Polysome-profiling in small tissue samples.

Polysome-profiling is commonly used to study translatomes and applies laborious extraction of efficiently translated mRNA (associated with >3 ribosomes) from a large volume across many fractions. This property makes polysome-profiling inconvenient for larger experimental designs or samples with low RNA amounts. To address this, we optimized a non-linear sucrose gradient which reproducibly enriches for efficiently translated mRNA in only one or two fractions, thereby reducing sample handling 5-10-fold. The technique generates polysome-associated RNA with a quality reflecting the starting material and, when coupled with smart-seq2 single-cell RNA sequencing, translatomes in small tissues from biobanks can be obtained. Translatomes acquired using optimized non-linear gradients resemble those obtained with the standard approach employing linear gradients. Polysome-profiling using optimized non-linear gradients in serum starved HCT-116 cells with or without p53 showed that p53 status associates with changes in mRNA abundance and translational efficiency leading to changes in protein levels. Moreover, p53 status also induced translational buffering whereby changes in mRNA levels are buffered at the level of mRNA translation. Thus, here we present a polysome-profiling technique applicable to large study designs, primary cells and frozen tissue samples such as those collected in biobanks.

eIF5A has a function in the cotranslational translocation of proteins into the ER.

The putative eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved and essential protein present in all organisms except bacteria. To be activated, eIF5A requires the conversion of a specific residue of lysine into hypusine. This hypusine modification occurs posttranslationally in two enzymatic steps, and the polyamine spermidine is the substrate. Despite having an essential function in translation elongation, the critical role played by eIF5A remains unclear. In addition to demonstrating genetic interactions with translation factors, eIF5A mutants genetically interact with mutations in YPT1, which encodes an essential protein involved in endoplasmic reticulum (ER)-to-Golgi vesicle transport. In this study, we investigated the correlation between the function of eIF5A in translation and secretion in yeast. The results of in vivo translocation assays and genetic interaction analyses suggest a specific role for eIF5A in the cotranslational translocation of proteins into the ER, but not in the posttranslational pathway. Additionally, we observed that a block in eIF5A activation up-regulates stress-induced chaperones, which also occurs when SRP function is lost. Finally, loss of eIF5A function affects binding of the ribosome-nascent chain complex to SRP. These results link eIF5A function in translation with a role of SRP in the cell and may help explain the dual effects of eIF5A in differential and general translation.

Translational control by eIF2α in neurons: Beyond the stress response.

The translation of mRNAs is a tightly controlled process that responds to multiple signaling pathways. In neurons, this control is also exerted locally due to the differential necessity of proteins in axons and dendrites. The phosphorylation of the alpha subunit of the translation initiation factor 2 (eIF2α) is one of the mechanisms of translational control. The phosphorylation of eIF2α has classically been viewed as a stress response, halting translation initiation. However, in the nervous system this type of regulation has been related to other mechanisms besides stress response, such as behavior, memory consolidation and nervous system development. Additionally, neurodegenerative diseases have a major stress component, thus eIF2α phosphorylation plays a preeminent role and its modulation is currently viewed as a new opportunity for therapeutic interventions. This review consolidates current information regarding eIF2α phosphorylation in neurons and its impact in neurodegenerative diseases. © 2016 Wiley Periodicals, Inc.