PTBP1 mediates Sertoli cell actin cytoskeleton organization by regulating alternative splicing of actin regulators.

Spermatogenesis is a biological process within the testis that produces haploid spermatozoa for the continuity of species. Sertoli cells are somatic cells in the seminiferous epithelium that orchestrate spermatogenesis. Cyclic reorganization of Sertoli cell actin cytoskeleton is vital for spermatogenesis, but the underlying mechanism remains largely unclear. Here, we report that RNA-binding protein PTBP1 controls Sertoli cell actin cytoskeleton reorganization by programming alternative splicing of actin cytoskeleton regulators. This splicing control enables ectoplasmic specializations, the actin-based adhesion junctions, to maintain the blood-testis barrier and support spermatid transport and transformation. Particularly, we show that PTBP1 promotes actin bundle formation by repressing the inclusion of exon 14 of Tnik, a kinase present at the ectoplasmic specialization. Our results thus reveal a novel mechanism wherein Sertoli cell actin cytoskeleton dynamics is controlled post-transcriptionally by utilizing functionally distinct isoforms of actin regulatory proteins, and PTBP1 is a critical regulatory factor in generating such isoforms.

Emerging roles of RNA-binding proteins in fatty liver disease.

A rampant and urgent global health issue of the 21st century is the emergence and progression of fatty liver disease (FLD), including alcoholic fatty liver disease and the more heterogenous metabolism-associated (or non-alcoholic) fatty liver disease (MAFLD/NAFLD) phenotypes. These conditions manifest as disease spectra, progressing from benign hepatic steatosis to symptomatic steatohepatitis, cirrhosis, and, ultimately, hepatocellular carcinoma. With numerous intricately regulated molecular pathways implicated in its pathophysiology, recent data have emphasized the critical roles of RNA-binding proteins (RBPs) in the onset and development of FLD. They regulate gene transcription and post-transcriptional processes, including pre-mRNA splicing, capping, and polyadenylation, as well as mature mRNA transport, stability, and translation. RBP dysfunction at every point along the mRNA life cycle has been associated with altered lipid metabolism and cellular stress response, resulting in hepatic inflammation and fibrosis. Here, we discuss the current understanding of the role of RBPs in the post-transcriptional processes associated with FLD and highlight the possible and emerging therapeutic strategies leveraging RBP function for FLD treatment. This article is categorized under: RNA in Disease and Development > RNA in Disease.

NK cell expansion requires HuR and mediates control of solid tumors and long-term virus infection.

Natural killer (NK) cells are lymphocytes capable of controlling tumors and virus infections through direct lysis and cytokine production. While both T and NK cells expand and accumulate in affected tissues, the role of NK cell expansion in tumor and viral control is not well understood. Here, we show that posttranscriptional regulation by the RNA-binding protein HuR is essential for NK cell expansion without negatively affecting effector functions. HuR-deficient NK cells displayed defects in the metaphase of the cell cycle, including decreased expression and alternative splicing of Ska2, a component of the spindle and kinetochore complex. HuR-dependent NK cell expansion contributed to long-term cytomegalovirus control and facilitated control of subcutaneous tumors but not tumor metastases in two independent tumor models. These results show that posttranscriptional regulation by HuR specifically affects NK cell expansion, which is required for the control of long-term virus infection and solid tumors, but not acute infection or tumor metastases, highlighting fundamental differences with antigen-specific T cell control.

The discovery, function, and regulation of epithelial splicing regulatory proteins (ESRP) 1 and 2.

Alternative splicing is a broad and evolutionarily conserved mechanism to diversify gene expression and functionality. The process relies on RNA binding proteins (RBPs) to recognize and bind target sequences in pre-mRNAs, which allows for the inclusion or skipping of various alternative exons. One recently discovered family of RBPs is the epithelial splicing regulatory proteins (ESRP) 1 and 2. Here, we discuss the structure and physiological function of the ESRPs in a variety of contexts. We emphasize the current understanding of their splicing activities, using the classic example of fibroblast growth factor receptor 2 mutually exclusive splicing. We also describe the mechanistic roles of ESRPs in coordinating the splicing and functional output of key signaling pathways that support the maintenance of, or shift between, epithelial and mesenchymal cell states. In particular, we highlight their functions in the development of mammalian limbs, the inner ear, and craniofacial structure while discussing the genetic and biochemical evidence that showcases their conserved roles in tissue regeneration, disease, and cancer pathogenesis.

PTBP1 controls intestinal epithelial regeneration through post-transcriptional regulation of gene expression.

The intestinal epithelial regeneration is driven by intestinal stem cells under homeostatic conditions. Differentiated intestinal epithelial cells, such as Paneth cells, are capable of acquiring multipotency and contributing to regeneration upon the loss of intestinal stem cells. Paneth cells also support intestinal stem cell survival and regeneration. We report here that depletion of an RNA-binding protein named polypyrimidine tract binding protein 1 (PTBP1) in mouse intestinal epithelial cells causes intestinal stem cell death and epithelial regeneration failure. Mechanistically, we show that PTBP1 inhibits neuronal-like splicing programs in intestinal crypt cells, which is critical for maintaining intestinal stem cell stemness. This function is achieved at least in part through promoting the non-productive splicing of its paralog PTBP2. Moreover, PTBP1 inhibits the expression of an AKT inhibitor PHLDA3 in Paneth cells and permits AKT activation, which presumably maintains Paneth cell plasticity and function in supporting intestinal stem cell niche. We show that PTBP1 directly binds to a CU-rich region in the 3' UTR of Phlda3, which we demonstrate to be critical for downregulating the mRNA and protein levels of Phlda3. Our results thus reveal the multifaceted in vivo regulation of intestinal epithelial regeneration by PTBP1 at the post-transcriptional level.

Splicing factor SRSF1 deficiency in the liver triggers NASH-like pathology and cell death.

Regulation of RNA processing contributes profoundly to tissue development and physiology. Here, we report that serine-arginine-rich splicing factor 1 (SRSF1) is essential for hepatocyte function and survival. Although SRSF1 is mainly known for its many roles in mRNA metabolism, it is also crucial for maintaining genome stability. We show that acute liver damage in the setting of targeted SRSF1 deletion in mice is associated with the excessive formation of deleterious RNA-DNA hybrids (R-loops), which induce DNA damage. Combining hepatocyte-specific transcriptome, proteome, and RNA binding analyses, we demonstrate that widespread genotoxic stress following SRSF1 depletion results in global inhibition of mRNA transcription and protein synthesis, leading to impaired metabolism and trafficking of lipids. Lipid accumulation in SRSF1-deficient hepatocytes is followed by necroptotic cell death, inflammation, and fibrosis, resulting in NASH-like liver pathology. Importantly, SRSF1-depleted human liver cancer cells recapitulate this pathogenesis, illustrating a conserved and fundamental role for SRSF1 in preserving genome integrity and tissue homeostasis. Thus, our study uncovers how the accumulation of detrimental R-loops impedes hepatocellular gene expression, triggering metabolic derangements and liver damage.

Cellular and molecular profiles of larval and adult Xenopus corneal epithelia resolved at the single-cell level.

Corneal Epithelial Stem Cells (CESCs) and their proliferative progeny, the Transit Amplifying Cells (TACs), are responsible for homeostasis and maintaining corneal transparency. Owing to our limited knowledge of cell fates and gene activity within the cornea, the search for unique markers to identify and isolate these cells remains crucial for ocular surface reconstruction. We performed single-cell RNA sequencing of corneal cells from larval and adult stages of Xenopus. Our results indicate that as the cornea develops and matures, there is an increase in cellular diversity, which is accompanied by a substantial shift in transcriptional profile, gene regulatory network and cell-cell communication dynamics. Our data also reveals several novel genes expressed in corneal cells and changes in gene expression during corneal differentiation at both developmental time-points. Importantly, we identify specific basal cell clusters in both the larval and adult cornea that comprise a relatively undifferentiated cell type and express distinct stem cell markers, which we propose are the putative larval and adult CESCs, respectively. This study offers a detailed atlas of single-cell transcriptomes in the frog cornea. In the future, this work will be useful to elucidate the function of novel genes in corneal epithelial homeostasis, wound healing and regeneration.

SimiC enables the inference of complex gene regulatory dynamics across cell phenotypes.

Single-cell RNA-Sequencing has the potential to provide deep biological insights by revealing complex regulatory interactions across diverse cell phenotypes at single-cell resolution. However, current single-cell gene regulatory network inference methods produce a single regulatory network per input dataset, limiting their capability to uncover complex regulatory relationships across related cell phenotypes. We present SimiC, a single-cell gene regulatory inference framework that overcomes this limitation by jointly inferring distinct, but related, gene regulatory dynamics per phenotype. We show that SimiC uncovers key regulatory dynamics missed by previously proposed methods across a range of systems, both model and non-model alike. In particular, SimiC was able to uncover CAR T cell dynamics after tumor recognition and key regulatory patterns on a regenerating liver, and was able to implicate glial cells in the generation of distinct behavioral states in honeybees. SimiC hence establishes a new approach to quantitating regulatory architectures between distinct cellular phenotypes, with far-reaching implications for systems biology.

Nuclear receptors FXR and SHP regulate protein N-glycan modifications in the liver.

Nuclear receptors farnesoid X receptor (FXR) and small heterodimer partner (SHP) are key regulators of metabolism. Here, we report a previously unknown function for the hepatic FXR-SHP axis in controlling protein N-linked glycosylation. Transcriptome analysis in liver-specific Fxr-Shp double knockout (LDKO) livers revealed induction of genes encoding enzymes in the N-glycosylation pathway, including Mgat5, Fut8, St3gal6, and St6gal1 FXR activation suppressed Mgat5, while Shp deletion induced St3gal6 and St6gal1 Increased percentages of core-fucosylated and triantennary glycan moieties were seen in LDKO livers, and proteins with the "hyperglycoforms" preferentially localized to exosomes and lysosomes. This up-regulation of N-glycosylation machinery was specific to the Golgi apparatus and not the endoplasmic reticulum. The increased glycan complexity in the LDKO correlated well with dilated unstacked Golgi ribbons and alterations in the secretion of albumin, cholesterol, and triglycerides. Our findings demonstrate a role for the FXR-SHP axis in maintaining glycoprotein diversity in the liver.

Cellular plasticity balances the metabolic and proliferation dynamics of a regenerating liver.

The adult liver has an exceptional ability to regenerate, but how it maintains its specialized functions during regeneration is unclear. Here, we used partial hepatectomy (PHx) in tandem with single-cell transcriptomics to track cellular transitions and heterogeneities of ∼22,000 liver cells through the initiation, progression, and termination phases of mouse liver regeneration. Our results uncovered that, following PHx, a subset of hepatocytes transiently reactivates an early-postnatal-like gene expression program to proliferate, while a distinct population of metabolically hyperactive cells appears to compensate for any temporary deficits in liver function. Cumulative EdU labeling and immunostaining of metabolic, portal, and central vein-specific markers revealed that hepatocyte proliferation after PHx initiates in the midlobular region before proceeding toward the periportal and pericentral areas. We further demonstrate that portal and central vein proximal hepatocytes retain their metabolically active state to preserve essential liver functions while midlobular cells proliferate nearby. Through combined analysis of gene regulatory networks and cell-cell interaction maps, we found that regenerating hepatocytes redeploy key developmental regulons, which are guided by extensive ligand-receptor-mediated signaling events between hepatocytes and nonparenchymal cells. Altogether, our study offers a detailed blueprint of the intercellular crosstalk and cellular reprogramming that balances the metabolic and proliferative requirements of a regenerating liver.

Unbiased proteomic screening identifies a novel role for the E3 ubiquitin ligase Nedd4-2 in translational suppression during ER stress.

Endoplasmic reticulum (ER) stress occurs when protein folding or maturation is disrupted. A malfunction in the ER stress response can lead to cell death and has been observed in many neurological diseases. However, how the ER stress response is regulated in neuronal cells remains largely unclear. Here, we studied an E3 ubiquitin ligase named neural precursor cell expressed developmentally down-regulated protein 4-like (Nedd4-2). Nedd4-2 is highly expressed in the brain and has a high affinity toward ubiquitinating membrane-bound proteins. We first utilized unbiased proteomic profiling with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) of isolated membrane fractions from mouse whole brains to identify novel targets of Nedd4-2. Through this screen, we found that the expression and ubiquitination of ribosomal proteins are regulated by Nedd4-2 and we confirmed an association between Nedd4-2 and ribosomes through ribosome sedimentation and polysome profiling. Further, we utilized immunoprecipitation and western blotting to show that induction of ER stress promotes an association between Nedd4-2 and ribosomal proteins, which is mediated through dephosphorylation of Nedd4-2 at serine-342. This increased interaction between Nedd4-2 and ribosomal proteins in turn mediates ER stress-associated translational suppression. In summary, the results of this study demonstrate a novel regulatory mechanism underlying the ER stress response and a novel function of Nedd4-2 in translational control. Our findings may shed light on neurological diseases in which the ER stress response or the function of Nedd4-2 is dysregulated.

Transcriptomic analysis across liver diseases reveals disease-modulating activation of constitutive androstane receptor in cholestasis.

BACKGROUND & AIMS: Liver diseases are caused by many factors, such as genetics, nutrition, and viruses. Therefore, it is important to delineate transcriptomic changes that occur in various liver diseases. METHODS: We performed high-throughput sequencing of mouse livers with diverse types of injuries, including cholestasis, diet-induced steatosis, and partial hepatectomy. Comparative analysis of liver transcriptome from mice and human samples of viral infections (HBV and HCV), alcoholic hepatitis (AH), non-alcoholic steatohepatitis (NASH), and biliary atresia revealed distinct and overlapping gene profiles associated with liver diseases. We hypothesised that discrete molecular signatures could be utilised to assess therapeutic outcomes. We focused on cholestasis to test and validate the hypothesis using pharmacological approaches. RESULTS: Here, we report significant overlap in the expression of inflammatory and proliferation-related genes across liver diseases. However, cholestatic livers were unique and displayed robust induction of genes involved in drug metabolism. Consistently, we found that constitutive androstane receptor (CAR) activation is crucial for the induction of the drug metabolic gene programme in cholestasis. When challenged, cholestatic mice were protected against zoxazolamine-induced paralysis and acetaminophen-induced hepatotoxicity. These protective effects were diminished upon inhibition of CAR activity. Further, drug metabolic genes were also induced in the livers from a subset of biliary atresia patients, but not in HBV and HCV infections, AH, or NASH. We also found a higher expression of CYP2B6, a CAR target, in the livers of biliary atresia patients, underscoring the clinical importance of our findings. CONCLUSIONS: Comparative transcriptome analysis of different liver disorders revealed specific induction of phase I and II metabolic genes in cholestasis. Our results demonstrate that CAR activation may lead to variations in drug metabolism and clinical outcomes in biliary atresia. LAY SUMMARY: Transcriptomic analysis of diverse liver diseases revealed alterations in common and distinct pathways. Specifically, in cholestasis, we found that detoxification genes and their activity are increased. Thus, cholestatic patients may have an unintended consequence on drug metabolism and not only have a beneficial effect against liver toxicity, but also may require adjustments to their therapeutic dosage.

Antagonism between splicing and microprocessor complex dictates the serum-induced processing of lnc-MIRHG for efficient cell cycle reentry.

Cellular quiescence and cell cycle reentry regulate vital biological processes such as cellular development and tissue homeostasis and are controlled by precise regulation of gene expression. The roles of long noncoding RNAs (lncRNAs) during these processes remain to be elucidated. By performing genome-wide transcriptome analyses, we identify differential expression of several hundreds of lncRNAs, including a significant number of the less-characterized class of microRNA-host-gene (MIRHG) lncRNAs or lnc-MIRHGs, during cellular quiescence and cell cycle reentry in human diploid fibroblasts. We observe that MIR222HG lncRNA displays serum-stimulated RNA processing due to enhanced splicing of the host nascent pri-MIR222HG transcript. The pre-mRNA splicing factor SRSF1 negatively regulates the microprocessor-catalyzed cleavage of pri-miR-222, thereby increasing the cellular pool of the mature MIR222HG Association of SRSF1 to pri-MIR222HG, including to a mini-exon, which partially overlaps with the primary miR-222 precursor, promotes serum-stimulated splicing over microRNA processing of MIR222HG Further, we observe that the increased levels of spliced MIR222HG in serum-stimulated cells promote the cell cycle reentry post quiescence in a microRNA-independent manner. MIR222HG interacts with DNM3OS, another lncRNA whose expression is elevated upon serum-stimulation, and promotes cell cycle reentry. The double-stranded RNA binding protein ILF3/2 complex facilitates MIR222HG:DNM3OS RNP complex assembly, thereby promoting DNM3OS RNA stability. Our study identifies a novel mechanism whereby competition between the splicing and microprocessor machinery modulates the serum-induced RNA processing of MIR222HG, which dictates cell cycle reentry.

Effect of PFOA on DNA Methylation and Alternative Splicing in Mouse Liver.

Perfluorooctanoic acid (PFOA) is a persistent organic pollutant prevalent in the environment and implicated in damage to the liver leading to a fatty liver phenotype called hepatocellular steatosis. Our goal is to provide a basis for PFOA-induced hepatocellular steatosis in relation to epigenetic alterations and mRNA splicing. Young adult female mice exposed to different concentrations of PFOA showed an increase in liver weight with decreased global DNA methylation (5-mC). At higher concentrations, the expression of DNA methyltransferase 3A (Dnmt3a) was significantly reduced and the expression of tet methycytosine dioxygenase 1 (Tet1) was significantly increased. There was no significant change in the other Dnmts and Tets. PFOA exposure significantly increased the expression of cell cycle regulators and anti-apoptotic genes. The expression of multiple genes involved in mTOR (mammalian target of rapamycin) signaling pathway were altered significantly with reduction in Pten (phosphatase and tensin homolog, primary inhibitor of mTOR pathway) expression. Multiple splicing factors whose protein but not mRNA levels affected by PFOA exposure were identified. The changes in protein abundance of the splicing factors was also reflected in altered splicing pattern of their target genes, which provided new insights on the previously unexplored mechanisms of PFOA-mediated hepatotoxicity and pathogenesis.

Complementary Oligonucleotide Conjugated Multicolor Carbon Dots for Intracellular Recognition of Biological Events.

By using complementary DNA sequences as surface ligands, we selectively allow two individual diffusing "dual-color" carbon dots to interact in situ and in vitro. Spontaneous nanoscale oxidation of surface-abundant nitroso-/nitro-functionalities leads to two distinctly colored carbon dots (CD) which are isolated by polarity driven chromatographic separation. Green- and red-emitting carbon dots (gCD and rCD) were decorated by complementary single-stranded DNAs which produce a marked increase in the fluorescence emission of the respective carbon dots. Mutual colloidal interactions are achieved through hybridization of complementary DNA base pairs attached to the respective particles, resulting in quenching of their photoluminescence. The observed post-hybridization quenching is presumably due to a combined effect from an aggregation of CDs post duplex DNA formation and close proximity of multicolored CDs, having overlapped spectral regions leading to a nonradiative energy transfer process possibly released as heat. This strategy may contribute to the rational design of mutually interacting carbon dots for a better control over the resulting assembly structure for studying different biological phenomenon including molecular cytogenetics. One of the newly synthesized CDs was successfully used to image intracellular location of GAPDH mRNA using an event of change in fluorescence intensity (FI) of CDs. This selectivity was introduced by conjugating an oligonucleotide harboring complementary sequence to GAPDH mRNA. FI of this conjugated carbon dot, rCD-GAPDH, was also found to decrease in the presence of Ca2+, varied in relation to H+ concentrations, and could serve as a tool to quantify the intracellular concentrations of Ca2+ and pH value (H+) which can give important information about cell survival. Therefore, CD-oligonucleotide conjugates could serve as efficient probes for cellular events and interventions.

Aberrant Expression of a Non-muscle RBFOX2 Isoform Triggers Cardiac Conduction Defects in Myotonic Dystrophy.

Myotonic dystrophy type 1 (DM1) is a multisystemic genetic disorder caused by the CTG repeat expansion in the 3'-untranslated region of DMPK gene. Heart dysfunctions occur in ∼80% of DM1 patients and are the second leading cause of DM1-related deaths. Herein, we report that upregulation of a non-muscle splice isoform of RNA-binding protein RBFOX2 in DM1 heart tissue-due to altered splicing factor and microRNA activities-induces cardiac conduction defects in DM1 individuals. Mice engineered to express the non-muscle RBFOX240 isoform in heart via tetracycline-inducible transgenesis, or CRISPR/Cas9-mediated genome editing, reproduced DM1-related cardiac conduction delay and spontaneous episodes of arrhythmia. Further, by integrating RNA binding with cardiac transcriptome datasets from DM1 patients and mice expressing the non-muscle RBFOX2 isoform, we identified RBFOX240-driven splicing defects in voltage-gated sodium and potassium channels, which alter their electrophysiological properties. Thus, our results uncover a trans-dominant role for an aberrantly expressed RBFOX240 isoform in DM1 cardiac pathogenesis.

Cellular and molecular basis of liver regeneration.

Recent advances in genetics and genomics have reinvigorated the field of liver regeneration. It is now possible to combine lineage-tracing with genome-wide studies to genetically mark individual liver cells and their progenies and detect precise changes in their genome, transcriptome, and proteome under normal versus regenerative settings. The recent use of single-cell RNA sequencing methodologies in model organisms has, in some ways, transformed our understanding of the cellular and molecular biology of liver regeneration. Here, we review the latest strides in our knowledge of general principles that coordinate regeneration of the liver and reflect on some conflicting evidence and controversies surrounding this topic. We consider the prominent mechanisms that stimulate homeostasis-related vis-à-vis injury-driven regenerative responses, highlight the likely cellular sources/depots that reconstitute the liver following various injuries and discuss the extrinsic and intrinsic signals that direct liver cells to proliferate, de-differentiate, or trans-differentiate while the tissue recovers from acute or chronic damage.

Epithelial splicing regulatory protein 2-mediated alternative splicing reprograms hepatocytes in severe alcoholic hepatitis.

Severe alcoholic hepatitis (SAH) is a deadly liver disease without an effective medical therapy. Although SAH mortality is known to correlate with hepatic accumulation of immature liver cells, why this occurs and how it causes death are unclear. Here, we demonstrate that expression of epithelial splicing regulatory protein 2 (ESRP2), an RNA-splicing factor that maintains the nonproliferative, mature phenotype of adult hepatocytes, was suppressed in both human SAH and various mouse models of SAH in parallel with the severity of alcohol consumption and liver damage. Inflammatory cytokines released by excessive alcohol ingestion reprogrammed adult hepatocytes into proliferative, fetal-like cells by suppressing ESRP2. Sustained loss of ESRP2 permitted reemergence of a fetal RNA-splicing program that attenuates the Hippo signaling pathway and thus allows fetal transcriptional regulators to accumulate in adult liver. We further showed that depleting ESRP2 in mice exacerbated alcohol-induced steatohepatitis, enabling surviving hepatocytes to shed adult hepatocyte functions and become more regenerative, but threatening overall survival by populating the liver with functionally immature hepatocytes. Our findings revealed a mechanism that explains why liver failure develops in patients with the clinical syndrome of SAH, suggesting that recovery from SAH might be improved by limiting adult-to-fetal reprogramming in hepatocytes.

Intrinsically cell-penetrating multivalent and multitargeting ligands for myotonic dystrophy type 1.

Developing highly active, multivalent ligands as therapeutic agents is challenging because of delivery issues, limited cell permeability, and toxicity. Here, we report intrinsically cell-penetrating multivalent ligands that target the trinucleotide repeat DNA and RNA in myotonic dystrophy type 1 (DM1), interrupting the disease progression in two ways. The oligomeric ligands are designed based on the repetitive structure of the target with recognition moieties alternating with bisamidinium groove binders to provide an amphiphilic and polycationic structure, mimicking cell-penetrating peptides. Multiple biological studies suggested the success of our multivalency strategy. The designed oligomers maintained cell permeability and exhibited no apparent toxicity both in cells and in mice at working concentrations. Furthermore, the oligomers showed important activities in DM1 cells and in a DM1 liver mouse model, reducing or eliminating prominent DM1 features. Phenotypic recovery of the climbing defect in adult DM1 Drosophila was also observed. This design strategy should be applicable to other repeat expansion diseases and more generally to DNA/RNA-targeted therapeutics.

Cell-type specific polysome profiling from mammalian tissues.

The regulation of gene expression occurs through complex relationships between transcription, processing, turnover, and translation, which are only beginning to be elucidated. We know that at least for certain messenger (m) RNAs, processing, modifications, and sequence elements can greatly influence their translational output through recognition by translation and turn-over machinery. Recently, we and others have combined high-throughput sequencing technologies with traditional biochemical methods of studying translation to extend our understanding of these relationships. Additionally, there is growing importance given to how these processes may be regulated across varied cell types as a means to achieve tissue-specific expression of proteins. Here, we provide an in-depth methodology for polysome profiling to dissect the composition of mRNAs and proteins that make up the translatome from both whole tissues and a specific cell type isolated from mammalian tissue. Also, we provide a detailed computational workflow for the analysis of the next-generation sequencing data generated from these experiments.