The folding of newly synthesized proteins to the native state is a major challenge within the crowded cellular environment, as non-productive interactions can lead to misfolding, aggregation and degradation. Cells cope with this challenge by coupling synthesis with polypeptide folding and by using molecular chaperones to safeguard folding cotranslationally. However, although most of the cellular proteome forms oligomeric assemblies, little is known about the final step of folding: the assembly of polypeptides into complexes. In prokaryotes, a proof-of-concept study showed that the assembly of heterodimeric luciferase is an organized cotranslational process that is facilitated by spatially confined translation of the subunits encoded on a polycistronic mRNA. In eukaryotes, however, fundamental differences-such as the rarity of polycistronic mRNAs and different chaperone constellations-raise the question of whether assembly is also coordinated with translation. Here we provide a systematic and mechanistic analysis of the assembly of protein complexes in eukaryotes using ribosome profiling. We determined the in vivo interactions of the nascent subunits from twelve hetero-oligomeric protein complexes of Saccharomyces cerevisiae at near-residue resolution. We find nine complexes assemble cotranslationally; the three complexes that do not show cotranslational interactions are regulated by dedicated assembly chaperones. Cotranslational assembly often occurs uni-directionally, with one fully synthesized subunit engaging its nascent partner subunit, thereby counteracting its propensity for aggregation. The onset of cotranslational subunit association coincides directly with the full exposure of the nascent interaction domain at the ribosomal tunnel exit. The action of the ribosome-associated Hsp70 chaperone Ssb is coordinated with assembly. Ssb transiently engages partially synthesized interaction domains and then dissociates before the onset of partner subunit association, presumably to prevent premature assembly interactions. Our study shows that cotranslational subunit association is a prevalent mechanism for the assembly of hetero-oligomers in yeast and indicates that translation, folding and the assembly of protein complexes are integrated processes in eukaryotes.

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Highlights d Ded1p phase-separates in response to heat and pH to form gel condensates d Condensation inactivates Ded1p and represses housekeeping mRNAs d Ded1p condensation promotes stress protein production and limits cell growth d Ded1p condensation is adapted to the maximum growth temperature of a species

Mutations in the RS1 gene that encodes the discoidin domain containing retinoschisin cause X-linked juvenile retinoschisis (XLRS), a common macular degeneration in males. Disorganization of retinal layers and electroretinogram abnormalities are hallmarks of the disease and are also found in mice deficient for the orthologous murine protein, indicating that retinoschisin is important for the maintenance of retinal cell integrity. Upon secretion, retinoschisin associates with plasma membranes of photoreceptor and bipolar cells, although the components by which the protein is linked to membranes in vivo are still unclear. Here, we show that retinoschisin fails to bind to phospholipids or unilamellar lipid vesicles. A recent proteomic approach identified the Na/K-ATPase subunits ATP1A3 and ATP1B2 as binding partners of retinoschisin. We analyzed mice deficient for retinoschisin (Rs1h(-/Y)) and ATP1B2 (Atp1b2(-/-)) to characterize the role of Na/K-ATPase interaction in the organization of retinoschisin on cellular membranes. We demonstrate that both the Na/K-ATPase and retinoschisin are significantly reduced in Atp1b2(-/-) retinas, suggesting that retinoschisin membrane association is severely impaired in the absence of ATP1A3 and ATP1B2 subunits. Conversely, the presence of ATP1A3 and ATP1B2 are obligatory for binding of exogenously applied retinoschisin to crude membranes. Also, co-expression of ATP1A3 and ATP1B2 is required for retinoschisin binding to intact Hek293 cells. Taken together, our data support a predominant role of Na/K-ATPase in anchoring retinoschisin to retinal cell surfaces. Furthermore, altered localization of ATP1A3 and ATP1B2 is a notable consequence of retinoschisin deficiency and thus may be an important downstream aspect of cellular pathology in XLRS.

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The rates at which domains fold and codons are translated are important factors in determining whether a nascent protein will co-translationally fold and function or misfold and malfunction. Here we develop a chemical kinetic model that calculates a protein domain's co-translational folding curve during synthesis using only the domain's bulk folding and unfolding rates and codon translation rates. We show that this model accurately predicts the course of co-translational folding measured in vivo for four different protein molecules. We then make predictions for a number of different proteins in yeast and find that synonymous codon substitutions, which change translation-elongation rates, can switch some protein domains from folding post-translationally to folding co-translationally—a result consistent with previous experimental studies. Our approach explains essential features of co-translational folding curves and predicts how varying the translation rate at different codon positions along a transcript's coding sequence affects this self-assembly process.

Analysis methods based on simulations and optimization have been previously developed to estimate relative translation rates from next-generation sequencing data. Translation involves molecules and chemical reactions, hence bioinformatics methods consistent with the laws of chemistry and physics are more likely to produce accurate results. Here, we derive simple equations based on chemical kinetic principles to measure the translation-initiation rate, transcriptome-wide elongation rate, and individual codon translation rates from ribosome profiling experiments. Our methods reproduce the known rates from ribosome profiles generated from detailed simulations of translation. By applying our methods to data from S . cerevisiae and mouse embryonic stem cells, we find that the extracted rates reproduce expected correlations with various molecular properties, and we also find that mouse embryonic stem cells have a global translation speed of 5.2 AA/s, in agreement with previous reports that used other approaches. Our analysis further reveals that a codon can exhibit up to 26-fold variability in its translation rate depending upon its context within a transcript. This broad distribution means that the average translation rate of a codon is not representative of the rate at which most instances of that codon are translated, and it suggests that translational regulation might be used by cells to a greater degree than previously thought.

Progressive telomere shortening occurs in somatic cells, and with increasing donor age a significant decline in telomere length has been shown in various postnatal tissues. In contrast, little is known about changes in telomere length during human fetal development. Therefore, we measured telomere length in the leukocyte fraction of umbilical cord blood samples from 15 preterm (Ͻ37 wk of gestation) and 11 full-term (Ͼ37 wk of gestation) neonates using the telomere restriction fragment assay. Whereas no differences in mean (Ϯ SD) telomere restriction fragment between the groups of preterm neonates (8512 Ϯ 523 bp) and full-term newborns (8323 Ϯ 503 bp) could be found, significantly longer telomeres (p ϭ 0.002) were found in very low birth weight preterm neonates when compared with low birth weight preterm neonates. In addition, a rapid and significant decline in mean telomere restriction fragment was observed between 27 and 32 wk of gestation (p ϭ 0.02, r ϭ 0.79) followed by a period of no significant loss of telomere repeats between 33 and 42 wk of gestation. These results are consistent with the known almost maximal proliferation rate of hematopoietic progenitor cells before 32 wk of gestation. The initial decrease in telomere restriction fragment could be caused by ontogenyrelated functional alterations of hematopoietic cells or differences in stem cell turnover or the rate of telomere loss per cell division. Telomeres play a major role in chromosome structure and function by protecting their degradation and apparently contributing to the attachment of chromosome ends to the nuclear envelope. Telomeres lose a portion of their noncoding repetitive DNA sequences with each cell division, and the ribonucleoprotein enzyme telomerase can compensate for this by synthesis of telomere repeats onto chromosomes (1-3). Despite the observation that some immortalized cell lines manifest stable telomeres without evidence of telomerase activity (4), telomerase-dependent telomere elongation seems to be the most important mechanism for maintaining telomere length in vitro and in vivo.In man telomerase is expressed during embryogenesis. Ulaner and Giudice (5) examined telomerase activity in various fetal tissues from 8 to 21 wk of gestation. They found that all tissues expressed telomerase at the earliest ages analyzed, followed by tissue-specific suppression during embryogenic development. After birth this enzyme is repressed in most somatic tissues (6), and high activity persists only in germline cells.Whereas in most postnatal somatic tissues telomerase is not expressed, low levels of this enzyme are detected in hematopoietic progenitor cells, activated lymphocytes (7), intestinal crypt cells (8), the basal layer of the skin (9), and during the proliferative phase in the premenopausal endometrium (10, 11). It is assumed that the low telomerase activity compensates the increased loss of telomere repeats associated with the high rate of cell turnover in these tissues. However, in contrast to germline and tumor cells, these low l...