Search

Translation modulates the timing and amplification of gene expression after transcription. Brain development requires uniquely complex gene expression patterns, but large-scale measurements of translation directly in the prenatal brain are lacking. We measure the reactants, synthesis, and products of translation spanning mouse neocortex neurogenesis, and discover a transient window of dynamic regulation at mid-gestation. Timed translation upregulation of chromatin binding proteins like Satb2, which is essential for neuronal subtype differentiation, restricts protein expression in neuronal lineages despite broad transcriptional priming in progenitors. In contrast, translation downregulation of ribosomal proteins sharply decreases ribosome number, coinciding with a major shift in protein synthesis dynamics at mid-gestation. Changing levels of eIF4EBP1, a direct inhibitor of ribosomal protein translation, are concurrent with ribosome downregulation and controls Satb2 fate acquisition during neuronal differentiation. Thus, the refinement of transcriptional programs by translation is central to the molecular logic of brain development. Modeling of the developmental neocortex translatome is provided as an open-source searchable resource: https://shiny.mdc-berlin.de/cortexomics/.

Transposons are parasitic genetic elements that frequently hijack vital cellular processes of their host. HMGXB4 is a known Wnt signaling-regulating HMG-box protein, previously identified as a host-encoded factor of Sleeping Beauty (SB) transposition. Here, we show that HMGXB4 is predominantly maternally expressed, and marks both germinal progenitor and somatic stem cells. SB piggybacks HMGXB4 to activate transposase expression and target transposition to germinal stem cells, thereby potentiating heritable transposon insertions. The HMGXB4 promoter is located within an active chromatin domain, offering multiple looping possibilities with neighboring genomic regions. HMGXB4 is activated by ERK2/MAPK1, ELK1 transcription factors, coordinating pluripotency and self-renewal pathways, but suppressed by the KRAB-ZNF/TRIM28 epigenetic repression machinery, also known to regulate transposable elements. At the post-translational level, SUMOylation regulates HMGXB4, which modulates binding affinity to its protein interaction partners and controls its transcriptional activator function via nucleolar compartmentalization. When expressed, HMGXB4 can participate in nuclear-remodeling protein complexes and transactivate target gene expression in vertebrates. Our study highlights HMGXB4 as an evolutionarily conserved host-encoded factor that assists Tc1/Mariner transposons to target the germline, which was necessary for their fixation and may explain their abundance in vertebrate genomes.

Aberrant expression of MYC transcription factor family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYCN induces massive lipid peroxidation on depletion of cysteine, the rate-limiting amino acid for glutathione (GSH) biosynthesis, and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and iron-dependent type of cell death. The high cysteine demand of MYCN-amplified childhood neuroblastoma is met by uptake and transsulfuration. When uptake is limited, cysteine usage for protein synthesis is maintained at the expense of GSH triggering ferroptosis and potentially contributing to spontaneous tumor regression in low-risk neuroblastomas. Pharmacological inhibition of both cystine uptake and transsulfuration combined with GPX4 inactivation resulted in tumor remission in an orthotopic MYCN-amplified neuroblastoma model. These findings provide a proof of concept of combining multiple ferroptosis targets as a promising therapeutic strategy for aggressive MYCN-amplified tumors.

Ribosome biogenesis is a fundamental multi-step cellular process in all domains of life that involves the production, processing, folding, and modification of ribosomal RNAs (rRNAs) and ribosomal proteins. To obtain insights into the still unexplored early assembly phase of the bacterial 50S subunit, we exploited a minimal in vitro reconstitution system using purified ribosomal components and scalable reaction conditions. Time-limited assembly assays combined with cryo-EM analysis visualizes the structurally complex assembly pathway starting with a particle consisting of ordered density for only ~500 nucleotides of 23S rRNA domain I and three ribosomal proteins. In addition, our structural analysis reveals that early 50S assembly occurs in a domain-wise fashion, while late 50S assembly proceeds incrementally. Furthermore, we find that both ribosomal proteins and folded rRNA helices, occupying surface exposed regions on pre-50S particles, induce, or stabilize rRNA folds within adjacent regions, thereby creating cooperativity.

Identifying the protein targets of drugs is an important but tedious process. Existing proteomic approaches enable unbiased target identification but lack the throughput needed to screen larger compound libraries. Here, we present a compound interaction screen on a photoactivatable cellulose membrane (CISCM) that enables target identification of several drugs in parallel. To this end, we use diazirine-based undirected photoaffinity labeling (PAL) to immobilize compounds on cellulose membranes. Functionalized membranes are then incubated with protein extract and specific targets are identified via quantitative affinity purification and mass spectrometry. CISCM reliably identifies known targets of natural products in less than three hours of analysis time per compound. In summary, we show that combining undirected photoimmobilization of compounds on cellulose with quantitative interaction proteomics provides an efficient means to identify the targets of natural products.

Inborn errors of immunity (IEIs) unveil regulatory pathways of human immunity. We describe a new IEI caused by mutations in the GTPase of the immune-associated protein 6 (GIMAP6) gene in patients with infections, lymphoproliferation, autoimmunity, and multiorgan vasculitis. Patients and Gimap6−/− mice show defects in autophagy, redox regulation, and polyunsaturated fatty acid (PUFA)–containing lipids. We find that GIMAP6 complexes with GABARAPL2 and GIMAP7 to regulate GTPase activity. Also, GIMAP6 is induced by IFN-γ and plays a critical role in antibacterial immunity. Finally, we observed that Gimap6−/− mice died prematurely from microangiopathic glomerulosclerosis most likely due to GIMAP6 deficiency in kidney endothelial cells.

Herpesviruses encode conserved protein kinases (CHPKs) to stimulate phosphorylation-sensitive processes during infection. How CHPKs bind to cellular factors and how this impacts their regulatory functions is poorly understood. Here, we use quantitative proteomics to determine cellular interaction partners of human herpesvirus (HHV) CHPKs. We find that CHPKs can target key regulators of transcription and replication. The interaction with Cyclin A and associated factors is identified as a signature of β-herpesvirus kinases. Cyclin A is recruited via RXL motifs that overlap with nuclear localization signals (NLS) in the non-catalytic N termini. This architecture is conserved in HHV6, HHV7 and rodent cytomegaloviruses. Cyclin A binding competes with NLS function, enabling dynamic changes in CHPK localization and substrate phosphorylation. The cytomegalovirus kinase M97 sequesters Cyclin A in the cytosol, which is essential for viral inhibition of cellular replication. Our data highlight a fine-tuned and physiologically important interplay between a cellular cyclin and viral kinases., Herpesviruses code for conserved protein kinases (CHPKs) that exert several regulatory functions by interacting with cellular factors. Here, the authors use affinity purification mass spectrometry (AP–MS) to identify differential interaction partners of CHPKs from seven different human herpesviruses, finding Cyclin A and associated factors as a specific signature of β-herpesvirus kinases.

Although new genes can arrive from modes other than duplication, few examples are well characterized. Given high expression in some human brain subregions and a putative link to psychological disorders [e.g., schizophrenia (SCZ)], suggestive of brain functionality, here we characterize piggyBac transposable element-derived 1 (PGBD1). PGBD1 is nonmonotreme mammal-specific and under purifying selection, consistent with functionality. The gene body of human PGBD1 retains much of the original DNA transposon but has additionally captured SCAN and KRAB domains. Despite gene body retention, PGBD1 has lost transposition abilities, thus transposase functionality is absent. PGBD1 no longer recognizes piggyBac transposon-like inverted repeats, nonetheless PGBD1 has DNA binding activity. Genome scale analysis identifies enrichment of binding sites in and around genes involved in neuronal development, with association with both histone activating and repressing marks. We focus on one of the repressed genes, the long noncoding RNA NEAT1, also dysregulated in SCZ, the core structural RNA of paraspeckles. DNA binding assays confirm specific binding of PGBD1 both in the NEAT1 promoter and in the gene body. Depletion of PGBD1 in neuronal progenitor cells (NPCs) results in increased NEAT1/paraspeckles and differentiation. We conclude that PGBD1 has evolved core regulatory functionality for the maintenance of NPCs. As paraspeckles are a mammal-specific structure, the results presented here show a rare example of the evolution of a novel gene coupled to the evolution of a contemporaneous new structure.

Pandemic influenza A virus (IAV) outbreaks occur when strains from animal reservoirs acquire the ability to infect and spread among humans. The molecular basis of this species barrier is incompletely understood. Here we combine metabolic pulse labeling and quantitative proteomics to monitor protein synthesis upon infection of human cells with a human- and a bird-adapted IAV strain and observe striking differences in viral protein synthesis. Most importantly, the matrix protein M1 is inefficiently produced by the bird-adapted strain. We show that impaired production of M1 from bird-adapted strains is caused by increased splicing of the M segment RNA to alternative isoforms. Strain-specific M segment splicing is controlled by the 3′ splice site and functionally important for permissive infection. In silico and biochemical evidence shows that avian-adapted M segments have evolved different conserved RNA structure features than human-adapted sequences. Thus, we identify M segment RNA splicing as a viral host range determinant., Avian influenza A virus (IAV) strains replicate poorly in mammalian hosts, but mechanisms underlying species restriction are incompletely understood. Here, Bogdanow et al. show that avian and mammalian adapted IAV strains have evolved different RNA structure features for regulation of M segment RNA splicing.

Telomere maintenance by telomerase activation or alternative lengthening of telomeres (ALT) is a major determinant of poor outcome in neuroblastoma. Here, we screen for ALT in primary and relapsed neuroblastomas (n = 760) and characterize its features using multi-omics profiling. ALT-positive tumors are molecularly distinct from other neuroblastoma subtypes and enriched in a population-based clinical sequencing study cohort for relapsed cases. They display reduced ATRX/DAXX complex abundance, due to either ATRX mutations (55%) or low protein expression. The heterochromatic histone mark H3K9me3 recognized by ATRX is enriched at the telomeres of ALT-positive tumors. Notably, we find a high frequency of telomeric repeat loci with a neuroblastoma ALT-specific hotspot on chr1q42.2 and loss of the adjacent chromosomal segment forming a neo-telomere. ALT-positive neuroblastomas proliferate slowly, which is reflected by a protracted clinical course of disease. Nevertheless, children with an ALT-positive neuroblastoma have dismal outcome., Alternative lengthening of telomeres (ALT) is associated with a poor outcome in neuroblastoma. Here, the authors find that ALT is associated with mutated ATRX and/or reduced protein abundance, frequent telomeric repeat loci and heterochromatic telomeric chromatin.

Very high risk neuroblastoma is characterised by increased MAPK signalling, and targeting MAPK signalling is a promising therapeutic strategy. We used a deeply characterised panel of neuroblastoma cell lines and found that the sensitivity to MEK inhibitors varied drastically between these cell lines. By generating quantitative perturbation data and mathematical modelling, we determined potential resistance mechanisms. We found that negative feedbacks within MAPK signalling and via the IGF receptor mediate re-activation of MAPK signalling upon treatment in resistant cell lines. By using cell-line specific models, we predict that combinations of MEK inhibitors with RAF or IGFR inhibitors can overcome resistance, and tested these predictions experimentally. In addition, phospho-proteomic profiling confirmed the cell-specific feedback effects and synergy of MEK and IGFR targeted treatment. Our study shows that a quantitative understanding of signalling and feedback mechanisms facilitated by models can help to develop and optimise therapeutic strategies. Our findings should be considered for the planning of future clinical trials introducing MEKi in the treatment of neuroblastoma., Author summary Only few targeted therapies are currently available to treat high-risk neuroblastoma. To address this issue we characterized the drug response of high risk neuroblastoma cell lines and correlated it with genomic and transcriptomic data. Particularly for MEK inhibition, we saw that our panel could be nicely separated into two groups of resistant and sensitive cell lines. Genomic and transcriptomic markers alone did not help to discriminate between responders and non-responders. We used signalling perturbation data to build cell line specific signalling models. Our models suggest that negative feedbacks within MAPK signalling lead to a stronger reactivation of MEK in MEKi resistant cell lines after MEK inhibition. Model analysis suggested that combining MEK inhibition with IGF1R or RAF inhibition could be an effective treatment and we characterised this combination using phosphoproteomics by mass-spectrometry and growth assays. Our study confirms the importance of quantitative understanding of signalling and may help plan future clinical trials involving MEK inhibition for the treatment of neuroblastoma.

The biological role of RNA-binding proteins in the secretory pathway is not well established. Here, we describe that human HDLBP/Vigilin directly interacts with more than 80% of ER-localized mRNAs. PAR-CLIP analysis reveals that these transcripts represent high affinity HDLBP substrates and are specifically bound in their coding sequences (CDS), in contrast to CDS/3’UTR-bound cytosolic mRNAs. HDLBP crosslinks strongly to long CU-rich motifs, which frequently reside in CDS of ER-localized mRNAs and result in high affinity multivalent interactions. In addition to HDLBP-ncRNA interactome, quantification of HDLBP-proximal proteome confirms association with components of the translational apparatus and the signal recognition particle. Absence of HDLBP results in decreased translation efficiency of HDLBP target mRNAs, impaired protein synthesis and secretion in model cell lines, as well as decreased tumor growth in a lung cancer mouse model. These results highlight a general function for HDLBP in the translation of ER-localized mRNAs and its relevance for tumor progression.

Mitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, which shapes the oxidative phosphorylation (OXPHOS) complexes essential for cellular energy metabolism. Despite the importance of mitochondrial translation control, it is challenging to identify and quantify the mitochondrial-encoded proteins due to their hydrophobic nature and low abundance. Here, we introduce a mass spectrometry-based proteomic method that combines biochemical isolation of mitochondria with pulse stable isotope labeling by amino acids in cell culture (pSILAC). Our method provides the highest protein identification rate with the shortest measurement time among currently available methods, enabling us to quantify 12 out of the 13 mitochondrial-encoded proteins. We applied this method to uncover the global picture of (post-)translational regulation of both mitochondrial- and nuclear-encoded subunits of OXPHOS complexes. We found that inhibition of mitochondrial translation led to degradation of orphan nuclear-encoded subunits that are considered to form subcomplexes with the mitochondrial-encoded subunits. The results also allowed us to infer the subcomplex members of each OXPHOS complex. This method should be readily applicable to study mitochondrial translation programs in many contexts, including oxidative stress and mitochondrial disease.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an ongoing global health threat with more than two million infected people since its emergence in late 2019. Detailed knowledge of the molecular biology of the infection is indispensable for understanding of the viral replication, host responses, and disease progression. We provide gene expression profiles of SARS-CoV and SARS-CoV-2 infections in three human cell lines (H1299, Caco-2 and Calu-3 cells), using bulk and single-cell transcriptomics. Small RNA profiling showed strong expression of the immunity and inflammation-associated microRNA miRNA-155 upon infection with both viruses. SARS-CoV-2 elicited approximately two-fold higher stimulation of the interferon response compared to SARS-CoV in the permissive human epithelial cell line Calu-3, and induction of cytokines such as CXCL10 or IL6. Single cell RNA sequencing data showed that canonical interferon stimulated genes such as IFIT2 or OAS2 were broadly induced, whereas interferon beta (IFNB1) and lambda (IFNL1-4) were expressed only in a subset of infected cells. In addition, temporal resolution of transcriptional responses suggested interferon regulatory factors (IRFs) activities precede that of nuclear factor-κB (NF-κB). Lastly, we identified heat shock protein 90 (HSP90) as a protein relevant for the infection. Inhibition of the HSP90 charperone activity by Tanespimycin/17-N-allylamino-17-demethoxygeldanamycin (17-AAG) resulted in a reduction of viral replication, and of TNF and IL1B mRNA levels. In summary, our study established in vitro cell culture models to study SARS-CoV-2 infection and identified HSP90 protein as potential drug target for therapeutic intervention of SARS-CoV-2 infection.

SUMMARYProtein synthesis must be finely tuned in the nervous system, as it represents an essential feature of neurodevelopmental gene expression, and dominant pathology in neurological disease. However, the architecture of ribosomal complexes in the developing mammalian brain has not been analyzed at high resolution. This study investigates the architecture of ribosomesex vivofrom the embryonic and perinatal mouse neocortex, revealing Ebp1 as a 60S peptide tunnel exit binding factor at near-atomic resolution by multiparticle cryo-electron microscopy. The impact of Ebp1 on the neuronal proteome was analyzed by pSILAC and BONCAT coupled mass spectrometry, implicating Ebp1 in neurite outgrowth proteostasis, within vivoembryonic Ebp1 knockdown resulting in dysregulation of neurite outgrowth. Our findings reveal Ebp1 as a central component of neocortical protein synthesis, and the 60S peptide tunnel exit as a focal point of gene expression control in the molecular specification of neuronal morphology.Graphical abstract

Recruitment of the human ribonucleolytic RNA exosome to nuclear polyadenylated (pA+) RNA is facilitated by the Poly(A) Tail eXosome Targeting (PAXT) connection. Besides its core dimer, formed by the exosome co-factor MTR4 and the ZFC3H1 protein, the PAXT connection remains poorly defined. By characterizing nuclear pA+-RNA bound proteomes as well as MTR4-ZFC3H1 containing complexes in conditions favoring PAXT assembly, we here uncover three additional proteins required for PAXT function: ZC3H3, RBM26 and RBM27 along with the known PAXT-associated protein, PABPN1. The zinc-finger protein ZC3H3 interacts directly with MTR4-ZFC3H1 and loss of any of the newly identified PAXT components results in the accumulation of PAXT substrates. Collectively, our results establish new factors involved in the turnover of nuclear pA+ RNA and suggest that these are limiting for PAXT activity.

Protein subcellular localization is fundamental to the establishment of the body axis, cell migration, synaptic plasticity, and a vast range of other biological processes. Protein localization occurs through three mechanisms: protein transport, mRNA localization, and local translation. However, the relative contribution of each process to neuronal polarity remains unknown. Using neurons differentiated from mouse embryonic stem cells, we analyze protein and RNA expression and translation rates in isolated cell bodies and neurites genome-wide. We quantify 7323 proteins and the entire transcriptome, and identify hundreds of neurite-localized proteins and locally translated mRNAs. Our results demonstrate that mRNA localization is the primary mechanism for protein localization in neurites that may account for half of the neurite-localized proteome. Moreover, we identify multiple neurite-targeted non-coding RNAs and RNA-binding proteins with potential regulatory roles. These results provide further insight into the mechanisms underlying the establishment of neuronal polarity., Subcellular localization of RNAs and proteins is important for polarized cells such as neurons. Here the authors differentiate mouse embryonic stem cells into neurons, and analyze the local transcriptome, proteome, and translated transcriptome in their cell bodies and neurites, providing a unique resource for future studies on neuronal polarity.

Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). We have developed the Phenol Toluol extraction (PTex) protocol that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium., RNA binding proteins are important regulators of RNA function. Here the authors describe a method for isolation of RNA-protein complexes that does not rely on a specific RNA sequence or motif, and demonstrate the approach by providing the global RNA-bound proteomes of human HEK293 cells and Salmonella Typhimurium.

Summary: Chlamydia trachomatis (Ctr) causes a range of infectious diseases and is epidemiologically associated with cervical and ovarian cancers. To obtain a panoramic view of Ctr-induced signaling, we performed global phosphoproteomic and transcriptomic analyses. We identified numerous Ctr phosphoproteins and Ctr-regulated host phosphoproteins. Bioinformatics analysis revealed that these proteins were predominantly related to transcription regulation, cellular growth, proliferation, and cytoskeleton organization. In silico kinase substrate motif analysis revealed that MAPK and CDK were the most overrepresented upstream kinases for upregulated phosphosites. Several of the regulated host phosphoproteins were transcription factors, including ETS1 and ERF, that are downstream targets of MAPK. Functional analysis of phosphoproteome and transcriptome data confirmed their involvement in epithelial-to-mesenchymal transition (EMT), a phenotype that was validated in infected cells, along with the essential role of ERK1/2, ETS1, and ERF for Ctr replication. Our data reveal the extent of Ctr-induced signaling and provide insights into its pro-carcinogenic potential. : Zadora et al. performed an integrated global phosphoproteomic and transcriptomic analysis, revealing a comprehensive map of Chlamydia-induced host cell signaling and identifying transcription factors ETS1 and ERF, which drive epithelial-to-mesenchymal transition. These insights provide mechanistic clues to Chlamydia pathogenesis and serve as an important resource for future studies. Keywords: Chlamydia trachomatis, signaling, human papillomavirus, transcription factors, cervical cancer, ovarian cancer, human primary cells

The principle of shotgun proteomics is to use peptide mass spectra in order to identify corresponding sequences in a protein database. The quality of peptide and protein identification and quantification critically depends on the sensitivity and specificity of this assignment process. Many peptides in proteomic samples carry biochemical modifications, and a large fraction of unassigned spectra arise from modified peptides. Spectra derived from modified peptides can erroneously be assigned to wrong amino acid sequences. However, the impact of this problem on proteomic data has not yet been investigated systematically. Here we use combinations of different database searches to show that modified peptides can be responsible for 20–50% of false positive identifications in deep proteomic data sets. These false positive hits are particularly problematic as they have significantly higher scores and higher intensities than other false positive matches. Furthermore, these wrong peptide assignments lead to hundreds of false protein identifications and systematic biases in protein quantification. We devise a “cleaned search” strategy to address this problem and show that this considerably improves the sensitivity and specificity of proteomic data. In summary, we show that modified peptides cause systematic errors in peptide and protein identification and quantification and should therefore be considered to further improve the quality of proteomic data annotation.

Eukaryotic gene expression requires the coordinated action of transcription factors, chromatin remodelling complexes and RNA polymerase. The conserved nuclear protein Akirin plays a central role in immune gene expression in insects and mammals, linking the SWI/SNF chromatin-remodelling complex with the transcription factor NFκB. Although nematodes lack NFκB, Akirin is also indispensable for the expression of defence genes in the epidermis of Caenorhabditis elegans following natural fungal infection. Through a combination of reverse genetics and biochemistry, we discovered that in C. elegans Akirin has conserved its role of bridging chromatin-remodellers and transcription factors, but that the identity of its functional partners is different since it forms a physical complex with NuRD proteins and the POU-class transcription factor CEH-18. In addition to providing a substantial step forward in our understanding of innate immune gene regulation in C. elegans, our results give insight into the molecular evolution of lineage-specific signalling pathways., Author summary When animals are infected, as part of their innate immune response, they switch on defence genes that encode proteins that help fight pathogens. We use the nematode Caenorhabditis elegans to understand the steps in this process. When infected, C. elegans can turn on clusters of antimicrobial peptide genes. We have discovered that the coordinated expression of these genes requires a particular chromatin remodelling complex (proteins that open up compact DNA), working in conjunction with a protein called Akirin. Akirin plays a central role in immune gene expression in insects and mammals, but we found that although it has conserved its role of bridging chromatin-remodellers and transcription factors needed for gene expression, the identity of its functional partners is different. Our findings represent a major advance in our understanding of innate immune gene regulation in C. elegans, and give insight into how biological mechanisms can evolve.

Targeted proteomic approaches like selected reaction monitoring (SRM) and parallel reaction monitoring (PRM) are increasingly popular because they enable sensitive and rapid analysis of a preselected set of proteins. However, developing targeted assays is tedious and requires the selection, synthesis and mass spectrometric analysis of candidate peptides before the actual samples can be analyzed. The SRMatlas and ProteomeTools projects recently published fragmentation spectra of synthetic peptides covering the entire human proteome. These datasets provide very valuable resources. However, extracting the relevant data for selected proteins of interest is not straightforward. For example, developing scheduled acquisition methods (i.e. analyzing specific peptides in defined elution time windows) is complicated and requires adjustments to specific chromatographic conditions employed. Here, we present Picky: a fast and easy to use online tool to design scheduled PRM/SRM assays (https://picky.mdc-berlin.de). Picky (i) automatically generates optimized and scheduled SRM/PRM assays for proteins of interest and (ii) provides means to validate the data via known fragmentation spectra of corresponding synthetic peptides.

Influenza A virus infections are a major cause for respiratory disease in humans, which affects all age groups and contributes substantially to global morbidity and mortality. IAV have a large natural host reservoir in avian species. However, many avian IAV strains lack adaptation to other hosts and hardly propagate in humans. While seasonal or pandemic influenza A virus (IAV) strains replicate efficiently in permissive human cells, many avian IAV cause abortive non-productive infections in these hosts despite successful cell entry. However, the precise reasons for these differential outcomes are poorly defined. We hypothesized that the distinct course of an IAV infection with a given virus strain is determined by the differential interplay between specific host and viral factors. By using Spike-in SILAC mass spectrometry-based quantitative proteomics we characterized sets of cellular factors whose abundance is specifically up- or down-regulated in the course of permissive vs. non-permissive IAV infection, respectively. This approach allowed for the definition and quantitative comparison of about 3500 proteins in human lung epithelial cells in response to seasonal or low-pathogenic avian H3N2 IAV. Many identified proteins were similarly regulated by both virus strains, but also 16 candidates with distinct changes in permissive vs. non-permissive infection were found. RNAi-mediated knockdown of these differentially regulated host factors identified Vpr binding protein (VprBP) as pro-viral host factor since its down-regulation inhibited efficient propagation of seasonal IAV while over-expression increased viral replication of both seasonal and avian IAV. These results not only show that there are similar differences in the overall changes during permissive and non-permissive imfluenza virus infections, but also provide a basis to evaluate VprBP as novel anti-IAV drug target.

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.

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.

Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity., The AAA+ ATPase p97 is an essential hexameric protein with multiple protein interaction partners and cellular functions. Here, the authors use interaction mapping to examine partner proteins of this large complex, and assess the effects of these proteins on the disassembly of the p97 complex.

Posttranscriptional gene regulation relies on hundreds of RNA binding proteins (RBPs) but the function of most RBPs is unknown. The human RBP HuR/ELAVL1 is a conserved mRNA stability regulator. We used PAR-CLIP, a recently developed method based on RNA-protein crosslinking, to identify transcriptome-wide ~26,000 HuR binding sites. These sites were on average highly conserved, enriched for HuR binding motifs and mainly located in 3' untranslated regions. Surprisingly, many sites were intronic, implicating HuR in mRNA processing. Upon HuR knockdown, mRNA levels and protein synthesis of thousands of target genes were downregulated, validating functionality. HuR and miRNA binding sites tended to reside nearby but generally did not overlap. Additionally, HuR knockdown triggered strong and specific upregulation of miR-7. In summary, we identified thousands of direct and functional HuR targets, found a human miRNA controlled by HuR, and propose a role for HuR in splicing.

The lectin Yos9p is a subunit of the HRD ligase that partakes in the degradation of aberrant glycoproteins from the ER. We demonstrate that a variant of Yos9p that has lost its ability to bind glycans enhances the elimination of non-glycosylated substrates indicating that Yos9p has an additional role in the degradation of non-glycosylated proteins., The HRD ubiquitin ligase recognizes and ubiquitylates proteins of the endoplasmic reticulum that display structural defects. Here, we apply quantitative proteomics to characterize the substrate spectrum of the HRD complex. Among the identified substrates is Erg3p, a glycoprotein involved in sterol synthesis. We characterize Erg3p and demonstrate that the elimination of Erg3p requires Htm1p and Yos9p, two proteins that take part in the glycan-dependent turnover of aberrant proteins. We further show that the HRD ligase also mediates the breakdown of Erg3p and CPY* engineered to lack N-glycans. The degradation of these nonglycosylated substrates is enhanced by a mutant variant of Yos9p that has lost its affinity for oligosaccharides, indicating that Yos9p has a previously unrecognized role in the quality control of nonglycosylated proteins.

There is increasing evidence that non-annotated short open reading frames (sORFs) can encode functional micropeptides, but computational identification remains challenging. We expand our published method and predict conserved sORFs in human, mouse, zebrafish, fruit fly and the nematode C. elegans. Isolating specific conservation signatures indicative of purifying selection on encoded amino acid sequence, we identify about 2000 novel sORFs 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. Encoded peptides have little homology to known proteins and are enriched in disordered regions and short interaction motifs. Published ribosome profiling data indicate translation for more than 100 of novel sORFs, and mass spectrometry data gives peptidomic evidence for more than 70 novel candidates. We thus provide a catalog of conserved micropeptides for functional validation in vivo.

CIDE-N domains mediate interactions between the DNase Dff40/CAD and its inhibitor Dff45/ICAD. In this study, we report that the CIDE-N protein Drep-2 is a novel synaptic protein important for learning and behavioral adaptation. Drep-2 was found at synapses throughout the Drosophila brain and was strongly enriched at mushroom body input synapses. It was required within Kenyon cells for normal olfactory short- and intermediate-term memory. Drep-2 colocalized with metabotropic glutamate receptors (mGluRs). Chronic pharmacological stimulation of mGluRs compensated for drep-2 learning deficits, and drep-2 and mGluR learning phenotypes behaved non-additively, suggesting that Drep 2 might be involved in effective mGluR signaling. In fact, Drosophila fragile X protein mutants, shown to benefit from attenuation of mGluR signaling, profited from the elimination of drep-2. Thus, Drep-2 is a novel regulatory synaptic factor, probably intersecting with metabotropic signaling and translational regulation. DOI: http://dx.doi.org/10.7554/eLife.03895.001, eLife digest Synapses are specialized structures that connect nerve cells to one another and allow information to be transmitted between the cells. Synapses are essential for learning and storing memories. Many proteins that regulate how signals are transmitted at synapses have already been studied. In this manner, much has been learned about their function in learning and memory. Cells can commit suicide by a process called apoptosis, also known as programmed cell death. Apoptosis is not only triggered in damaged cells but is also necessary for an organism to develop correctly. In fruit flies, the protein Drep-2 is a member of a family of proteins that degrade the DNA of cells that undergo apoptosis. Andlauer et al. found no evidence that Drep-2 plays a role in apoptosis, but have now found Drep-2 at the synapses of the brain of the fruit fly Drosophila. Drep-2 could be observed in close proximity to another type of protein called metabotropic glutamate receptors. Metabotropic glutamate receptors and their signaling pathways are important for regulating certain changes to the synapses that mediate learning processes. Indeed, Andlauer et al. found that flies that have lost the gene that produces Drep-2 were unable to remember smells when these were paired with a punishment. Stimulating the regulatory glutamate receptors with drugs helped to overcome learning deficits that result from the lack of Drep-2. Alterations in the production of a protein called FMRP cause fragile X syndrome in humans, the most common form of hereditary mental disability originating from a single gene defect. Flies lacking the FMRP protein show learning deficits that are very similar to the ones seen in flies that cannot produce Drep-2. However, Andlauer et al. observed that flies lacking both Drep-2 and FMRP can learn normally. Exactly how Drep-2 works in synapses to help with memory formation remains to be discovered, although there are indications that it boosts the effects of signaling from the glutamate receptors and counteracts FMRP. Further research will be needed to establish whether the mammalian proteins related to Drep-2 perform similar roles in the brains of mammals. DOI: http://dx.doi.org/10.7554/eLife.03895.002

We studied the dynamics of the proteome of influenza virus A/PR/8/34 (H1N1) infected Madin-Darby canine kidney cells up to 12 hours post infection by mass spectrometry based quantitative proteomics using the approach of stable isotope labeling by amino acids in cell culture (SILAC). We identified 1311 cell proteins and, apart from the proton channel M2, all major virus proteins. Based on their abundance two groups of virus proteins could be distinguished being in line with the function of the proteins in genesis and formation of new virions. Further, the data indicate a correlation between the amount of proteins synthesized and their previously determined copy number inside the viral particle. We employed bioinformatic approaches such as functional clustering, gene ontology, and pathway (KEGG) enrichment tests to uncover co- regulated cellular protein sets, assigned the individual subsets to their biological function, and determined their interrelation within the progression of viral infection. For the first time we are able to describe dynamic changes of the cellular and, of note, the viral proteome in a time dependent manner simultaneously. Through cluster analysis, time dependent patterns of protein abundances revealed highly dynamic up- and/or down-regulation processes. Taken together our study provides strong evidence that virus infection has a major impact on the cell status at the protein level.

Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. ‘Cut and paste’ DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, Sleeping Beauty (SB) and piggyBac (PB) that are widely used for genome manipulation in vertebrate species. SB originates from fish and PB from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither SB nor PB is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1), a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the SB transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both SB and PB were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, SB is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for PB., Author Summary Transposons (“jumping genes”) are ubiquitous, mobile genetic elements that make up significant fraction of genomes, and are best described as molecular parasites. During ‘cut and paste’ transposition, the excised transposon relocates from one genomic location to another. Here we focus on the molecular events following excision of two eukaryotic DNA transposons, Sleeping Beauty and piggyBac. Both transposons are primarily used in a cellular environment that is different from their original hosts, thereby offering a new model to study host-parasite interaction in higher organisms. In the last decade, they have been developed into a technology platform for vertebrate genetics, including gene discovery, transgenesis, gene therapy and stem cell manipulation. Despite the wide range of their application, relatively little is known about their molecular mechanism in vertebrates. We show that these elements are not capable of self-avoidance, as a significant portion of the excised transposons integrates into its own genome in a suicidal process. Despite mechanistic differences, both transposons are affected similarly, and larger transposons are particularly vulnerable. We propose that transposons might recruit phylogenetically conserved cellular factors in a new host that protects against self-disruption. Suboptimal conditions in a new environment could generate abnormal, genotoxic transposition reactions, and should be monitored.

Chromatin immunoprecipitation and sequencing (ChIP-seq) provides a static snap-shot of DNA-associated proteins which fails to reflect the dynamics of the DNA-bound proteome. Now, Catic and co-workers combine ubiquitin ChIP-seq and proteasome inhibitors to map sites of DNA-associated protein degradation on a genome-wide scale. They identify an ubiquitin ligase which targets a transcriptional repressor for destruction by the proteasome, thus activating transcription of specific genes. These findings reveal that the ubiquitin proteasome system actively regulates transcription.

Contains fulltext : 139221.pdf (Publisher’s version ) (Open Access) Mutations in the gene encoding the RNA-binding protein RBM20 have been implicated in dilated cardiomyopathy (DCM), a major cause of chronic heart failure, presumably through altering cardiac RNA splicing. Here, we combined transcriptome-wide crosslinking immunoprecipitation (CLIP-seq), RNA-seq, and quantitative proteomics in cell culture and rat and human hearts to examine how RBM20 regulates alternative splicing in the heart. Our analyses revealed the presence of a distinct RBM20 RNA-recognition element that is predominantly found within intronic binding sites and linked to repression of exon splicing with RBM20 binding near 3' and 5' splice sites. Proteomic analysis determined that RBM20 interacts with both U1 and U2 small nuclear ribonucleic particles (snRNPs) and suggested that RBM20-dependent splicing repression occurs through spliceosome stalling at complex A. Direct RBM20 targets included several genes previously shown to be involved in DCM as well as genes not typically associated with this disease. In failing human hearts, reduced expression of RBM20 affected alternative splicing of several direct targets, indicating that differences in RBM20 expression may affect cardiac function. Together, these findings identify RBM20-regulated targets and provide insight into the pathogenesis of human heart failure. 01 augustus 2014

Gene expression is a multistep process that involves the transcription, translation and turnover of messenger RNAs and proteins. Although it is one of the most fundamental processes of life, the entire cascade has never been quantified on a genome-wide scale. Here we simultaneously measured absolute mRNA and protein abundance and turnover by parallel metabolic pulse labelling for more than 5,000 genes in mammalian cells. Whereas mRNA and protein levels correlated better than previously thought, corresponding half-lives showed no correlation. Using a quantitative model we have obtained the first genome-scale prediction of synthesis rates of mRNAs and proteins. We find that the cellular abundance of proteins is predominantly controlled at the level of translation. Genes with similar combinations of mRNA and protein stability shared functional properties, indicating that half-lives evolved under energetic and dynamic constraints. Quantitative information about all stages of gene expression provides a rich resource and helps to provide a greater understanding of the underlying design principles.

A report on the Proteomic Forum 2009 conference, Berlin, Germany, 29 March to 2 April, 2009.

SummaryPhosphatidylinositol 3′ OH kinase (PI3K) signaling and FOXO transcription factors play opposing roles at several B cell developmental stages. We show here abundant nuclear FOXO1 expression in the proliferative compartment of the germinal center (GC), its dark zone (DZ), and PI3K activity, downregulating FOXO1, in the light zone (LZ), where cells are selected for further differentiation. In the LZ, however, FOXO1 was expressed in a fraction of cells destined for DZ reentry. Upon FOXO1 ablation or induction of PI3K activity, GCs lost their DZ, owing at least partly to downregulation of the chemokine receptor CXCR4. Although this prevented proper cyclic selection of cells in GCs, somatic hypermutation and proliferation were maintained. Class switch recombination was partly lost due to a failure of switch region targeting by activation-induced deaminase (AID).

Despite advances in multi-modal treatment approaches, clinical outcomes of patients suffering from PAX3-FOXO1 fusion oncogene-expressing alveolar rhabdomyosarcoma (ARMS) remain dismal. Here we show that PAX3-FOXO1-expressing ARMS cells are sensitive to pharmacological ataxia telangiectasia and Rad3 related protein (ATR) inhibition. Expression of PAX3-FOXO1 in muscle progenitor cells is not only sufficient to increase sensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibit increased sensitivity to structurally diverse inhibitors of ATR. Mechanistically, ATR inhibition leads to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increases sensitivity of ARMS cells to PARP1 inhibition in vitro, and combined treatment with ATR and PARP1 inhibitors induces complete regression of primary patient-derived ARMS xenografts in vivo. Lastly, a genome-wide CRISPR activation screen (CRISPRa) in combination with transcriptional analyses of ATR inhibitor resistant ARMS cells identifies the RAS-MAPK pathway and its targets, the FOS gene family, as inducers of resistance to ATR inhibition. Our findings provide a rationale for upcoming biomarker-driven clinical trials of ATR inhibitors in patients suffering from ARMS.