Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling.

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

Expanded roles and divergent regulation of FAMA in Brachypodium and Arabidopsis stomatal development.

Stomata, cellular valves found on the surfaces of aerial plant tissues, present a paradigm for studying cell fate and patterning in plants. A highly conserved core set of related basic helix-loop-helix (bHLH) transcription factors regulates stomatal development across diverse species. We characterized BdFAMA in the temperate grass Brachypodium distachyon and found this late-acting transcription factor was necessary and sufficient for specifying stomatal guard cell fate, and unexpectedly, could also induce the recruitment of subsidiary cells in the absence of its paralogue, BdMUTE. The overlap in function is paralleled by an overlap in expression pattern and by unique regulatory relationships between BdMUTE and BdFAMA. To better appreciate the relationships among the Brachypodium stomatal bHLHs, we used in vivo proteomics in developing leaves and found evidence for multiple shared interaction partners. We reexamined the roles of these genes in Arabidopsis thaliana by testing genetic sufficiency within and across species, and found that while BdFAMA and AtFAMA can rescue stomatal production in Arabidopsis fama and mute mutants, only AtFAMA can specify Brassica-specific myrosin idioblasts. Taken together, our findings refine the current models of stomatal bHLH function and regulatory feedback among paralogues within grasses as well as across the monocot/dicot divide.

Enhancer activation via TCP and HD-ZIP and repression by Dof transcription factors mediate giant cell-specific expression.

Proper cell-type identity relies on highly coordinated regulation of gene expression. Regulatory elements such as enhancers can produce cell type-specific expression patterns, but the mechanisms underlying specificity are not well understood. We previously identified an enhancer region capable of driving specific expression in giant cells, which are large, highly endoreduplicated cells in the Arabidopsis thaliana sepal epidermis. In this study, we use the giant cell enhancer as a model to understand the regulatory logic that promotes cell type-specific expression. Our dissection of the enhancer revealed that giant cell specificity is mediated primarily through the combination of two activators and one repressor. HD-ZIP and TCP transcription factors are involved in the activation of expression throughout the epidermis. High expression of HD-ZIP transcription factor genes in giant cells promoted higher expression driven by the enhancer in giant cells. Dof transcription factors repressed the activity of the enhancer such that only giant cells maintained enhancer activity. Thus, our data are consistent with a conceptual model whereby cell type-specific expression emerges from the combined activities of three transcription factor families activating and repressing expression in epidermal cells.

SPINDLY mediates O-fucosylation of hundreds of proteins and sugar-dependent growth in Arabidopsis.

The recent discovery of SPINDLY (SPY)-catalyzed protein O-fucosylation revealed a novel mechanism for regulating nucleocytoplasmic protein functions in plants. Genetic evidence indicates the important roles of SPY in diverse developmental and physiological processes. However, the upstream signal controlling SPY activity and the downstream substrate proteins O-fucosylated by SPY remain largely unknown. Here, we demonstrated that SPY mediates sugar-dependent growth in Arabidopsis (Arabidopsis thaliana). We further identified hundreds of O-fucosylated proteins using lectin affinity chromatography followed by mass spectrometry. All the O-fucosylation events quantified in our proteomic analyses were undetectable or dramatically decreased in the spy mutants, and thus likely catalyzed by SPY. The O-fucosylome includes mostly nuclear and cytosolic proteins. Many O-fucosylated proteins function in essential cellular processes, phytohormone signaling, and developmental programs, consistent with the genetic functions of SPY. The O-fucosylome also includes many proteins modified by O-linked N-acetylglucosamine (O-GlcNAc) and by phosphorylation downstream of the target of rapamycin (TOR) kinase, revealing the convergence of these nutrient signaling pathways on key regulatory functions such as post-transcriptional/translational regulation and phytohormone responses. Our study identified numerous targets of SPY/O-fucosylation and potential nodes of crosstalk among sugar/nutrient signaling pathways, enabling future dissection of the signaling network that mediates sugar regulation of plant growth and development.

Mapping the signaling network of BIN2 kinase using TurboID-mediated biotin labeling and phosphoproteomics.

Elucidating enzyme-substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically difficult because enzyme-substrate interactions tend to be transient. Here, we demonstrate that TurboID-based proximity labeling (TbPL) effectively and specifically captures the substrates of kinases and phosphatases. TbPL-mass spectrometry (TbPL-MS) identified over 400 proximal proteins of Arabidopsis thaliana BRASSINOSTEROID-INSENSITIVE2 (BIN2), a member of the GLYCOGEN SYNTHASE KINASE 3 (GSK3) family that integrates signaling pathways controlling diverse developmental and acclimation processes. A large portion of the BIN2-proximal proteins showed BIN2-dependent phosphorylation in vivo or in vitro, suggesting that these are BIN2 substrates. Protein-protein interaction network analysis showed that the BIN2-proximal proteins include interactors of BIN2 substrates, revealing a high level of interactions among the BIN2-proximal proteins. Our proteomic analysis establishes the BIN2 signaling network and uncovers BIN2 functions in regulating key cellular processes such as transcription, RNA processing, translation initiation, vesicle trafficking, and cytoskeleton organization. We further discovered significant overlap between the GSK3 phosphorylome and the O-GlcNAcylome, suggesting an evolutionarily ancient relationship between GSK3 and the nutrient-sensing O-glycosylation pathway. Our work presents a powerful method for mapping PTM networks, a large dataset of GSK3 kinase substrates, and important insights into the signaling network that controls key cellular functions underlying plant growth and acclimation.

SECRET AGENT O-GlcNAcylates Hundreds of Proteins Involved in Diverse Cellular Processes in Arabidopsis.

O-GlcNAcylation is a critical post-translational modification of proteins observed in both plants and animals and plays a key role in growth and development. While considerable knowledge exists about over 3000 substrates in animals, our understanding of this modification in plants remains limited. Unlike animals, plants possess two putative homologs: SECRET AGENT (SEC) and SPINDLY, with SPINDLY also exhibiting O-fucosylation activity. To investigate the role of SEC as a major O-GlcNAc transferase in plants, we utilized lectin-weak affinity chromatography enrichment and stable isotope labeling in Arabidopsis labeling, quantifying at both MS1 and MS2 levels. Our findings reveal a significant reduction in O-GlcNAc levels in the sec mutant, indicating the critical role of SEC in mediating O-GlcNAcylation. Through a comprehensive approach, combining higher-energy collision dissociation and electron-transfer high-energy collision dissociation fragmentation with substantial fractionations, we expanded our GlcNAc profiling, identifying 436 O-GlcNAc targets, including 227 new targets. The targets span diverse cellular processes, suggesting broad regulatory functions of O-GlcNAcylation. The expanded targets also enabled exploration of crosstalk between O-GlcNAcylation and O-fucosylation. We also examined electron-transfer high-energy collision dissociation fragmentation for site assignment. This report advances our understanding of O-GlcNAcylation in plants, facilitating further research in this field.

The Quantitative Biotinylproteomics Studies Reveal a WInd-Related Kinase 1 (Raf-Like Kinase 36) Functioning as an Early Signaling Component in Wind-Induced Thigmomorphogenesis and Gravitropism.

Wind is one of the most prevalent environmental forces entraining plants to develop various mechano-responses, collectively called thigmomorphogenesis. Largely unknown is how plants transduce these versatile wind force signals downstream to nuclear events and to the development of thigmomorphogenic phenotype or anemotropic response. To identify molecular components at the early steps of the wind force signaling, two mechanical signaling-related phosphoproteins, identified from our previous phosphoproteomic study of Arabidopsis touch response, mitogen-activated protein kinase kinase 1 (MKK1) and 2 (MKK2), were selected for performing in planta TurboID (ID)-based quantitative proximity-labeling (PL) proteomics. This quantitative biotinylproteomics was separately performed on MKK1-ID and MKK2-ID transgenic plants, respectively, using the genetically engineered TurboID biotin ligase expression transgenics as a universal control. This unique PTM proteomics successfully identified 11 and 71 MKK1 and MKK2 putative interactors, respectively. Biotin occupancy ratio (BOR) was found to be an alternative parameter to measure the extent of proximity and specificity between the proximal target proteins and the bait fusion protein. Bioinformatics analysis of these biotinylprotein data also found that TurboID biotin ligase favorably labels the loop region of target proteins. A WInd-Related Kinase 1 (WIRK1), previously known as rapidly accelerated fibrosarcoma (Raf)-like kinase 36 (RAF36), was found to be a putative common interactor for both MKK1 and MKK2 and preferentially interacts with MKK2. Further molecular biology studies of the Arabidopsis RAF36 kinase found that it plays a role in wind regulation of the touch-responsive TCH3 and CML38 gene expression and the phosphorylation of a touch-regulated PATL3 phosphoprotein. Measurement of leaf morphology and shoot gravitropic response of wirk1 (raf36) mutant revealed that the WIRK1 gene is involved in both wind-triggered rosette thigmomorphogenesis and gravitropism of Arabidopsis stems, suggesting that the WIRK1 (RAF36) protein probably functioning upstream of both MKK1 and MKK2 and that it may serve as the crosstalk point among multiple mechano-signal transduction pathways mediating both wind mechano-response and gravitropism.

Uncovering the proximal proteome of CTR1 through TurboID-mediated proximity labeling.

Protein-protein interactions play a crucial role in driving cellular processes and enabling appropriate physiological responses in organisms. The plant hormone ethylene signaling pathway is complex and regulated by the spatiotemporal regulation of its signaling molecules. Constitutive Triple Response 1 (CTR1), a key negative regulator of the pathway, regulates the function of Ethylene-Insensitive 2 (EIN2), a positive regulator of ethylene signaling, at the endoplasmic reticulum (ER) through phosphorylation. Our recent study revealed that CTR1 can also translocate from the ER to the nucleus in response to ethylene and positively regulate ethylene responses by stabilizing EIN3. To gain further insights into the role of CTR1 in plants, we used TurboID-based proximity labeling and mass spectrometry to identify the proximal proteomes of CTR1 in Nicotiana benthamiana. The identified proximal proteins include known ethylene signaling components, as well as proteins involved in diverse cellular processes such as mitochondrial respiration, mRNA metabolism, and organelle biogenesis. Our study demonstrates the feasibility of proximity labeling using the N. benthamiana transient expression system and identifies the potential interactors of CTR1 in vivo, uncovering the potential roles of CTR1 in a wide range of cellular processes.

TRIPP Is a Plant-Specific Component of the Arabidopsis TRAPPII Membrane Trafficking Complex with Important Roles in Plant Development.

How the membrane trafficking system spatially organizes intracellular activities and intercellular signaling networks in plants is not well understood. Transport Protein Particle (TRAPP) complexes play key roles in the selective delivery of membrane vesicles to various subcellular compartments in yeast and animals but remain to be fully characterized in plants. Here, we investigated TRAPP complexes in Arabidopsis (Arabidopsis thaliana) using immunoprecipitation followed by quantitative mass spectrometry analysis of AtTRS33, a conserved core component of all TRAPP complexes. We identified 14 AtTRS33-interacting proteins, including homologs of all 13 TRAPP components in mammals and a protein that has homologs only in multicellular photosynthetic organisms and is thus named TRAPP-Interacting Plant Protein (TRIPP). TRIPP specifically associates with the TRAPPII complex through binary interactions with two TRAPPII-specific subunits. TRIPP colocalized with a subset of TRS33 compartments and trans-Golgi network markers in a TRS33-dependent manner. Loss-of-function tripp mutants exhibited dwarfism, sterility, partial photomorphogenesis in the dark, reduced polarity of the auxin transporter PIN2, incomplete cross wall formation, and altered localization of a TRAPPII-specific component. Therefore, TRIPP is a plant-specific component of the TRAPPII complex with important functions in trafficking, plant growth, and development.

Plant U-Box40 Mediates Degradation of the Brassinosteroid-Responsive Transcription Factor BZR1 in Arabidopsis Roots.

Brassinosteroid (BR) regulates a wide range of physiological responses through the activation of BRASSINAZOLE RESISTANT1 (BZR1), whose activity is tightly controlled by its phosphorylation status and degradation. Although BZR1 appears to be degraded in distinct ways in response to different hormonal or environmental cues, little is known about how BR signaling regulates its degradation. Here we show that the BR-regulated U-box protein PUB40 mediates the proteasomal degradation of BZR1 in a root-specific manner in Arabidopsis (Arabidopsis thaliana). BZR1 levels were strongly reduced by plant U-box40 (PUB40) overexpression, whereas the pub39 pub40 pub41 mutant accumulated much more BZR1 than wild type in roots. The bzr1-1D gain-of-function mutation reduced the interaction with PUB40, which suppressed PUB40-mediated BZR1 degradation in roots. The cell layer-specific expression of PUB40 in roots helps induce selective BZR1 accumulation in the epidermal layer. Both BR treatment and loss-of-function of PUB40 expanded BZR1 accumulation to most cell layers. In addition, BZR1 accumulation increased the resistance of pub39 pub40 pub41 to low inorganic phosphate availability, as observed in bzr1-1D BRASSINOSTEROID-INSENSITIVE2-induced phosphorylation of PUB40, which mainly occurs in roots, gives rise to BZR1 degradation through enhanced binding of PUB40 to BZR1 and PUB40's stability. Our results suggest a molecular mechanism of root-specific BZR1 degradation regulated by BR signaling.

Mutual Regulation of Receptor-Like Kinase SIT1 and B'κ-PP2A Shapes the Early Response of Rice to Salt Stress.

The receptor-like kinase SIT1 acts as a sensor in rice (Oryza sativa) roots, relaying salt stress signals via elevated kinase activity to enhance salt sensitivity. Here, we demonstrate that Protein Phosphatase 2A (PP2A) regulatory subunit B'κ constrains SIT1 activity under salt stress. B'κ-PP2A deactivates SIT1 directly by dephosphorylating the kinase at Thr515/516, a salt-induced phosphorylation site in the activation loop that is essential for SIT1 activity. B'κ overexpression suppresses the salt sensitivity of rice plants expressing high levels of SIT1, thereby contributing to salt tolerance. B'κ functions in a SIT1 kinase-dependent manner. During early salt stress, activated SIT1 phosphorylates B'κ; this not only enhances its binding with SIT1, it also promotes B'κ protein accumulation via Ser502 phosphorylation. Consequently, by blocking SIT1 phosphorylation, B'κ inhibits and fine-tunes SIT1 activity to balance plant growth and stress adaptation.

Proteomic analysis reveals O-GlcNAc modification on proteins with key regulatory functions in Arabidopsis.

Genetic studies have shown essential functions of O-linked N-acetylglucosamine (O-GlcNAc) modification in plants. However, the proteins and sites subject to this posttranslational modification are largely unknown. Here, we report a large-scale proteomic identification of O-GlcNAc-modified proteins and sites in the model plant Arabidopsis thaliana Using lectin weak affinity chromatography to enrich modified peptides, followed by mass spectrometry, we identified 971 O-GlcNAc-modified peptides belonging to 262 proteins. The modified proteins are involved in cellular regulatory processes, including transcription, translation, epigenetic gene regulation, and signal transduction. Many proteins have functions in developmental and physiological processes specific to plants, such as hormone responses and flower development. Mass spectrometric analysis of phosphopeptides from the same samples showed that a large number of peptides could be modified by either O-GlcNAcylation or phosphorylation, but cooccurrence of the two modifications in the same peptide molecule was rare. Our study generates a snapshot of the O-GlcNAc modification landscape in plants, indicating functions in many cellular regulation pathways and providing a powerful resource for further dissecting these functions at the molecular level.

N-Glycopeptide Profiling in Arabidopsis Inflorescence.

This study presents the first large-scale analysis of plant intact glycopeptides. Using wheat germ agglutinin lectin weak affinity chromatography to enrich modified peptides, followed by electron transfer dissociation (ETD)(1) fragmentation tandem mass spectrometry, glycan compositions on over 1100 glycopeptides from 270 proteins found in Arabidopsis inflorescence tissue were characterized. While some sites were only detected with a single glycan attached, others displayed up to 16 different glycoforms. Among the identified glycopeptides were four modified in nonconsensus glycosylation motifs. While most of the modified proteins are secreted, membrane, endoplasmic reticulum (ER), or Golgi-localized proteins, surprisingly, N-linked sugars were detected on a protein predicted to be cytosolic or nuclear.

Multisite light-induced phosphorylation of the transcription factor PIF3 is necessary for both its rapid degradation and concomitant negative feedback modulation of photoreceptor phyB levels in Arabidopsis.

Plants constantly monitor informational light signals using sensory photoreceptors, which include the phytochrome (phy) family (phyA to phyE), and adjust their growth and development accordingly. Following light-induced nuclear translocation, photoactivated phy molecules bind to and induce rapid phosphorylation and degradation of phy-interacting basic Helix Loop Helix (bHLH) transcription factors (PIFs), such as PIF3, thereby regulating the expression of target genes. However, the mechanisms underlying the signal-relay process are still not fully understood. Here, using mass spectrometry, we identify multiple, in vivo, light-induced Ser/Thr phosphorylation sites in PIF3. Using transgenic expression of site-directed mutants of PIF3, we provide evidence that a set of these phosphorylation events acts collectively to trigger rapid degradation of the PIF3 protein in response to initial exposure of dark-grown seedlings to light. In addition, we show that phyB-induced PIF3 phosphorylation is also required for the known negative feedback modulation of phyB levels in prolonged light, potentially through codegradation of phyB and PIF3. This mutually regulatory intermolecular transaction thus provides a mechanism with the dual capacity to promote early, graded, or threshold regulation of the primary, PIF3-controlled transcriptional network in response to initial light exposure, and later, to attenuate global sensitivity to the light signal through reductions in photoreceptor levels upon prolonged exposure.

Impact of alternative splicing on Arabidopsis proteome.

Alternative splicing is an important regulatory process in eukaryotes. In plants, the major form of alternative splicing is intron retention. Despite its importance, the global impact of AS on the Arabidopsis proteome has not been investigated. In this study, we address this gap by performing a comprehensive integrated analysis of how changes in AS can affect the Arabidopsis proteome using mutants that disrupt ACINUS and PININ, two evolutionarily conserved alternative splicing factors. We used tandem mass tagging (TMT) with real-time search MS3 (RTS-SPS-MS3) coupled with extensive sample fractionations to achieve very high coverage and accurate protein quantification. We then integrated our proteomic data with transcriptomic data to assess how transcript changes and increased intron retention (IIR) affect the proteome. For differentially expressed transcripts, we have observed a weak to moderate correlation between transcript changes and protein changes. Our studies revealed that some IIRs have no effect on either transcript or protein levels, while some IIRs can significantly affect protein levels. Surprisingly, we found that IIRs have a much smaller effect on increasing protein diversity. Notably, the increased intron retention events detected in the double mutant are also detected in the WT under various biotic or abiotic stresses. We further investigated the characteristics of the retained introns. Our extensive proteomic data help to guide the phenotypic analysis and reveal that collective protein changes contribute to the observed phenotypes of the increased anthocyanin, pale green, reduced growth, and short root observed in the acinus pnn double mutant. Overall, our study provides insight into the intricate regulatory mechanism of intron retention and its impact on protein abundance in plants.

Spatially resolved proteomics of the Arabidopsis stomatal lineage identifies polarity complexes for cell divisions and stomatal pores.

Cell polarity is used to guide asymmetric divisions and create morphologically diverse cells. We find that two oppositely oriented cortical polarity domains present during the asymmetric divisions in the Arabidopsis stomatal lineage are reconfigured into polar domains marking ventral (pore-forming) and outward-facing domains of maturing stomatal guard cells. Proteins that define these opposing polarity domains were used as baits in miniTurboID-based proximity labeling. Among differentially enriched proteins, we find kinases, putative microtubule-interacting proteins, and polar SOSEKIs with their effector ANGUSTIFOLIA. Using AI-facilitated protein structure prediction models, we identify potential protein-protein interaction interfaces among them. Functional and localization analyses of the polarity protein OPL2 and its putative interaction partners suggest a positive interaction with mitotic microtubules and a role in cytokinesis. This combination of proteomics and structural modeling with live-cell imaging provides insights into how polarity is rewired in different cell types and cell-cycle stages.

Next-generation mapping of the salicylic acid signaling hub and transcriptional cascade.

For over 60 years, salicylic acid (SA) has been known as a plant immune signal required for both basal and systemic acquired resistance (SAR). SA activates these immune responses by reprogramming up to 20% of the transcriptome through the function of NPR1. However, components in the NPR1-signaling hub, which appears as nuclear condensates, and the NPR1- signaling cascade remained elusive due to difficulties in studying transcriptional cofactors whose chromatin associations are often indirect and transient. To overcome this challenge, we applied TurboID to divulge the NPR1-proxiome, which detected almost all known NPR1-interactors as well as new components of transcription-related complexes. Testing of new components showed that chromatin remodeling and histone demethylation contribute to SA-induced resistance. Globally, NPR1-proxiome shares a striking similarity to GBPL3-proxiome involved in SA synthesis, except associated transcription factors (TFs), suggesting that common regulatory modules are recruited to reprogram specific transcriptomes by transcriptional cofactors, like NPR1, through binding to unique TFs. Stepwise greenCUT&RUN analyses showed that, upon SA-induction, NPR1 initiates the transcriptional cascade primarily through association with TGA TFs to induce expression of secondary TFs, predominantly WRKYs. WRKY54 and WRKY70 then play a major role in inducing immune-output genes without interacting with NPR1 at the chromatin. Moreover, a loss of NPR1 condensate formation decreases its chromatin-association and transcriptional activity, indicating the importance of condensates in organizing the NPR1- signaling hub and initiating the transcriptional cascade. This study demonstrates how combinatorial applications of TurboID and stepwise greenCUT&RUN transcend traditional genetic methods to globally map signaling hubs and transcriptional cascades.

Workflow enhancement of TurboID-mediated proximity labeling for SPY signaling network mapping.

TurboID-based proximity labeling coupled to mass spectrometry (PL-MS) has emerged as a powerful tool for mapping protein-protein interactions in both plant and animal systems. Despite advances in sensitivity, PL-MS studies can still suffer from false negatives, especially when dealing with low abundance bait proteins and their transient interactors. Protein-level enrichment for biotinylated proteins is well developed and popular, but direct detection of biotinylated proteins by peptide-level enrichment and the difference in results between direct and indirect detection remain underexplored. To address this gap, we compared and improved enrichment and data analysis methods using TurboID fused to SPY, a low-abundance O-fucose transferase, using an AAL-enriched SPY target library for cross-referencing. Our results showed that MyOne and M280 streptavidin beads significantly outperformed antibody beads for peptide-level enrichment, with M280 performing best. In addition, while a biotin concentration ≤ 50 µM is recommended for protein-level enrichment in plants, higher biotin concentrations can be used for peptide-level enrichment, allowing us to improve detection and data quality. FragPipe's MSFragger protein identification and quantification software outperformed Maxquant and Protein Prospector for SPY interactome enrichment due to its superior detection of biotinylated peptides. Our improved washing protocols for protein-level enrichment mitigated bead collapse issues, improving data quality, and reducing experimental time. We found that the two enrichment methods provided complementary results and identified a total of 160 SPY-TurboID-enriched interactors, including 60 previously identified in the AAL-enriched SPY target list and 100 additional novel interactors. SILIA quantitative proteomics comparing WT and spy-4 mutants showed that SPY affects the protein levels of some of the identified interactors, such as nucleoporin proteins. We expect that our improvement will extend beyond TurboID to benefit other PL systems and hold promise for broader applications in biological research.

ZNF574 is a Quality Control Factor For Defective Ribosome Biogenesis Intermediates.

Eukaryotic ribosome assembly is an intricate process that involves four ribosomal RNAs, 80 ribosomal proteins, and over 200 biogenesis factors that take part in numerous interdependent steps. This complexity creates a large genetic space in which pathogenic mutations can occur. Dead-end ribosome intermediates that result from biogenesis errors are rapidly degraded, affirming the existence of quality control pathway(s) that monitor ribosome assembly. However, the factors that differentiate between on-path and dead-end intermediates are unknown. We engineered a system to perturb ribosome assembly in human cells and discovered that faulty ribosomes are degraded via the ubiquitin proteasome system. We identified ZNF574 as a key component of a novel quality control pathway, which we term the Ribosome Assembly Surveillance Pathway (RASP). Loss of ZNF574 results in the accumulation of faulty biogenesis intermediates that interfere with global ribosome production, further emphasizing the role of RASP in protein homeostasis and cellular health.