Protocol for detecting and quantifying hATF4-HA in non-stress versus stress conditions using automated and quantitative Jess western blotting.

Activating transcription factor 4 (ATF4) is a key player in the integrated stress response, whose expression is subject to tight translational control. Studying its stress-provoked induction, accompanied by the general translational shutdown, is intricate because the expression of reference genes declines rapidly, and finding appropriate normalization controls is challenging. We present a protocol for human hemagglutinin-tagged ATF4 (hATF4-HA) detection and high-throughput quantification in non-stress versus stress conditions using automated and quantitative western blotting.

Perturbations in eIF3 subunit stoichiometry alter expression of ribosomal proteins and key components of the MAPK signaling pathways.

Protein synthesis plays a major role in homeostasis and when dysregulated leads to various pathologies including cancer. To this end, imbalanced expression of eukaryotic translation initiation factors (eIFs) is not only a consequence but also a driver of neoplastic growth. eIF3 is the largest, multi-subunit translation initiation complex with a modular assembly, where aberrant expression of one subunit generates only partially functional subcomplexes.

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.

Impacts of yeast Tma20/MCTS1, Tma22/DENR and Tma64/eIF2D on translation reinitiation and ribosome recycling.

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

Stem-loop-induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control.

Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response, leading cells toward adaptation or death. ATF4's induction under stress was thought to be due to delayed translation reinitiation, where the reinitiation-permissive upstream open reading frame 1 (uORF1) plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes.

RIP-seq reveals RNAs that interact with RNA polymerase and primary sigma factors in bacteria.

Bacteria have evolved structured RNAs that can associate with RNA polymerase (RNAP). Two of them have been known so far-6S RNA and Ms1 RNA but it is unclear if any other types of RNAs binding to RNAP exist in bacteria. To identify all RNAs interacting with RNAP and the primary σ factors, we have established and performed native RIP-seq in Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis.

Stem-loop induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control.

ATF4 is a master transcriptional regulator of the integrated stress response leading cells towards adaptation or death. ATF4's induction under stress was thought to be mostly due to delayed translation reinitiation, where the reinitiation-permissive uORF1 plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes.

Short tRNA anticodon stem and mutant eRF1 allow stop codon reassignment.

Cognate tRNAs deliver specific amino acids to translating ribosomes according to the standard genetic code, and three codons with no cognate tRNAs serve as stop codons. Some protists have reassigned all stop codons as sense codons, neglecting this fundamental principle. Here we analyse the in-frame stop codons in 7,259 predicted protein-coding genes of a previously undescribed trypanosomatid, Blastocrithidia nonstop.

Selective footprinting of 40S and 80S ribosome subpopulations (Sel-TCP-seq) to study translation and its control.

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.

eIF4G is retained on ribosomes elongating and terminating on short upstream ORFs to control reinitiation in yeast.

Translation reinitiation is a gene-specific translational control mechanism. It is characterized by the ability of short upstream ORFs to prevent full ribosomal recycling and allow the post-termination 40S subunit to resume traversing downstream for the next initiation event. It is well known that variable transcript-specific features of various uORFs and their prospective interactions with initiation factors lend them an unequivocal regulatory potential.

Increased expression of tryptophan and tyrosine tRNAs elevates stop codon readthrough of reporter systems in human cell lines.

Regulation of translation via stop codon readthrough (SC-RT) expands not only tissue-specific but also viral proteomes in humans and, therefore, represents an important subject of study. Understanding this mechanism and all involved players is critical also from a point of view of prospective medical therapies of hereditary diseases caused by a premature termination codon. tRNAs were considered for a long time to be just passive players delivering amino acid residues according to the genetic code to ribosomes without any active regulatory roles.

Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes.

Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells.

Adapted formaldehyde gradient cross-linking protocol implicates human eIF3d and eIF3c, k and l subunits in the 43S and 48S pre-initiation complex assembly, respectively.

One of the key roles of the 12-subunit eukaryotic translation initiation factor 3 (eIF3) is to promote the formation of the 43S and 48S pre-initiation complexes (PICs). However, particular contributions of its individual subunits to these two critical initiation reactions remained obscure. Here, we adapted formaldehyde gradient cross-linking protocol to translation studies and investigated the efficiency of the 43S and 48S PIC assembly in knockdowns of individual subunits of human eIF3 known to produce various partial subcomplexes.

uS3/Rps3 controls fidelity of translation termination and programmed stop codon readthrough in co-operation with eIF3.

Ribosome was long considered as a critical yet passive player in protein synthesis. Only recently the role of its basic components, ribosomal RNAs and proteins, in translational control has begun to emerge. Here we examined function of the small ribosomal protein uS3/Rps3, earlier shown to interact with eukaryotic translation initiation factor eIF3, in termination.

Binding of eIF3 in complex with eIF5 and eIF1 to the 40S ribosomal subunit is accompanied by dramatic structural changes.

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.

Dynamics of the Pollen Sequestrome Defined by Subcellular Coupled Omics.

Reproduction success in angiosperm plants depends on robust pollen tube growth through the female pistil tissues to ensure successful fertilization. Accordingly, there is an apparent evolutionary trend to accumulate significant reserves during pollen maturation, including a population of stored mRNAs, that are utilized later for a massive translation of various proteins in growing pollen tubes. Here, we performed a thorough transcriptomic and proteomic analysis of stored and translated transcripts in three subcellular compartments of tobacco (), long-term storage EDTA/puromycin-resistant particles, translating polysomes, and free ribonuclear particles, throughout tobacco pollen development and in in vitro-growing pollen tubes.