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Eliscovich, C.

Publications and source records attributed to Eliscovich, C..

7 recordsLinked to original sources

A novel subset of hepatocytes is simultaneously gluconeogenic and de novo lipogenic in the fed state and is naturally insulin resistant

It is generally accepted that hepatic gluconeogenesis, the synthesis of glucose from non-carbohydrate substrates is active in the fasted state and inactive in the fed state. In contrast, de novo lipogenesis is active in the fed state and is inactive in the fasted state. Here, we used targeted single cell RNA-seq, HCR RNA-FISH, and PrimeFlow in normal physiological mouse liver, and identified a subpopulation of periportal hepatocytes that simultaneously co-express both gluconeogenic and lipogenic genes in the fed state. Euglycemic-hyperinsulinemic clamps further demonstrated that this novel hepatocyte subpopulation is naturally insulin resistant. Spatial metabolic imaging coupled with stable isotope tracing analyses revealed individual hepatocytes that simultaneously undergo both gluconeogenesis and de novo lipogenesis. These dual-positive hepatocytes were also present in human hepatocytes from humanized mouse livers. Moreover, the number of dual-positive hepatocytes increased in high-fat diet-fed mice, suggesting a paradigm shift in our understanding of how the liver becomes insulin resistant.

molecular biology↗

Identification and classification of abundant RNA-binding proteins in the mouse lens and interactions of Carhsp1, Igf2bp1/ZBP1, and Ybx1 with crystallin and β-actin mRNAs

RNA-binding proteins (RBPs) are critical regulators of mRNAs controlling all processes such as RNA transcription, transport, localization, translation, mRNA:ncRNA interactions, and decay. Cellular differentiation is driven by tissue-specific and/or tissue-preferred expression of proteins needed for the optimal function of mature cells, tissues and organs. Lens fiber cell differentiation is marked by high levels of expression of crystallin genes encoding critical proteins for lens transparency and light refraction. Herein we performed proteomic and transcriptomic analyses of RBPs in differentiating mouse lenses to identify the most abundant RBPs and establish dynamic changes of their expression in differentiating lens. Expression analyses include highly abundant RBPs, including Carhsp1, Igf2bp1/ZBP1, Ybx1, Pabpc1, Ddx39, and Rbm38. Binding sites of Carhsp1, Ybx1, and Igf2bp1/ZBP1 were predicted in various crystallin and {beta}-actin mRNAs. Immunoprecipitations using antibodies against Carhsp1, Igf2bp1/ZBP1, and Ybx1 confirmed their interactions with A-, B-, and {gamma}A-crystallin mRNAs. A combination of single molecule RNA FISH (smFISH) and immunofluorescence was used to probe in vivo interactions of these RBPs with A-, B-crystallin, and {beta}-actin mRNAs in cytoplasm and nucleoplasm of cultured mouse lens epithelial cells. Together, these results open new avenues to perform comprehensive genetic, cell, and molecular biology studies of individual RBPs in the lens.

developmental biology↗

Continuous nucleolar ribosomal RNA synthesis in differentiating lens fiber cells until abrupt nuclear degradation required for ocular lens transparency

Cellular differentiation requires highly coordinate action of all three transcriptional systems to produce rRNAs, mRNAs, and various "short" and "long" non-coding RNAs by RNA Polymerase I, II, and III systems, respectively. The RNA Polymerase I catalyzes transcription of about 400 copies of rDNA genes generating 18S, 5.8S, and 28S rRNA molecules from the individual primary transcript. Lens fiber cell differentiation is a unique process to study transcriptional mechanisms of individual crystallin genes as their very high transcriptional outputs are directly comparable only to globin genes in erythrocytes. Importantly, both terminally differentiated lens fiber cells and mammalian erythrocytes degrade their nuclei though by different mechanisms. In lens, generation of organelle-free zone (OFZ) includes degradation of mitochondria, endoplasmic reticulum, Golgi apparatus, and nuclei; nevertheless, very little is known about their nucleoli and rRNA transcription. Here, using RNA fluorescence in situ hybridization (FISH) we evaluated nascent rRNA transcription during the entire process of lens fiber cell differentiation. The lens fiber cell nuclei undergo morphological changes prior their denucleation, including chromatin condensation; remarkably, the nascent rRNA transcription persists in all nuclei next to the OFZ. The changes in both nuclei and nucleoli shape and microarchitecture were evaluated by immunofluorescence to detect fibrillarin, nucleolin, UBF, and other nuclear proteins. These studies demonstrate for the first time that highly condensed lens fiber cell nuclei have the capacity to support rRNA transcription. Thus, "late" production of rRNA molecules and consequently the ribosomes contribute to the terminal translational mechanisms to produce maximal quantities of the crystallin proteins.

developmental biology↗

Spatial hepatocyte plasticity of gluconeogenesis during the metabolic transitions between fed, fasted and starvation states

The liver acts as a master regulator of metabolic homeostasis in part by performing gluconeogenesis. This process is dysregulated in type 2 diabetes, leading to elevated hepatic glucose output. The parenchymal cells of the liver (hepatocytes) are heterogeneous, existing on an axis between the portal triad and the central vein, and perform distinct functions depending on location in the lobule. Here, using single cell analysis of hepatocytes across the liver lobule, we demonstrate that gluconeogenic gene expression (Pck1 and G6pc) is relatively low in the fed state and gradually increases first in the periportal hepatocytes during the initial fasting period. As the time of fasting progresses, pericentral hepatocyte gluconeogenic gene expression increases, and following entry into the starvation state, the pericentral hepatocytes show similar gluconeogenic gene expression to the periportal hepatocytes. Similarly, pyruvate-dependent gluconeogenic activity is approximately 10-fold higher in the periportal hepatocytes during the initial fasting state but only 1.5-fold higher in the starvation state. In parallel, starvation suppresses canonical beta-catenin signaling and modulates expression of pericentral and periportal glutamine synthetase and glutaminase, resulting in an enhanced pericentral glutamine-dependent gluconeogenesis. These findings demonstrate that hepatocyte gluconeogenic gene expression and gluconeogenic activity are highly spatially and temporally plastic across the liver lobule, underscoring the critical importance of using well-defined feeding and fasting conditions to define the basis of hepatic insulin resistance and glucose production.

molecular biology↗

Co-translational sorting enables a single mRNA to generate distinct polysomes with different localizations and protein fates

{beta}-catenin is a multi-functional protein playing essential roles in tissue homeostasis and cancer. It bridges E-cadherin to the cytoskeleton and also activates transcription in response to Wnt. Plasma membrane {beta}-catenin is stable whereas without Wnt, cytoplasmic {beta}-catenin is degraded by the destruction complex, composed of APC and Axin. Here, we show that APC and Axin associate with many mRNAs and that this occurs via the nascent protein chains. Notably, APC and Axin bind {beta}-catenin mRNAs present as either single polysome or polysome condensates, and co-translational interactions constitute the major fraction of their binding to the {beta}-catenin protein. Remarkably, E-cadherin also binds {beta}-catenin co-translationally, and {beta}-catenin mRNAs localize either with APC in the cytosol or E-cadherin at the plasma membrane. Thus, co-translational interactions sort {beta}-catenin mRNAs into distinct polysome populations that spatially segregate in cells and synthesize proteins with different functions. Co-translational polysome sorting provides a mechanism to regulate the fate of multi-functional proteins.

molecular biology↗

Visualization of early RNA replication kinetics of SARS-CoV-2 by using single molecule RNA-FISH

SARS-CoV-2 infection has caused a major global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood including the kinetics of formation of plus stranded genomic and subgenomic RNAs (gRNA and sgRNA) starting from the RNA from the first virus that enters the cell. We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA in infected cells and carried out a time course analysis to determine the kinetics of their replication. We visualized the single molecules of gRNA within the cytoplasm of infected cells 30 minutes post-infection and detected the co-expression of gRNA and sgRNA within two hours post-infection. Furthermore, we observed the formation of a replication organelle (RO) from a single RNA, which led to the formation of multiple ROs within the same cells. Single molecule analysis indicated that while gRNA resided in the center of these ROs, the sgRNAs were found to radiate and migrate out of these structures. Our results also indicated that after the initial delay, there was a rapid but asynchronous replication, and the gRNA and sgRNAs dispersed throughout the cell within 4-5 hours post-infection forming multiple ROs that filled the entire cytoplasm. These results provide insight into the kinetics of early post-entry events of SARS-CoV-2 and the formation of RO, which will help to understand the molecular events associated with viral infection and facilitate the identification of new therapeutic targets that can curb the virus at a very early stage of replication to combat COVID-19. Author SummarySARS-CoV-2 infection continues to be a global burden. Soon after the entry, SARS-CoV-2 replicates by an elaborate process, producing genomic and subgenomic RNAs (gRNA and sgRNAs) within specialized structures called replication organelles (RO). Many questions including the timing of multiplication of gRNA and sgRNA, the generation, subcellular localization, and function of the ROs, and the mechanism of vRNA synthesis within ROs is not completely understood. Here, we have developed probes and methods to simultaneously detect the viral gRNA and a sgRNA at single cell single molecule resolution and have employed a method to scan thousands of cells to visualize the early kinetics of gRNA and sgRNA synthesis soon after the viral entry into the cell. Our results reveal that the replication is asynchronous and ROs are rapidly formed from a single RNA that enters the cell within 2 hours, which multiply to fill the entire cell cytoplasm within ~4 hours after infection. Furthermore, our studies provide a first glimpse of the gRNA and sgRNA synthesis within ROs at single molecule resolution. Our studies may facilitate the development of drugs that inhibit the virus at the earliest possible stages of replication to minimize the pathogenic impact of viral infection.

microbiology↗

The structural basis for RNA selectivity by the IMP family of RNA binding proteins

The Igf2 mRNA binding proteins (ZBP1/IMP1, IMP2, IMP3) are highly conserved post-transcriptional regulators of RNA stability, localization and translation. They play important roles in cell migration, neural development, metabolism and cancer cell survival. The knockout phenotypes of individual IMP proteins suggest that each family member regulates a unique pool of RNAs, yet evidence and an underlying mechanism for this is lacking. Here, we combine SELEX and NMR spectroscopy to demonstrate that the major RNA binding domains of the two most distantly related IMPs (ZBP1 and IMP2) bind to different consensus sequences and regulate targets consistent with their knockout phenotypes and roles in disease. We find that the targeting specificity of each IMP is determined by few amino acids in their variable loops. As variable loops often differ amongst KH domain paralogs, we hypothesize that this is a general mechanism for evolving specificity and regulation of the transcriptome.

biochemistry↗