RNA-sequencing protocols can quantify gene expression regulation from transcription to protein synthesis. Ribosome profiling (Ribo-seq) maps the positions of translating ribosomes over the entire ...transcriptome. We have developed RiboTaper (available at https://ohlerlab.mdc-berlin.de/software/), a rigorous statistical approach that identifies translated regions on the basis of the characteristic three-nucleotide periodicity of Ribo-seq data. We used RiboTaper with deep Ribo-seq data from HEK293 cells to derive an extensive map of translation that covered open reading frame (ORF) annotations for more than 11,000 protein-coding genes. We also found distinct ribosomal signatures for several hundred upstream ORFs and ORFs in annotated noncoding genes (ncORFs). Mass spectrometry data confirmed that RiboTaper achieved excellent coverage of the cellular proteome. Although dozens of novel peptide products were validated in this manner, few of the currently annotated long noncoding RNAs appeared to encode stable polypeptides. RiboTaper is a powerful method for comprehensive de novo identification of actively used ORFs from Ribo-seq data.
Do young and old protein molecules have the same probability to be degraded? We addressed this question using metabolic pulse-chase labeling and quantitative mass spectrometry to obtain degradation ...profiles for thousands of proteins. We find that >10% of proteins are degraded non-exponentially. Specifically, proteins are less stable in the first few hours of their life and stabilize with age. Degradation profiles are conserved and similar in two cell types. Many non-exponentially degraded (NED) proteins are subunits of complexes that are produced in super-stoichiometric amounts relative to their exponentially degraded (ED) counterparts. Within complexes, NED proteins have larger interaction interfaces and assemble earlier than ED subunits. Amplifying genes encoding NED proteins increases their initial degradation. Consistently, decay profiles can predict protein level attenuation in aneuploid cells. Together, our data show that non-exponential degradation is common, conserved, and has important consequences for complex formation and regulation of protein abundance.
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•Global pulse-chase experiments identify non-exponentially degraded (NED) proteins•NED proteins become more stable with age•Many NED proteins are over-synthesized subunits of multiprotein complexes•Decay profiles can predict steady-state protein levels in aneuploid cells
The ability to track protein degradation kinetics across the mammalian proteome reveals that a subset of proteins is less likely to be degraded the more time has passed since synthesis. Effects of genetic copy-number variation can be predicted by the properties of these proteins.
Emerging evidence indicates that heterogeneity in ribosome composition can give rise to specialized functions. Until now, research mainly focused on differences in core ribosomal proteins and ...associated factors. The effect of posttranslational modifications has not been studied systematically. Analyzing ribosome heterogeneity is challenging because individual proteins can be part of different subcomplexes (40S, 60S, 80S, and polysomes). Here we develop polysome proteome profiling to obtain unbiased proteomic maps across ribosomal subcomplexes. Our method combines extensive fractionation by sucrose gradient centrifugation with quantitative mass spectrometry. The high resolution of the profiles allows us to assign proteins to specific subcomplexes. Phosphoproteomics on the fractions reveals that phosphorylation of serine 38 in RPL12/uL11, a known mitotic CDK1 substrate, is strongly depleted in polysomes. Follow-up experiments confirm that RPL12/uL11 phosphorylation regulates the translation of specific subsets of mRNAs during mitosis. Together, our results show that posttranslational modification of ribosomal proteins can regulate translation.
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•High-resolution polysome proteome profiling across ribosomal subcomplexes•Core ribosome of monosomes and polysomes does not differ significantly•Systematic identification of proteins associated with actively translating ribosomes•Phosphorylation of RPL12/uL11 regulates translation during mitosis
Multiple regulatory layers shape gene expression. Imami et al. show that protein production in mitosis can be regulated by phosphorylation of the ribosomal protein RPL12/uL11.
There is increasing evidence that transcripts or transcript regions annotated as non-coding can harbor functional short open reading frames (sORFs). Loss-of-function experiments have identified ...essential developmental or physiological roles for a few of the encoded peptides (micropeptides), but genome-wide experimental or computational identification of functional sORFs remains challenging.
Here, we expand our previously developed method and present results of an integrated computational pipeline for the identification of conserved sORFs in human, mouse, zebrafish, fruit fly, and the nematode C. elegans. Isolating specific conservation signatures indicative of purifying selection on amino acid (rather than nucleotide) sequence, we identify about 2,000 novel small ORFs located in the untranslated regions of canonical mRNAs or on transcripts annotated as non-coding. Predicted sORFs show stronger conservation signatures than those identified in previous studies and are sometimes conserved over large evolutionary distances. The encoded peptides have little homology to known proteins and are enriched in disordered regions and short linear interaction motifs. Published ribosome profiling data indicate translation of more than 100 novel sORFs, and mass spectrometry data provide evidence for more than 70 novel candidates.
Taken together, we identify hundreds of previously unknown conserved sORFs in major model organisms. Our computational analyses and integration with experimental data show that these sORFs are expressed, often translated, and sometimes widely conserved, in some cases even between vertebrates and invertebrates. We thus provide an integrated resource of putatively functional micropeptides for functional validation in vivo.
How do cells maintain relative proportions of protein complex components? Advances in quantitative, genome-wide measurements have begun to shed light onto the roles of protein synthesis and ...degradation in establishing the precise proportions in living cells: on the one hand, ribosome profiling studies indicate that proteins are already produced in the correct relative proportions. On the other hand, proteomic studies found that many complexes contain subunits that are made in excess and subsequently degraded. Here, we discuss these seemingly contradictory findings, emerging principles, and remaining open questions. We conclude that establishing precise protein levels involves both coordinated synthesis and post-translational fine-tuning via protein degradation.
Plants have an unusual plastid-localized NADP-thioredoxin reductase C (NTRC) containing both an NADP-thioredoxin reductase (NTR) and a thioredoxin (Trx) domain in a single polypeptide. Although NTRC ...is known to supply reductant for detoxifying hydrogen peroxide in the dark, its other functions are unknown. We now report that NTRC plays a previously unrecognized role in the redox regulation of ADP-glucose pyrophosphorylase (AGPase), a central enzyme of starch synthesis. When supplied NADPH, NTRC activated AGPase in vitro in a redox reaction that required the active site cysteines of both domains of the enzyme. In leaves, AGPase was activated in planta either by light or external feeding of sucrose in the dark. Leaves of an Arabidopsis NTRC KO mutant showed a decrease both in the extent of redox activation of AGPase and in the enhancement of starch synthesis either in the light (by 40-60%) or in the dark after treatment with external sucrose (by almost 100%). The light-dependent activation of AGPase in isolated chloroplasts, by contrast, was unaffected. In nonphotosynthetic tissue (roots), KO of NTRC decreased redox activation of AGPase and starch synthesis in response to light or external sucrose by almost 90%. The results provide biochemical and genetic evidence for a role of NTRC in regulating starch synthesis in response to either light or sucrose. The data also suggest that the Trx domain of NTRC and, to a lesser extent, free Trxs linked to ferredoxin enable amyloplasts of distant sink tissues to sense light used in photosynthesis by leaf chloroplasts and adjust heterotrophic starch synthesis accordingly.
The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of ...particular polyubiquitin signals remain enigmatic. The homologous ubiquitin‐conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin‐binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63‐linked polyubiquitin and facilitates the selective assembly of K48/K63‐branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis.
SYNOPSIS
Although ubiquitin‐conjugating enzymes Ubc1 and Ube2K have been studied in diverse biological contexts, the function of their UBA domain has remained poorly understood. Here, the UBA domain is found to facilitate the selective targeting of K63‐linked polyubiquitin chains, resulting in formation of branched K48/K63‐linked ubiquitin conjugates.
UBA domain binding to an individual ubiquitin moiety in a K63‐linked chain enables reaction of the catalytic UBC domain with the adjacent proximal ubiquitin molecule.
This process involves close association of the UBA and UBC domains, which are connected through a flexible linker region.
The formation of K48/K63‐branched polyubiquitin is conserved from yeast to humans.
Proteomics and genetic data indicate that branched chain formation by Ubc1 regulates resistance to heat stress and DNA replication stress.
Binding of K63‐linked ubiquitin chains via a tightly‐linked accessory domains allows two homologous human and yeast E2 enzymes to add further K48‐linked moieties and build branched conjugates.
Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop ...quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six Rho GTPases (Cdc42, Rac1, RhoA, RhoB, RhoC, and RhoD), depending on their nucleotide loading state. The method works with cell line or tissue-derived protein lysates in combination with SILAC-based or label-free quantification, respectively. We demonstrate that qGAP identifies known and novel binding partners that can be validated in an independent assay. Our interaction network for six Rho GTPases contains many novel binding partners, reveals highly promiscuous interaction of several effectors, and mirrors evolutionary relationships among Rho GTPases.
Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis and cancer invasion. Unlike cargo-specific clathrin-mediated endocytosis, the clathrin- and ...dynamin-independent endocytic pathway (CLIC-GEEC, CG pathway) is considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids. While the core molecular players of CG-endocytosis have been recently defined, evidence of cargo-specific adaptors or selective uptake of proteins for the pathway are lacking. Here we identify the actin-binding protein Swiprosin-1 (Swip1, EFHD2) as a cargo-specific adaptor for CG-endocytosis. Swip1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery-Arf1, IRSp53 and actin-and is critical for integrin endocytosis. Through this function, Swip1 supports integrin-dependent cancer-cell migration and invasion, and is a negative prognostic marker in breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG pathway and a role for specific adaptors in recruitment into this endocytic route.
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