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.

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.

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.

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.

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.

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.

A screen for regeneration-associated silencer regulatory elements in zebrafish.

Regeneration involves gene expression changes explained in part by context-dependent recruitment of transcriptional activators to distal enhancers. Silencers that engage repressive transcriptional complexes are less studied than enhancers and more technically challenging to validate, but they potentially have profound biological importance for regeneration. Here, we identified candidate silencers through a screening process that examined the ability of DNA sequences to limit injury-induced gene expression in larval zebrafish after fin amputation. A short sequence (s1) on chromosome 5 near several genes that reduce expression during adult fin regeneration could suppress promoter activity in stable transgenic lines and diminish nearby gene expression in knockin lines. High-resolution analysis of chromatin organization identified physical associations of s1 with gene promoters occurring preferentially during fin regeneration, and genomic deletion of s1 elevated the expression of these genes after fin amputation. Our study provides methods to identify "tissue regeneration silencer elements" (TRSEs) with the potential to reduce unnecessary or deleterious gene expression during regeneration.

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.

Cell-Membrane Coated Nanoparticles for Tumor Delineation and Qualitative Estimation of Cancer Biomarkers at Single Wavelength Excitation in Murine and Phantom Models.

Real-time guidance through fluorescence imaging improves the surgical outcomes of tumor resections, reducing the chances of leaving positive margins behind. As tumors are heterogeneous, it is imperative to interrogate multiple overexpressed cancer biomarkers with high sensitivity and specificity to improve surgical outcomes. However, for accurate tumor delineation and ratiometric detection of tumor biomarkers, current methods require multiple excitation wavelengths to image multiple biomarkers, which is impractical in a clinical setting. Here, we have developed a biomimetic platform comprising near-infrared fluorescent semiconducting polymer nanoparticles (SPNs) with red blood cell membrane (RBC) coating, capable of targeting two representative cell-surface biomarkers (folate, αυß3 integrins) using a single excitation wavelength for tumor delineation during surgical interventions. We evaluate our single excitation ratiometric nanoparticles in in vitro tumor cells, ex vivo tumor-mimicking phantoms, and in vivo mouse xenograft tumor models. Favorable biological properties (improved biocompatibility, prolonged blood circulation, reduced liver uptake) are complemented by superior spectral features: (i) specific fluorescence enhancement in tumor regions with high tumor-to-normal tissue (T/NT) ratios in ex vivo samples and (ii) estimation of cell-surface tumor biomarkers with single wavelength excitation providing insights about cancer progression (metastases). Our single excitation, dual output approach has the potential to differentiate between the tumor and healthy regions and simultaneously provide a qualitative indicator of cancer progression, thereby guiding surgeons in the operating room with the resection process.

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.

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.

Promiscuous DNA cleavage by HpyAII endonuclease is modulated by the HNH catalytic residues.

Helicobacter pylori is a carcinogenic bacterium that is responsible for 5.5% of all human gastric cancers. H. pylori codes for an unusually large number of restriction-modification (R-M) systems and several of them are strain-specific and phase-variable. HpyAII is a novel Type IIs phase-variable restriction endonuclease present in 26695 strain of H. pylori. We show that HpyAII prefers two-site substrates over one-site substrates for maximal cleavage activity. HpyAII is less stringent in metal ion requirement and shows higher cleavage activity with Ni2+ over Mg2+. Mutational analysis of the putative residues of the HNH motif of HpyAII confirms that the protein has an active HNH site for the cleavage of DNA. However, mutation of the first Histidine residue of the HNH motif to Alanine does not abolish the enzymatic activity, but instead causes loss of fidelity compared with wildtype HpyAII. Previous studies have shown that mutation of the first Histidine residue of the HNH motif of all other known HNH motif motif-containing enzymes completely abolishes enzymatic activity. We found, in the case of HpyAII, mutation of an active site residue leads to the loss of endonuclease fidelity. The present study provides further insights into the evolution of restriction enzymes.

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.

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.

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.

Alternative splicing rewires Hippo signaling pathway in hepatocytes to promote liver regeneration.

During liver regeneration, most new hepatocytes arise via self-duplication; yet, the underlying mechanisms that drive hepatocyte proliferation following injury remain poorly defined. By combining high-resolution transcriptome and polysome profiling of hepatocytes purified from quiescent and toxin-injured mouse livers, we uncover pervasive alterations in messenger RNA translation of metabolic and RNA-processing factors, which modulate the protein levels of a set of splicing regulators. Specifically, downregulation of the splicing regulator ESRP2 activates a neonatal alternative splicing program that rewires the Hippo signaling pathway in regenerating hepatocytes. We show that production of neonatal splice isoforms attenuates Hippo signaling, enables greater transcriptional activation of downstream target genes, and facilitates liver regeneration. We further demonstrate that ESRP2 deletion in mice causes excessive hepatocyte proliferation upon injury, whereas forced expression of ESRP2 inhibits proliferation by suppressing the expression of neonatal Hippo pathway isoforms. Thus, our findings reveal an alternative splicing axis that supports regeneration following chronic liver injury.

Advances in analyzing RNA diversity in eukaryotic transcriptomes: peering through the Omics lens.

Alternative splicing, polyadenylation, and chemical modifications of RNA generate astonishing complexity within eukaryotic transcriptomes. The last decade has brought numerous advances in sequencing technologies that allow biologists to investigate these phenomena with greater depth and accuracy while reducing time and cost. A commensurate development in biochemical techniques for the enrichment and analysis of different RNA variants has accompanied the advancement of global sequencing analysis platforms. Here, we present a detailed overview of the latest biochemical methods, along with bioinformatics pipelines that have aided in identifying different RNA variants. We also highlight the ongoing developments and challenges associated with RNA variant detection and quantification, including sample heterogeneity and isolation, as well as 'Omics' big data handling.