The IRESite (http://www.iresite.org) presents carefully curated experimental evidence of many eukaryotic viral and cellular internal ribosome entry site (IRES) regions. At the time of submission, IRESite stored >600 records. The IRESite gradually evolved into a robust tool providing (i) biologically meaningful information regarding the IRESs and their experimental background (including annotation of IRES secondary structures and IRES trans-acting factors) as well as (ii) thorough concluding remarks to stored database entries and regularly updated evaluation of the reported IRES function. A substantial portion of the IRESite data results purely from in-house bioinformatic analyses of currently available sequences, in silico attempts to repeat published cloning experiments, DNA sequencing and restriction endonuclease verification of received plasmid DNA. We also present a newly implemented tool for displaying RNA secondary structures and for searching through the structures currently stored in the database. The Supplementary Data contains an updated list of reported IRESs.

Eukaryotic translation initiation factor 4E (eIF4E) plays a pivotal role in the control of cap-dependent translation initiation, modulates the fate of specific mRNAs, occurs in processing bodies (PBs) and is required for formation of stress granules (SGs). In this study, we focused on the subcellular localization of a representative compendium of eIF4E protein isoforms, particularly on the less studied members of the human eIF4E protein family, eIF4E2 and eIF4E3. We showed that unlike eIF4E1, its less studied isoform eIF4E3_A, encoded by human chromosome 3, localized to stress granules but not PBs upon both heat shock and arsenite stress. Furthermore, we found that eIF4E3_A interacts with human translation initiation factors eIF4G1, eIF4G3 and PABP1 in vivo and sediments into the same fractions as canonical eIF4E1 during polysome analysis in sucrose gradients. Contrary to this finding, the truncated human eIF4E3 isoform, eIF4E3_B, showed no localization to SGs and no binding to eIF4G. We also highlighted that eIF4E2 may exhibit distinct functions under different stresses as it readily localizes to P-bodies during arsenite and heat stresses, whereas it is redirected to stress granules only upon heat shock. We extended our study to a number of protein variants, arising from alternative mRNA splicing, of each of the three eIF4E isoforms. Our results surprisingly uncovered differences in the ability of eIF4E1_1 and eIF4E1_3 to form stress granules in response to cellular stresses. Our comparison of all three human eIF4E isoforms and their protein variants enriches the intriguing spectrum of roles attributed to the eukaryotic initiation translation factors of the 4E family, which exhibit a distinctive localization within different RNA granules under different stresses. The localization of eIF4E3_A to stress granules, but not to processing bodies, along with its binding to eIF4G and PABP1 suggests a role of human eIF4E3_A in translation initiation rather than its involvement in a translational repression and mRNA decay and turnover. The localization of eIF4E2 to stress granules under heat shock but not arsenite stress indicates its distinct function in cellular response to these stresses and points to the variable protein content of SGs as a consequence of different stress insults.

Rck2 is a mitogen-activated protein kinase-activated protein kinase in yeast implicated in translational regulation. rck2Δ mutants are mildly sensitive to oxidative stress, a condition that causes dissociation of actively translating ribosomes (polysomes). In rck2Δ cells, polysomes are lost to an even higher degree than in the wild-type upon stress. Cells overexpressing the catalytically inactive rck2-kd allele are highly sensitive to oxidative stress. In such cells, dissociation of polysomes upon stress was instead greatly delayed. The protein synthesis rate decreased to a similar degree as in wild-type cells, however, indicating that in rck2-kd cells, the polysome complexes were inactive. Array analyses of total and polysome-associated mRNAs revealed major deregulation of the translational machinery in rck2 mutant cells. This involves transcripts for cytosolic ribosomal proteins and for processing and assembly of ribosomes. In rck2Δ cells, weakly transcribed mRNAs associate more avidly with polysomes than in wild-type cells, whereas the opposite holds true for rck2-kd cells. This is consistent with perturbed regulation of translation elongation, which is predicted to alter the ratio between mRNAs with and without strong entry sites at ribosomes. We infer that imbalances in the translational apparatus are a major reason for the inability of these cells to respond to stress.

Translation is critical for development as transcription in the oocyte and early embryo is silenced. To illustrate the translational changes during meiosis and consecutive two mitoses of the oocyte and early embryo, we performed a genome-wide translatome analysis. Acquired data showed significant and uniform activation of key translational initiation and elongation axes specific to M-phases. Although global protein synthesis decreases in M-phases, translation initiation and elongation activity increases in a uniformly fluctuating manner, leading to qualitative changes in translation regulation via the mTOR1/4F/eEF2 axis. Overall, we have uncovered a highly dynamic and oscillatory pattern of translational reprogramming that contributes to the translational regulation of specific mRNAs with different modes of polysomal occupancy/translation that are important for oocyte and embryo developmental competence. Our results provide new insights into the regulation of gene expression during oocyte meiosis as well as the first two embryonic mitoses and show how temporal translation can be optimized. This study is the first step towards a comprehensive analysis of the molecular mechanisms that not only control translation during early development, but also regulate translation-related networks employed in the oocyte-to-embryo transition and embryonic genome activation.