While there is growing realization that sleep is important for memory consolidation, most evidence remains correlative and the causal role of specific sleep events remains unclear. Moreover, there is a substantial gap between detailed electrophysiological animal research and non-invasive cognitive human research – where recording neural activity in the medial temporal lobe is particularly challenging. The proposed research aims to understand the causal role that sleep plays in memory consolidation. Upon informed consent, epilepsy patients at UCLA implanted with intracranial electrodes for clinical monitoring will participate in learning/memory paradigms combined with recordings and intracranial electrical stimulation during sleep. I will address two specific aims: (1) Determine which sleep activities correlate best with learning and memory improvements: I hypothesize that for declarative memory, NREM sleep will be best correlated with learning, and that coordinated coupling between neocortical slow waves, sleep spindles and hippocampal ripples, co-occurring with reactivation of neuronal ensembles that were selectively engaged in the learning task, will be maximally correlated with successful learning. (2) Elucidate the causal mechanisms through intracranial electrical stimulation. I hypothesize that electrical stimulation in the neocortex that mimics or locks to endogenous slow wave up-states and hippocampal ripples will be effective in promoting memory consolidation. This proposal has the potential to generate a breakthrough in our understanding of the coordination between neocortex and hippocampus during sleep and how sleep causally aids human memory consolidation.

The muscular systems of humans and chimpanzees differ in a variety of traits, with human muscles built for dexterity and endurance, whereas chimpanzee muscles are built for strength. However, the genetic basis of this variation is little understood. Here, I propose to use a novel system of hybrid human-chimpanzee tetraploid induced pluripotent cells to study how the regulation of genes affecting muscle physiology has evolved along our lineage. This hybrid cell line offers a unique and unprecedented opportunity to tease out genetic from environmental factors, as well as cis- from trans-acting factors. I will first differentiate the parental and hybrid cells into skeletal myocytes, the main cells determining fitness capabilities. Then, I will identify allele-specific expression by comparing the gene expression levels of the hybrids to their parental cells. This will allow me to establish the first catalogue of cis- and trans-regulatory divergence in muscle cells. Finally, I offer to pick ten genes, where it is most probable to identify the variant the drives their diverged regulation, and to genetically edit them to investigate the phenotypic effect of each sequence change. This novel system will allow me to circumnavigate a key ethical barrier in great ape studies – obtaining great ape cells. Moreover, this system and pipeline could be used to study a variety of cells, thus establishing a novel approach to identify the regulatory variants that drove species-specific traits.

While hematopoietic stem cell (HSC) transplantation has become the standard of care for treating many hematologic diseases, wider use is restrained by immunologic incompatibility and inefficient reconstitution of hematopoiesis from allogeneic sources. Pluripotent stem cells (PSCs) promise an attractive alternative source of HSCs, but current PSC-derived HSCs display fetal phenotypes that lead to stem cell exhaustion and immature progeny function. We hypothesize that human PSC-derived definitive hemogenic endothelium (HE) can be efficiently converted to HSCs with adult-like functionality. To achieve this, we first propose to develop a robust reporter system that differentially marks fetal versus adult HSCs. By combining this with recently-developed technology to transgenically convert HE into fetal HSC-like cells, we can execute in vivo targeted and high-throughput genetic screens to identify TFs and pathways that regulate HSC maturity. We will subsequently find small molecules or morphogens that modulate activity of these regulatory factors to establish transgene-free conditions for the specification of adult-like HSCs. To ensure physiological relevance of the resultant cells, they will be assessed for long-term engraftment capability, functionality of their terminally-differentiated cells and appropriate response to stress conditions in vivo. This proposal will provide proof-of-concept that adult functionality can be conferred to PSC-derived cells, provide a sustainable platform for modeling adult hematologic diseases, and most importantly, bring us one step closer towards clinical grade PSC-derived HSCs for transplantation.

Tissue stem cells (SCs) depend upon their niche microenvironment, suggesting that spatially localized signals play key roles in determining SC identity and plasticity. Evidence from the hematopoietic system suggests that vasculature is a critical niche component. Vasculature also governs tumor-initiating SCs of squamous cell carcinomas. The spatial and temporal organization of capillary networks in healthy dermis suggests that vasculature also impacts normal hair follicle SC (HFSC) behavior, a notion further fueled by hair growth sensitivity to systemic changes. Despite several recent advances, the molecular basis by which a SC can sustain regeneration is still poorly understood. Here, I aim to perform the first spatiotemporal screening to dissect the molecular crosstalk between HFSCs and vasculature remodeling, and elucidate how crosstalk dictates ‘stemness’ identity to meet regenerative demands during normal hair cycling and wound-repair. Using mouse as model, I aim to tackle these objectives by: (1) Mapping and defining the spatial and temporal localization of the skin vasculature network relative to HFSCs during development and hair cycling. (2) Elucidate the molecular crosstalk between vasculature and HFSCs, and how it changes during capillary network remodeling. (3) Dissect mechanistically how vasculature-mediated signaling influences SC physiology during homeostasis and wound-repair by using in utero lentiviral delivery to skin epithelium of HFSC-specific activation of Cas9/guide gene silencing. By understanding how the vasculature dynamically redistributes and reshapes the niche, I hope to uncover new avenues for therapeutics in skin regeneration and wound-repair.

Cell survival requires efficient adaptation to external changes. Bacteria use complex signaling networks to sense and respond to these changes, particularly nutrient availability. The current knowledge of bacterial responses to carbon starvation is based mainly on E. coli and involves small molecule second messengers like cAMP. However, intracellular responses to starvation and cAMP regulation may differ in other species. The alpha-proteobacterium Caulobacter crescentus is a powerful model for understanding how intracellular signals are transduced and impact cell-cycle progression in bacteria. Carbon starvation somehow prevents cell differentiation and a G1-S transition during the Caulobacter cell cycle. This differentiation step is normally triggered by the synthesis of a regulator called SpmX. I propose that the absence of a carbon source prevents SpmX synthesis through the inhibition of its transcriptional regulator, TacA. I will use ChIP-qPCR and in vivo phosphorylation assays to test this model and dissect the mechanisms that temporally regulate SpmX. Further, I hypothesize that, like E. coli, the main intracellular signal following carbon starvation in Caulobacter is cAMP, and that cAMP directly regulates TacA activation. I will measure cAMP intracellular levels using a FRET-based method and assess the impact of cAMP on TacA activity and SpmX synthesis. I will also use capture-compound mass spectrometry to identify the cAMP interactome in Caulobacter. I anticipate that this study will provide new insights into cAMP-dependent cellular responses and nutrient-sensing mechanisms in Caulobacter, which may differ significantly from what is known in E. coli.

During development, embryonic tissues develop into organs of stereotyped size and shape. Morphogens are secreted from discrete regions in developing organs and form spatial concentration gradients that guide gene activation, pattern formation and tissue growth. Morphogen gradients scale with tissue size, ensuring that morphological patterns remain proportionate in organs of different size. The mechanisms of morphogen dispersal and scaling are not yet clear. Two models for morphogen spreading attracted much attention in the field: i) fast extracellular diffusion versus ii) cycles of internalization and recycling (transcytosis). I will develop a generalized theory of transport that captures extracellular diffusion and transcytosis simultaneously: I will consider extracellular diffusion, morphogen/receptor binding/unbinding and various endocytic trafficking steps. This novel theoretical method will uncover the parameter space where either extracellular diffusion or transcytosis dominates the dynamics of dispersal. I will also explore whether both phenomena could contribute significantly to the length scale of the steady-state gradient. My model system will be the Decapentaplegic (Dpp) gradient in the fly wing. Using novel experimental endocytic assays from the host lab, I will parameterize Dpp trafficking dynamics to establish the prevalence of extracellular diffusion versus transcytosis. Unpublished work from the host lab shows that trafficking is spatially heterogeneous in the wing. I will upgrade my theory to a heterogeneous scenario with the potential to yield, as an emerging property, a novel mechanism of gradient scaling.

The random selection of an allele to be exclusively expressed affects gene expression and any possible phenotypes related to the allele. This may be one of the key mechanisms through which phenotypic variation is established. Estimates of the prevalence of autosomal random monoallelic expression (aRME) vary widely between different experiments and cell types and importantly, the frequencies of fixed and dynamic aRME have not been addressed in the same experiments. I aim to address the balance between fixed and dynamic aRME in an in vivo as well as in vitro model. By combining single cell, allele sensitive transcriptomics approaches, which have been developed in the Sandberg lab, combined with an inducible cell-lineage reporter system, I will be uniquely able to characterize the extent of both fixed and dynamic aRME. We hypothesize that the balance between fixed and dynamic aRME depends on the stabilization of expression on a given allele. According to that model, fixed as well as dynamic aRME is caused by stochastic expression that is then stabilized to different extents. By studying this in vivo we will be able to characterize the stability of aRME in different tissues where aRME stabilization may differ (eg. neurons vs. fibroblasts). The in vitro model on the other hand would give me the possibility to perturb the system and study the mechanisms that establish and stabilize aRME. Additionally, the data resulting from the experiments I propose will also provide a novel and unique insight into the relationship between gene expression and cell clonality in a wide range of mouse tissues and cell types.

Chemical probes are small molecules that can perturb the activity of a target macromolecule, and thus can be used to understand the biological roles of receptors. Currently, identifying a chemical probe for one specific target requires screening many chemicals to find ones that bind that target. If one screen could be used to find chemical probes for many targets, one could dramatically increase the rate of probe discovery.

Our first goal is to identify novel chemical probes for approximately 40 receptors in S. cerevisiae. This will be done using a novel high throughput metabolomics approach. Recent advances in the Sauer lab allow for the speedy generation of a metabolomic fingerprint of the effect of a compound treatment on the cell. Therefore it should be possible to adopt an innovative approach to chemical screening where compounds are identified that are able to mimic the metabolomic profile of yeast where receptors are genetically perturbed. This will allow us to find chemical probes for those targets. Thus, we will be able to identify many chemical probes for many receptors from a single chemical screen.

Our second goal will be to investigate the roles of these receptors in vivo using our chemical probes. We will investigate what specific metabolomic and transcriptomic changes individual S. cerevisiae nutrient sensors elicit when they activated by a non-metabolizable ligand. This is of interest because it can isolate what the cellular response to receptor activation is, and potentially show why it is adaptive for the cell to respond in that way. Additionally, we will use them to investigate the biological roles of poorly characterized receptors in S. cerevisiae.

The neural circuits underlying vision share a lot of structural and functional similarities between Drosophila and mammals, with motion detection being the best-studied visual modality. The highly stereotyped columnar and layered organization of Drosophila optic lobe neuropils (the visual processing centres) allows a precise study of circuit wiring. Two parallel motion channels have been described in Drosophila, one for detecting moving light edges (ON pathway) and one for detecting moving dark edges (OFF pathway). The main components of these channels have been identified, comprising neurons in the lamina, medulla, lobula and lobula plate neuropils. However, little is known about the specific mechanisms leading to the orderly and stereotypic assembly of these cell types to form functional circuits. In this proposal we offer to study by live imaging the spatiotemporal dynamics of motion detection circuit assembly and the determinants of such formation. It has been proposed that circuits are assembled in the order of neuron generation. To test this hypothesis, we will analyse the assembly order of ON and OFF motion circuit components and study how the developmental specification of cell types is linked to circuit formation. Furthermore, we will investigate whether there are neurons with pioneer roles in circuit assembly, the influence of neuronal soma positioning on axonal and dendritic layer targeting, and the role of spontaneous activity in circuit wiring. Altogether, this project will advance our understanding of the mechanisms governing functional circuitry assembly, which is likely to have broader implications for other neuronal systems.

Demography (i.e. a population’s history of size changes, gene flow, and divergence events) affects patterns of genetic diversity, and has implications for studying evolution, adaptations, and disease. However, demographic inference is complicated by “ghost populations,” unobserved populations in the past that admixed with the ancestors of sampled populations. Inferring this hidden structure is challenging both because the number of ghost populations is unknown, and also because signals of cryptic structure may be distorted by other evolutionary forces. I will address this problem by developing a flexible method capable of estimating unknown population structure jointly with other sources of genetic diversity. Our method will combine models from Coalescent theory with machine learning techniques for inferring graph structure, and will estimate the number of ghost populations, their demographic history, and the distribution of fitness effects for mutations arising in these ghost populations. Furthermore, our approach will allow us to estimate allele frequencies in ancestral populations, which will be useful for identifying adaptive introgressed regions from ghost populations, and for addressing questions such as the adaptive consequences of gene flow from ghost populations. We will apply our new method to diverse human whole-genomes to shed light on open questions in recent human evolutionary history, such as the relationship between modern African populations and the out-of-Africa population that gave rise to modern Europeans and Asians, or the extent to which variants selected in particular populations have spread more broadly by admixture and introgression.

Propagation of a-synuclein in the brain is the hallmark feature in the pathogenesis of Parkinson’s disease (PD). Patients with PD exhibit marked differences in the rate of a-synuclein propagation, but the reasons for this variability are unknown. I hypothesize that the rate of a-synuclein propagation varies because it is affected by various genetic modifiers. In addition, I hypothesize that individuals with PD are mosaics for a-synuclein mutations in the brain, a type of genetic variation that is missed by standard screening methods, which further impacts on a-synuclein propagation. In order to investigate these hypotheses, I am going to generate human iPSc-derived neuronal cultures expressing a construct in which mutant a-synuclein that is fused with mCherry on its C-terminus is also fused to a GFP tag on its N-terminus through a 2A self-cleaving peptide. Neuronal cultures expressing wild type (WT) a-synuclein fused with BFP on its C-terminus will also be generated. I am then going to mix these neuronal cultures to generate cultures of various mosaic percentages for the a-synuclein mutation. This system will enable the identification of single neurons that have uptaken WT or mutant a-synuclein through propagation based on their positivity to GFP, mCherry and/or BFP. I am then going to use a CRISPR-Cas9 high throughput screening system combined with high content imaging techniques developed at the host institution to assess the effect of thousands of genetic factors on the rate of propagation in the mosaic neuronal cultures, followed by RNA sequencing to determine molecular signatures that underlie the propagation process and that are specific for each genetic modifier.

My project aims to identify neuronal circuits that are spared with severe spinal cord injury and function as motor command relay pathways during reactivation of the lumbar spinal cord. I will use a neuroprosthetic platform that supports bipedal stepping and implant an electro-chemotrode over the lumbar spinal cord of mice. First, I will optogenetically activate motor cortex projections in severely contused mice and assess whether immediate will-powered motor control is regained over completely paralysed hindlimbs during spinal stimulations (Aim 1). Next, I will use a combination of conventional tracers, such as Fast blue, and viral tracers that are regulated by a Cre promoter in vGluT2 Cre or 5-HT Cre transgenic mice to label and identify brainstem projections to the lumbar spinal cord. Moreover, I will prove these brainstem projections are required to rely motor cortex commands by chemogenetically silencing them with the DREADD technology, which should abolish any leg motor function (Aim 2). Mice will undergo 8 weeks of actively engaging neurorehabilitation with spinal electrochemical stimulation. I will assess recovery of will-powered voluntary motor function and remodeling of spared pathways (Aim 3). I will laser-microdissect cell bodies of motor command relay neurons in the brainstem and assess the transcriptional profile with RNA sequencing to identify which molecular cues are involved in circuit reorganization (Aim 4). Taken together, these results will establish the causal relationship between circuit reorganisation of residual descending projections and voluntary motor control with electrochemical neurorehabilitation in severe spinal contusion injuries.

Cell division and differentiation are strictly coordinated during development. Although it is known that differences in S-phase length are essential for embryogenesis our understanding of its causes and consequences is extremely poor. The second cell division of C. elegans is stringently asynchronous and is well-suited to study developmental control of replication regulation in a multi-cellular organism.
Studying replication initiation is limited by the availability of specific assays. I will develop a novel live assay to visualise the recruitment of replication factors towards origins in single cells within the C. elegans embryo. With the LacO/LacI system I will generate functional origins and measure the binding of fluorescently tagged replication factors by confocal imaging dissecting consecutive steps of DNA replication. This assay will permit to follow the dynamics of replication factors in real-time and to reveal changes in S-phase control during development. On this basis, I will perform an RNAi-based high-throughput screen for novel regulators of genome duplication in the C. elegans embryo. This unique screen implemented in a whole organism will reveal novel interactions between DNA replication and developmental signalling pathways. In a complementary approach I will generate mutants bypassing the normal cell cycle regulation in the embryo. I will examine potential changes in cell fate adoption through transcriptional analysis and will measure differences in their sites of replication. This approach will clarify if replication control plays a direct role in differentiation and illustrate a physiological role for S-phase differences in development.

It has been postulated that cancer stem cells (CSC), an identifiable subpopulation of cancer cell that can differentiate and repopulate cancer, exist. In this context, cancer cells follow strict hierarchy with CSC being at the apex of it. However, depending on the subtype and malignancy of cancer, cell fate plasticity had been observed where non-CSC dedifferentiates to acquire CSC function. However, the nature and regulation of CSC formation and plasticity has been difficult to understand in vivo. For example, what kind of niche signaling is responsible for cancer cell plasticity? What is the epigenetic or transcriptomic changes in cancer cell plasticity? In this project, I aim to address these questions in vivo using highly novel technique involving transgenic mice carrying recombinase-based state machine (RSM), which enables each individual cell within cancer to keep a memory of its history and sequence of changes in cell fate. The proposal will involve generation and validation of mammalian RSM that can label each cell with different cell fate history (e.g. original CSC and non-CSC that had dedifferentiated to CSC) with different fluorescent proteins. I will then cross the transgenic mice harboring RSM with mouse models of breast cancer and colon cancer can accurately identify heterogeneity in individual cancer cell fate in vivo and in situ. By examining the relative locations of cancer cell fate plasticity in intact tumor mass, and FACS sorting of cells with different fate history for transcriptomic or epigenetic analysis, I aim to dissect the extracellular factors and intracellular signaling events that govern cancer cell plasticity in vivo.

Direction selectivity is a feature of selected neurons in the visual cortex that is conserved in evolution. The emergence of cortical direction selectivity has been extensively studied but its circuit mechanism is still debated. My goal is to understand the circuit mechanism of cortical direction selectivity that is independent of retinal direction selectivity. I plan to approach this by using two animal models in which retinal direction selectivity has been disrupted. I will investigate the neuronal circuits of those cortical direction-selective neurons which retain their specificity in these animal models by performing single-cell-initiated functionalized rabies tracing and single-cell electrophysiological recordings. I will ask the three following questions: Do axon terminals of the principal cells of the lateral geniculate nucleus (LGN) fully lose direction selectivity after the abolishment of retinal direction selectivity, or could cortical feedback from layer 6 entrain direction selectivity in a subset of LGN cell axon terminals? What is the synaptic input profile underlying retina-independent cortical direction selectivity in pyramidal neurons? What is the contribution of the local presynaptic networks to the direction-selective tuning of pyramidal neurons with retina-independent cortical direction selectivity? This work will provide insights into how cortical selectively for environmental features arises.

Eukaryotic genomes are organized into a highly-compacted nucleoprotein complex known as chromatin. Hi-C and high-resolution microscopy studies reveal organization of chromosomes at multiple levels, from chromosome territories, to MB-scale functional domains that are spatially separated from each other, to shorter contact domains often called topological associated domains (TADs). While genetic studies in cell culture coupled with Hi-C or high-resolution microscopy reveal key roles for a variety of factors in higher-order chromatin folding, our understanding of chromatin fiber folding largely derives from biochemical studies in vitro. Mostly, these in vitro studies rely on artificial, homogenous chromatin templates that do not reflect the heterogeneous local folding properties of in vivo chromatin.
The aim of this project is to approach higher-order chromatin folding and chromosome organization biochemically. Micro-C detects internucleosomal interactions, which refer to local chromosome folding, by identifying nucleosomal DNA sequences and is therefore equally suitable for in vivo and in vitro studies. I will use chromatin prepared from cells (ex vivo) and in vivo-like in vitro reconstituted chromatin for chromatin compaction studies. With Micro-C I will be able to measure autonomous salt-dependent chromatin folding driven by all endogenous factors (ex vivo chromatin) or solely by histone-DNA interactions. Biochemical manipulation will allow to remove or test individual candidate factors.
This biochemical approach will help to decipher the regulatory role of higher-order chromatin organization on DNA templated processes, such as gene regulation.

Synthetic approaches toward redesigning cellular circuits and signaling networks have accelerated our understanding of sophisticated cellular behaviors. In particular, exploiting optogenetic approaches on neurons to manipulate neuronal activities have significantly expanded our comprehension on the nature of neuronal circuitry, whereas those approaches were applied much less in immune cells. Indeed, repurposed and/or engineered immune cells have been successfully used as cancer treatments, but they are only sometimes successful and can have harmful side effects. We hypothesize that these attempts have been limited because they have been restricted to a limited number of wild-type like signaling inputs. Here, through evolution-based approaches we intend to engineer chimeric antigen receptors (CARs) in T cells as a model system to answer a simple, yet, fundamental question regarding finding the maximal potency and unique phenotypes of T cell-based immune responses. Also, we offer a generalizable design scheme for safely controlling CARs through an optogenetic module. Newly engineered CARs could be of great use in eradicating cancer, and unique combinations and sequences of T cell signaling components could provide unexplained mechanisms and further understanding of the evolutionary nature of designed immune system. In order to achieve the goal, we build CAR intracellular domain libraries, and with a properly designed activity-based selection strategy we anticipate to find novel classes of CARs with extraordinary potency of immune responses.

It has been a long-standing question what mechanisms counter antibiotic resistance in nature. Are there any widespread multidrug strategies in soil bacteria for avoiding, inhibiting, or bypassing resistance mechanisms against antibiotics, whose biosynthetic pathways evolved a hundred million years ago, but still exist across natural microbial communities? One of the well-known examples of naturally occurring combination strategies is the co-production of beta-lactam antibiotics and beta-lactamase inhibitor in Streptomyces spp. which has led to the development of a potent drug combination for clinical use known as Augmentin. However, our knowledge on this issue is extremely limited due to the shortage of systematic investigations. It is important to emphasize that the extraordinary capacity of soil bacteria to produce multiple natural products may reflect an ecological advantage for them. Therefore, I hypothesize that a systematic investigation of the combinatorial biosynthesis of secondary metabolites, including antibiotics and non-ribosomal antimicrobial peptides, could reveal novel drug combinations. First, using state-of-the-art computational approaches I will explore the co-occurrence of biosynthetic genes of known secondary metabolites and antibiotics in the genome sequence of soil microorganisms which presumably co-evolved under strong selection pressure. Second, I will explore the capacity of novel candidate combinations i) to restore susceptibility of the resistant bacteria, ii) to synergistically inhibit bacterial growth or iii) to invert the selective advantage of resistance mutations.

Plants have evolved sophisticated mechanisms to recognize non-self molecules, allowing them to deploy effective immune reactions against a myriad of pathogens. Sometimes, however, plants mistakenly identify their own molecules as foreign and induce autoimmunity, causing severe growth defects including plant death. Autoimmunity is a hallmark of a syndrome that is seen in certain intra- and interspecific hybrids and that is known as hybrid necrosis. A particularly interesting case in Arabidopsis thaliana is that of the RPP7 and RPW8 loci, where three different pairs of alleles from different wild strains interact in F1 hybrids to cause hybrid necrosis. RPP7, which confers resistance against oomycetes, encodes members of the well-studied family of NLR immune receptors. The biochemical function of the RPW8 coiled-coil domain proteins that primarily confer resistance to fungi is less well understood than that of NLRs. I propose to reveal the mechanisms by which interactions between RPP7 and RPW8 proteins activate immune responses, taking advantage of the allele-specificity of these interactions. By interpreting these findings in the context of normal immunity, my work will not only advance our understanding of genetic incompatibility and autoimmunity, but will also contribute to optimizing combinations of favorable immune alleles in crop breeding.

Cells are constantly making millions of proteins that need to be synthesized correctly, folded, trafficked, and assembled to their functional state. All of these steps are monitored by the cells for mistakes or failures, and these failed products are degraded to avoid their accumulation. These quality control (QC) pathways are emerging as major contributors to neurodegeneration. The newly discovered ribosome-associated quality control (RQC) pathway represents one of the most cleverly imposed QC pathways to allow cotranslational surveillance of erroneous translation products. Recent studies have identified the factors involved in RQC pathway and defined its primary steps: recognition and splitting of stalled ribosomes into subunits, signaling of the heat shock response, ubiquitination of nascent chains, and extraction of polyubiquitinated nascent chain from the 60S subunit for degradation. I propose to dissect the mechanism of two unresolved steps of the RQC pathway and define its endogenous clients. Aim1 will investigate an essential step in signaling the heat shock response: the addition of C-terminal Alanine and Threonine (CAT) tails to stalled nascent chains. I will reconstitute this unusual mRNA-independent polypeptide elongation reaction in vitro to identify the minimum required components, define the energy source, and order the primary steps of this process. Aim2 will reconstitute nascent chain extraction in vitro to determine how a stalled polypeptide is delivered from the ribosome to the proteasome. Aim3 will identify the physiological clients of the RQC by trapping in vivo intermediates and identifying associated mRNAs or partially synthesized nascent chains.