Bacillus subtilis remains translationally active after CRISPRi-mediated replication initiation arrest.

Initiation of bacterial DNA replication takes place at the origin of replication (oriC), a region characterized by the presence of multiple DnaA boxes that serve as the binding sites for the master initiator protein DnaA. This process is tightly controlled by modulation of the availability or activity of DnaA and oriC during development or stress conditions. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium Bacillus subtilis. We successfully arrested replication in B. subtilis by employing a clustered regularly interspaced short palindromic repeats interference (CRISPRi) approach to specifically target the key DnaA boxes 6 and 7, preventing DnaA binding to oriC. In this way, other functions of DnaA, such as a transcriptional regulator, were not significantly affected. When replication initiation was halted by this specific artificial and early blockage, we observed that non-replicating cells continued translation and cell growth, and the initial replication arrest did not induce global stress conditions such as the SOS response.IMPORTANCEAlthough bacteria constantly replicate under laboratory conditions, natural environments expose them to various stresses such as lack of nutrients, high salinity, and pH changes, which can trigger non-replicating states. These states can enable bacteria to (i) become tolerant to antibiotics (persisters), (ii) remain inactive in specific niches for an extended period (dormancy), and (iii) adjust to hostile environments. Non-replicating states have also been studied because of the possibility of repurposing energy for the production of additional metabolites or proteins. Using clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeting bacterial replication initiation sequences, we were able to successfully control replication initiation in Bacillus subtilis. This precise approach makes it possible to study non-replicating phenotypes, contributing to a better understanding of bacterial adaptive strategies.

Automated Phosphopeptide Enrichment for Gram-Positive Bacteria.

Protein phosphorylation in prokaryotes has gained more attention in recent years as several studies linked it to regulatory and signaling functions, indicating importance similar to protein phosphorylation in eukaryotes. Studies on bacterial phosphorylation have so far been conducted using manual or HPLC-supported phosphopeptide enrichment, whereas automation of phosphopeptide enrichment has been established in eukaryotes, allowing for high-throughput sampling. To facilitate the prospect of studying bacterial phosphorylation on a systems level, we here established an automated Ser/Thr/Tyr phosphopeptide enrichment workflow on the Agilent AssayMap platform. We present optimized buffer conditions for TiO2 and Fe(III)-NTA-IMAC cartridge-based enrichment and the most advantageous, species-specific loading amounts for Streptococcus pyogenes, Listeria monocytogenes, and Bacillus subtilis. For higher sample amounts (≥250 µg), we observed superior performance of the Fe(III)-NTA cartridges, whereas for lower sample amounts (≤100 µg), TiO2-based enrichment is equally efficient. Both cartridges largely enriched the same set of phosphopeptides, suggesting no improvement of peptide yield by the complementary use of the two cartridges. Our data represent, to the best of our knowledge, the largest phosphoproteome identified in a single study for each of these bacteria.

Time-Resolved Proteome Analysis of Listeria monocytogenes during Infection Reveals the Role of the AAA+ Chaperone ClpC for Host Cell Adaptation.

The cellular proteome comprises all proteins expressed at a given time and defines an organism's phenotype under specific growth conditions. The proteome is shaped and remodeled by both protein synthesis and protein degradation. Here, we developed a new method which combines metabolic and chemical isobaric peptide labeling to simultaneously determine the time-resolved protein decay and de novo synthesis in an intracellular human pathogen. We showcase this method by investigating the Listeria monocytogenes proteome in the presence and absence of the AAA+ chaperone protein ClpC. ClpC associates with the peptidase ClpP to form an ATP-dependent protease complex and has been shown to play a role in virulence development in L. monocytogenes. However, the mechanism by which ClpC is involved in the survival and proliferation of intracellular L. monocytogenes remains elusive. Employing this new method, we observed extensive proteome remodeling in L. monocytogenes upon interaction with the host, supporting the hypothesis that ClpC-dependent protein degradation is required to initiate bacterial adaptation mechanisms. We identified more than 100 putative ClpC target proteins through their stabilization in a clpC deletion strain. Beyond the identification of direct targets, we also observed indirect effects of the clpC deletion on the protein abundance in diverse cellular and metabolic pathways, such as iron acquisition and flagellar assembly. Overall, our data highlight the crucial role of ClpC for L. monocytogenes adaptation to the host environment through proteome remodeling. IMPORTANCE Survival and proliferation of pathogenic bacteria inside the host depend on their ability to adapt to the changing environment. Profiling the underlying changes on the bacterial proteome level during the infection process is important to gain a better understanding of the pathogenesis and the host-dependent adaptation processes. The cellular protein abundance is governed by the interplay between protein synthesis and decay. The direct readout of these events during infection can be accomplished using pulsed stable-isotope labeling by amino acids in cell culture (SILAC). Combining this approach with tandem-mass-tag (TMT) labeling enabled multiplexed and time-resolved bacterial proteome quantification during infection. Here, we applied this integrated approach to investigate protein turnover during the temporal progression of adaptation of the human pathogen L. monocytogenes to its host on a system-wide scale. Our experimental approach can easily be transferred to probe the proteome remodeling in other bacteria under a variety of perturbations.

Role of Premycofactocin Synthase in Growth, Microaerophilic Adaptation, and Metabolism of Mycobacterium tuberculosis.

Mycofactocin is a new class of peptide-derived redox cofactors present in a selected group of bacteria including Mycobacterium tuberculosis. Mycofactocin biosynthesis requires at least six genes, including mftD, encoding putative lactate dehydrogenase, which catalyzes the penultimate biosynthetic step. Cellular functions remained unknown until recent reports on the significance of mycofactocin in primary alcohol metabolism. Here, we show that mftD transcript levels were increased in hypoxia-adapted M. tuberculosis; however, mftD functionality was found likely dispensable for l-lactate metabolism. Targeted deletion of mftD reduced the survival of M. tuberculosis in in vitro and in vivo hypoxia models but increased the bacterial growth in glucose-containing broth as well as in the lungs and spleens, albeit modestly, of aerosol-infected C57BL/6J mice. The cause of this growth advantage remains unestablished; however, the mftD-deficient M. tuberculosis strain had reduced NAD(H)/NADP(H) levels and glucose-6-phosphate dehydrogenase activity with no impairment in phthiocerol dimycocerosate lipid synthesis. An ultrastructural examination of parental and mycofactocin biosynthesis gene mutants in M. tuberculosis, M. marinum, and M. smegmatis showed no altered cell morphology and size except the presence of outer membrane-bound fibril-like features only in a mutant subpopulation. A cell surface-protein analysis of M. smegmatis mycofactocin biosynthesis mutants with trypsin revealed differential abundances of a subset of proteins that are known to interact with mycofactocin and their homologs that can enhance protein aggregation or amyloid-like fibrils in riboflavin-starved eukaryotic cells. In sum, phenotypic analyses of the mutant strain implicate the significance of MftD/mycofactocin in M. tuberculosis growth and persistence in its host. IMPORTANCE Characterization of proteins with unknown functions is a critical research priority as the intracellular growth and metabolic state of Mycobacterium tuberculosis, the causative agent of tuberculosis, remain poorly understood. Mycofactocin is a peptide-derived redox cofactor present in almost all mycobacterial species; however, its functional relevance in M. tuberculosis pathogenesis and host survival has never been studied experimentally. In this study, we examine the phenotypes of an M. tuberculosis mutant strain lacking a key mycofactocin biosynthesis gene in in vitro and disease-relevant mouse models. Our results pinpoint the multifaceted role of mycofactocin in M. tuberculosis growth, hypoxia adaptation, glucose metabolism, and redox homeostasis. This evidence strongly implies that mycofactocin could fulfill specialized biochemical functions that increase the survival fitness of mycobacteria within their specific niche.

Maternal exercise conveys protection against NAFLD in the offspring via hepatic metabolic programming.

Maternal exercise (ME) during pregnancy has been shown to improve metabolic health in offspring and confers protection against the development of non-alcoholic fatty liver disease (NAFLD). However, its underlying mechanism are still poorly understood, and it remains unclear whether protective effects on hepatic metabolism are already seen in the offspring early life. This study aimed at determining the effects of ME during pregnancy on offspring body composition and development of NAFLD while focusing on proteomic-based analysis of the hepatic energy metabolism during developmental organ programming in early life. Under an obesogenic high-fat diet (HFD), male offspring of exercised C57BL/6J-mouse dams were protected from body weight gain and NAFLD in adulthood (postnatal day (P) 112). This was associated with a significant activation of hepatic AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor alpha (PPARα) and PPAR coactivator-1 alpha (PGC1α) signaling with reduced hepatic lipogenesis and increased hepatic ß-oxidation at organ programming peak in early life (P21). Concomitant proteomic analysis revealed a characteristic hepatic expression pattern in offspring as a result of ME with the most prominent impact on Cholesterol 7 alpha-hydroxylase (CYP7A1). Thus, ME may offer protection against offspring HFD-induced NAFLD by shaping hepatic proteomics signature and metabolism in early life. The results highlight the potential of exercise during pregnancy for preventing the early origins of NAFLD.

Expanding the Depth and Sensitivity of Cross-Link Identification by Differential Ion Mobility Using High-Field Asymmetric Waveform Ion Mobility Spectrometry.

In cross-linking mass spectrometry (XL-MS), the depth and sensitivity of cross-link detection is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of cross-links, here, we present a gas-phase separation strategy using high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in the gas phase using the FAIMS device, we increase the number of cross-link identifications by 22% for a medium complex sample and 59% for strong cation exchange-fractionated HEK293T cell lysate in XL-MS experiments using disuccinimidyl sulfoxide (DSSO) cross-linker. When disuccinimidyl suberate (DSS) cross-linker is in use, we are able to boost cross-link identification by 89% for the medium and 100% for the high complex sample compared to the analyses without FAIMS. Furthermore, we show that, for medium complex samples, FAIMS enables the collection of single-shot XL-MS data with a comparable depth to the corresponding sample fractionated by chromatography-based approaches. Altogether, we demonstrate FAIMS is highly beneficial for XL-MS studies by expanding the proteome coverage of cross-links while improving the efficiency and confidence of cross-link identification.

CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex.

The exon junction complex (EJC) is an essential constituent and regulator of spliced messenger ribonucleoprotein particles (mRNPs) in metazoans. As a core component of the EJC, CASC3 was described to be pivotal for EJC-dependent nuclear and cytoplasmic processes. However, recent evidence suggests that CASC3 functions differently from other EJC core proteins. Here, we have established human CASC3 knockout cell lines to elucidate the cellular role of CASC3. In the knockout cells, overall EJC composition and EJC-dependent splicing are unchanged. A transcriptome-wide analysis reveals that hundreds of mRNA isoforms targeted by nonsense-mediated decay (NMD) are upregulated. Mechanistically, recruiting CASC3 to reporter mRNAs by direct tethering or via binding to the EJC stimulates mRNA decay and endonucleolytic cleavage at the termination codon. Building on existing EJC-NMD models, we propose that CASC3 equips the EJC with the persisting ability to communicate with the NMD machinery in the cytoplasm. Collectively, our results characterize CASC3 as a peripheral EJC protein that tailors the transcriptome by promoting the degradation of EJC-dependent NMD substrates.

A salvage pathway maintains highly functional respiratory complex I.

Regulation of the turnover of complex I (CI), the largest mitochondrial respiratory chain complex, remains enigmatic despite huge advancement in understanding its structure and the assembly. Here, we report that the NADH-oxidizing N-module of CI is turned over at a higher rate and largely independently of the rest of the complex by mitochondrial matrix protease ClpXP, which selectively removes and degrades damaged subunits. The observed mechanism seems to be a safeguard against the accumulation of dysfunctional CI arising from the inactivation of the N-module subunits due to attrition caused by its constant activity under physiological conditions. This CI salvage pathway maintains highly functional CI through a favorable mechanism that demands much lower energetic cost than de novo synthesis and reassembly of the entire CI. Our results also identify ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability.

Mitochondria-Endoplasmic Reticulum Contacts in Reactive Astrocytes Promote Vascular Remodeling.

Astrocytes have emerged for playing important roles in brain tissue repair; however, the underlying mechanisms remain poorly understood. We show that acute injury and blood-brain barrier disruption trigger the formation of a prominent mitochondrial-enriched compartment in astrocytic endfeet, which enables vascular remodeling. Integrated imaging approaches revealed that this mitochondrial clustering is part of an adaptive response regulated by fusion dynamics. Astrocyte-specific conditional deletion of Mitofusin 2 (Mfn2) suppressed perivascular mitochondrial clustering and disrupted mitochondria-endoplasmic reticulum (ER) contact sites. Functionally, two-photon imaging experiments showed that these structural changes were mirrored by impaired mitochondrial Ca2+ uptake leading to abnormal cytosolic transients within endfeet in vivo. At the tissue level, a compromised vascular complexity in the lesioned area was restored by boosting mitochondrial-ER perivascular tethering in MFN2-deficient astrocytes. These data unmask a crucial role for mitochondrial dynamics in coordinating astrocytic local domains and have important implications for repairing the injured brain.

Alterations of redox and iron metabolism accompany the development of HIV latency.

HIV-1 persists in a latent form during antiretroviral therapy, mainly in CD4+ T cells, thus hampering efforts for a cure. HIV-1 infection is accompanied by metabolic alterations, such as oxidative stress, but the effect of cellular antioxidant responses on viral replication and latency is unknown. Here, we show that cells survive retroviral replication, both in vitro and in vivo in SIVmac-infected macaques, by upregulating antioxidant pathways and the intertwined iron import pathway. These changes are associated with remodeling of promyelocytic leukemia protein nuclear bodies (PML NBs), an important constituent of nuclear architecture and a marker of HIV-1 latency. We found that PML NBs are hyper-SUMOylated and that PML protein is degraded via the ubiquitin-proteasome pathway in productively infected cells, before latency establishment and after reactivation. Conversely, normal numbers of PML NBs were restored upon transition to latency or by decreasing oxidative stress or iron content. Our results highlight antioxidant and iron import pathways as determinants of HIV-1 latency and support their pharmacologic inhibition as tools to regulate PML stability and impair latency establishment.

The alarmones (p)ppGpp are part of the heat shock response of Bacillus subtilis.

Bacillus subtilis cells are well suited to study how bacteria sense and adapt to proteotoxic stress such as heat, since temperature fluctuations are a major challenge to soil-dwelling bacteria. Here, we show that the alarmones (p)ppGpp, well known second messengers of nutrient starvation, are also involved in the heat stress response as well as the development of thermo-resistance. Upon heat-shock, intracellular levels of (p)ppGpp rise in a rapid but transient manner. The heat-induced (p)ppGpp is primarily produced by the ribosome-associated alarmone synthetase Rel, while the small alarmone synthetases RelP and RelQ seem not to be involved. Furthermore, our study shows that the generated (p)ppGpp pulse primarily acts at the level of translation, and only specific genes are regulated at the transcriptional level. These include the down-regulation of some translation-related genes and the up-regulation of hpf, encoding the ribosome-protecting hibernation-promoting factor. In addition, the alarmones appear to interact with the activity of the stress transcription factor Spx during heat stress. Taken together, our study suggests that (p)ppGpp modulates the translational capacity at elevated temperatures and thereby allows B. subtilis cells to respond to proteotoxic stress, not only by raising the cellular repair capacity, but also by decreasing translation to concurrently reduce the protein load on the cellular protein quality control system.

A protein-RNA interaction atlas of the ribosome biogenesis factor AATF.

AATF is a central regulator of the cellular outcome upon p53 activation, a finding that has primarily been attributed to its function as a transcription factor. Recent data showed that AATF is essential for ribosome biogenesis and plays a role in rRNA maturation. AATF has been implicated to fulfil this role through direct interaction with rRNA and was identified in several RNA-interactome capture experiments. Here, we provide a first comprehensive analysis of the RNA bound by AATF using CLIP-sequencing. Interestingly, this approach shows predominant binding of the 45S pre-ribosomal RNA precursor molecules. Furthermore, AATF binds to mRNAs encoding for ribosome biogenesis factors as well as snoRNAs. These findings are complemented by an in-depth analysis of the protein interactome of AATF containing a large set of proteins known to play a role in rRNA maturation with an emphasis on the protein-RNA-complexes known to be required for the generation of the small ribosomal subunit (SSU). In line with this finding, the binding sites of AATF within the 45S rRNA precursor localize in close proximity to the SSU cleavage sites. Consequently, our multilayer analysis of the protein-RNA interactome of AATF reveals this protein to be an important hub for protein and RNA interactions involved in ribosome biogenesis.

The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells.

BACKGROUND: RNA-binding proteins (RBPs) are fundamental regulators of cellular biology that affect all steps in the generation and processing of RNA molecules. Recent evidence suggests that regulation of RBPs that modulate both RNA stability and translation may have a profound effect on the proteome. However, regulation of RBPs in clinically relevant experimental conditions has not been studied systematically. METHODS: We used RNA interactome capture, a method for the global identification of RBPs to characterize the global RNA-binding proteome (RBPome) associated with polyA-tailed RNA species in murine ciliated epithelial cells of the inner medullary collecting duct. To study regulation of RBPs in a clinically relevant condition, we analyzed hypoxia-associated changes of the RBPome. RESULTS: We identified >1000 RBPs that had been previously found using other systems. In addition, we found a number of novel RBPs not identified by previous screens using mouse or human cells, suggesting that these proteins may be specific RBPs in differentiated kidney epithelial cells. We also found quantitative differences in RBP-binding to mRNA that were associated with hypoxia versus normoxia. CONCLUSIONS: These findings demonstrate the regulation of RBPs through environmental stimuli and provide insight into the biology of hypoxia-response signaling in epithelial cells in the kidney. A repository of the RBPome and proteome in kidney tubular epithelial cells, derived from our findings, is freely accessible online, and may contribute to a better understanding of the role of RNA-protein interactions in kidney tubular epithelial cells, including the response of these cells to hypoxia.

Noncanonical Modulation of the eIF2 Pathway Controls an Increase in Local Translation during Neural Wiring.

Local translation is rapidly regulated by extrinsic signals during neural wiring, but its control mechanisms remain elusive. Here we show that the extracellular cue Sema3A induces an initial burst in local translation that precisely controls phosphorylation of the translation initiation factor eIF2α via the unfolded protein response (UPR) kinase PERK. Strikingly, in contrast to canonical UPR signaling, Sema3A-induced eIF2α phosphorylation bypasses global translational repression and underlies an increase in local translation through differential activity of eIF2B mediated by protein phosphatase 1. Ultrasensitive proteomics analysis of axons reveals 75 proteins translationally controlled via the Sema3A-p-eIF2α pathway. These include proteostasis- and actin cytoskeleton-related proteins but not canonical stress markers. Finally, we show that PERK signaling is needed for directional axon migration and visual pathway development in vivo. Thus, our findings reveal a noncanonical eIF2 signaling pathway that controls selective changes in axon translation and is required for neural wiring.

Mechanism suppressing H3K9 trimethylation in pluripotent stem cells and its demise by polyQ-expanded huntingtin mutations.

Pluripotent stem cells are invaluable resources to study development and disease, holding a great promise for regenerative medicine. Here we use human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) from patients with Huntington's disease (HD-iPSCs) to shed light into the normal function of huntingtin (HTT) and its demise in disease. We find that HTT binds ATF7IP, a regulator of the histone H3 methyltransferase SETDB1. HTT inhibits the interaction of the ATF7IP-SETDB1 complex with other heterochromatin regulators and transcriptional repressors, maintaining low levels of H3K9 trimethylation (H3K9me3) in hESCs. Loss of HTT promotes global increased H3K9me3 levels and enrichment of H3K9me3 marks at distinct genes, including transcriptional regulators of neuronal differentiation. Although these genes are normally expressed at low amounts in hESCs, HTT knockdown (KD) reduces their induction during neural differentiation. Notably, mutant expanded polyglutamine repeats in HTT diminish its interaction with ATF7IP-SETDB1 complex and trigger H3K9me3 in HD-iPSCs. Conversely, KD of ATF7IP in HD-iPSCs reduces H3K9me3 alterations and ameliorates gene expression changes in their neural counterparts. Taken together, our results indicate ATF7IP as a potential target to correct aberrant H3K9me3 levels induced by mutant HTT.

Rapid Cue-Specific Remodeling of the Nascent Axonal Proteome.

Axonal protein synthesis and degradation are rapidly regulated by extrinsic signals during neural wiring, but the full landscape of proteomic changes remains unknown due to limitations in axon sampling and sensitivity. By combining pulsed stable isotope labeling of amino acids in cell culture with single-pot solid-phase-enhanced sample preparation, we characterized the nascent proteome of isolated retinal axons on an unparalleled rapid timescale (5 min). Our analysis detects 350 basally translated axonal proteins on average, including several linked to neurological disease. Axons stimulated by different cues (Netrin-1, BDNF, Sema3A) show distinct signatures with more than 100 different nascent protein species up- or downregulated within the first 5 min followed by further dynamic remodeling. Switching repulsion to attraction triggers opposite regulation of a subset of common nascent proteins. Our findings thus reveal the rapid remodeling of the axonal proteomic landscape by extrinsic cues and uncover a logic underlying attraction versus repulsion.

Single-nephron proteomes connect morphology and function in proteinuric kidney disease.

In diseases of many parenchymatous organs, heterogeneous deterioration of individual functional units determines the clinical prognosis. However, the molecular characterization at the level of such individual subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Proteinuric glomerular kidney diseases are frequent and assorted diseases affecting a fraction of glomeruli and their draining tubules to variable extents, and for which no specific treatment exists. Here, we developed and applied a mass spectrometry-based methodology to investigate heterogeneity of proteomes from individually isolated nephron segments from mice with proteinuric kidney disease. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinuria) and LAMP1, a lysosomal protein. This module was associated with a loss of podocyte marker proteins while genetic ablation of LAMP1-correlated lysosomal proteases could ameliorate glomerular damage in vivo. Furthermore, proteomic analyses of individual glomeruli from patients with genetic sclerotic and non-sclerotic proteinuric diseases revealed increased abundance of lysosomal proteins, in combination with a decreased abundance of mutated gene products. Thus, altered protein homeostasis (proteostasis) is a conserved key mechanism in proteinuric kidney diseases. Moreover, our technology can capture intra-individual variability in diseases of the kidney and other tissues at a sub-biopsy scale.

Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap.

This protocol is an extension to: Nat. Protoc. 8, 491-500 (2013); doi:10.1038/nprot.2013.020; published online 14 February 2013RBDmap is a method for identifying, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native interactions with RNA. In brief, cells are irradiated with UV light to induce protein-RNA cross-links. Following stringent denaturing washes, the resulting covalently linked protein-RNA complexes are purified with oligo(dT) magnetic beads. After elution, RBPs are subjected to partial proteolysis, in which the protein regions still bound to the RNA and those released to the supernatant are separated by a second oligo(dT) selection. After sample preparation and mass-spectrometric analysis, peptide intensity ratios between the RNA-bound and released fractions are used to determine the RNA-binding regions. As a Protocol Extension, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol (for the RNA interactome capture method) describes how to identify the active RBPs in cultured cells, whereas this Protocol Extension also enables the identification of the RNA-binding domains of RBPs. The experimental workflow takes 1 week plus 2 additional weeks for proteomics and data analysis. Notably, RBDmap presents numerous advantages over classic methods for determining RNA-binding domains: it produces proteome-wide, high-resolution maps of the protein regions contacting the RNA in a physiological context and can be adapted to different biological systems and conditions. Because RBDmap relies on the isolation of polyadenylated RNA via oligo(dT), it will not provide RNA-binding information on proteins interacting exclusively with nonpolyadenylated transcripts. Applied to HeLa cells, RBDmap uncovered 1,174 RNA-binding sites in 529 proteins, many of which were previously unknown.

Quantitative Map of Proteome Dynamics during Neuronal Differentiation.

Neuronal differentiation is a multistep process that shapes and re-shapes neurons by progressing through several typical stages, including axon outgrowth, dendritogenesis, and synapse formation. To systematically profile proteome dynamics throughout neuronal differentiation, we took cultured rat hippocampal neurons at different developmental stages and monitored changes in protein abundance using a combination of stable isotope labeling and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Almost one third of all 4,500 proteins quantified underwent a more than 2-fold expression change during neuronal differentiation, indicating extensive remodeling of the neuron proteome. To highlight the strength of our resource, we studied the neural-cell-adhesion molecule 1 (NCAM1) and found that it stimulates dendritic arbor development by promoting actin filament growth at the dendritic growth cone. We anticipate that our quantitative map of neuronal proteome dynamics is a rich resource for further analyses of the many identified proteins in various neurodevelopmental processes.

Comprehensive Identification of RNA-Binding Domains in Human Cells.

Mammalian cells harbor more than a thousand RNA-binding proteins (RBPs), with half of these employing unknown modes of RNA binding. We developed RBDmap to determine the RNA-binding sites of native RBPs on a proteome-wide scale. We identified 1,174 binding sites within 529 HeLa cell RBPs, discovering numerous RNA-binding domains (RBDs). Catalytic centers or protein-protein interaction domains are in close relationship with RNA-binding sites, invoking possible effector roles of RNA in the control of protein function. Nearly half of the RNA-binding sites map to intrinsically disordered regions, uncovering unstructured domains as prevalent partners in protein-RNA interactions. RNA-binding sites represent hot spots for defined posttranslational modifications such as lysine acetylation and tyrosine phosphorylation, suggesting metabolic and signal-dependent regulation of RBP function. RBDs display a high degree of evolutionary conservation and incidence of Mendelian mutations, suggestive of important functional roles. RBDmap thus yields profound insights into native protein-RNA interactions in living cells.