Multiple aspects of mRNA translation are subject to regulation. Here we present a ribosome footprinting protocol to determine the location and composition of 40S and 80S ribosome complexes on endogenous mRNAs transcriptome-wide in vivo in yeast and mammalian cells. We present an extension of the translation complex profiling (TCP-seq) protocol, originally developed in yeast, by including an immunoprecipitation step to assay the location of both 40S and 80S ribosome complexes containing proteins of interest. This yields information on where along mRNAs the ribosome-bound protein of interest joins the ribosome to act, and where it leaves again, thereby monitoring the sequential steps of translation and the roles of various translation factors therein. Rapid fixation of live cells ensures the integrity of all translation complexes bound to mRNA at native positions. Two procedures are described, differing mainly in the fixation conditions and the library preparation. Depending on the research question, either procedure offers advantages. Execution of a Sel-TCP-seq experiment takes 5-10 working days, and initial data analysis can be completed within 2 days.

• MeSH
• RNA, Messenger genetics   MeSH
• Protein Biosynthesis * MeSH
• Ribosomes genetics   MeSH
• Saccharomyces cerevisiae * genetics   MeSH
• Mammals genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• RNA, Messenger MeSH

We employed virus-like elements (VLEs) pGKL1,2 from Kluyveromyces lactis as a model to investigate the previously neglected transcriptome of the broader group of yeast cytoplasmic linear dsDNA VLEs. We performed 5' and 3' RACE analyses of all pGKL1,2 mRNAs and found them not 3' polyadenylated and containing frequently uncapped 5' poly(A) leaders that are not complementary to VLE genomic DNA. The degree of 5' capping and/or 5' mRNA polyadenylation is specific to each gene and is controlled by the corresponding promoter region. The expression of pGKL1,2 transcripts is independent of eIF4E and Pab1 and is enhanced in lsm1Δ and pab1Δ strains. We suggest a model of primitive pGKL1,2 gene expression regulation in which the degree of 5' mRNA capping and 5' non-template polyadenylation, together with the presence of negative regulators such as Pab1 and Lsm1, play important roles. Our data also support a hypothesis of a close relationship between yeast linear VLEs and poxviruses.

• Keywords
• Lsm1, Pab1, eIF4E, linear cytoplasmic plasmid, pGKL, poly(A) leader, poxvirus, virus-like element,
• Publication type
• Journal Article MeSH

eIF3 is a large multiprotein complex serving as an essential scaffold promoting binding of other eIFs to the 40S subunit, where it coordinates their actions during translation initiation. Perhaps due to a high degree of flexibility of multiple eIF3 subunits, a high-resolution structure of free eIF3 from any organism has never been solved. Employing genetics and biochemistry, we previously built a 2D interaction map of all five yeast eIF3 subunits. Here we further improved the previously reported in vitro reconstitution protocol of yeast eIF3, which we cross-linked and trypsin-digested to determine its overall shape in 3D by advanced mass-spectrometry. The obtained cross-links support our 2D subunit interaction map and reveal that eIF3 is tightly packed with its WD40 and RRM domains exposed. This contrasts with reported cryo-EM structures depicting eIF3 as a molecular embracer of the 40S subunit. Since the binding of eIF1 and eIF5 further fortified the compact architecture of eIF3, we suggest that its initial contact with the 40S solvent-exposed side makes eIF3 to open up and wrap around the 40S head with its extended arms. In addition, we mapped the position of eIF5 to the region below the P- and E-sites of the 40S subunit.

• MeSH
• Cryoelectron Microscopy MeSH
• Eukaryotic Initiation Factor-1 chemistry   genetics   metabolism   MeSH
• Eukaryotic Initiation Factor-3 chemistry   genetics   metabolism   MeSH
• Eukaryotic Initiation Factor-5 chemistry   genetics   metabolism   MeSH
• Peptide Chain Initiation, Translational * MeSH
• Ribosome Subunits, Small, Eukaryotic genetics   metabolism   MeSH
• Models, Molecular MeSH
• Protein Domains MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   ultrastructure   MeSH
• Protein Binding MeSH
• Binding Sites genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Eukaryotic Initiation Factor-1 MeSH
• Eukaryotic Initiation Factor-3 MeSH
• Eukaryotic Initiation Factor-5 MeSH
• Saccharomyces cerevisiae Proteins MeSH