA, involves yeast Tma20/Tma22 heterodimer and Tma64, counterparts of mammalian MCTS1/DENR and eIF2D. MCTS1/DENR enhance reinitiation (REI) at short upstream open reading frames (uORFs) harboring penultimate codons that confer heightened dependence on these factors in bulk 40S recycling. Tma factors, by contrast, inhibited REI at particular uORFs in extracts; however, their roles at regulatory uORFs in vivo were unknown.