Systematic exploration of dynamic splicing networks reveals conserved multistage regulators of neurogenesis.

Alternative splicing (AS) is a critical regulatory layer; yet, factors controlling functionally coordinated splicing programs during developmental transitions are poorly understood. Here, we employ a screening strategy to identify factors controlling dynamic splicing events important for mammalian neurogenesis. Among previously unknown regulators, Rbm38 acts widely to negatively control neural AS, in part through interactions mediated by the established repressor of splicing, Ptbp1. Puf60, a ubiquitous factor, is surprisingly found to promote neural splicing patterns. This activity requires a conserved, neural-differential exon that remodels Puf60 co-factor interactions. Ablation of this exon rewires distinct AS networks in embryonic stem cells and at different stages of mouse neurogenesis. Single-cell transcriptome analyses further reveal distinct roles for Rbm38 and Puf60 isoforms in establishing neuronal identity. Our results describe important roles for previously unknown regulators of neurogenesis and establish how an alternative exon in a widely expressed splicing factor orchestrates temporal control over cell differentiation.

A highly conserved program of neuronal microexons is misregulated in autistic brains.

Alternative splicing (AS) generates vast transcriptomic and proteomic complexity. However, which of the myriad of detected AS events provide important biological functions is not well understood. Here, we define the largest program of functionally coordinated, neural-regulated AS described to date in mammals. Relative to all other types of AS within this program, 3-15 nucleotide "microexons" display the most striking evolutionary conservation and switch-like regulation. These microexons modulate the function of interaction domains of proteins involved in neurogenesis. Most neural microexons are regulated by the neuronal-specific splicing factor nSR100/SRRM4, through its binding to adjacent intronic enhancer motifs. Neural microexons are frequently misregulated in the brains of individuals with autism spectrum disorder, and this misregulation is associated with reduced levels of nSR100. The results thus reveal a highly conserved program of dynamic microexon regulation associated with the remodeling of protein-interaction networks during neurogenesis, the misregulation of which is linked to autism.

Sugar defeats the Hippo: Glycogen regulation of the Hippo pathway in liver.

Liver glycogen is famous for glucose storage, but new work by Liu et al. (2021) now reveals that it's been hiding a few secrets and can directly promote liver enlargement and tumorigenesis by sequestering the tumor-suppressive Hippo signaling pathway.

Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration.

Stroma in the tumor microenvironment plays a critical role in cancer progression, but how it promotes metastasis is poorly understood. Exosomes are small vesicles secreted by many cell types and enable a potent mode of intercellular communication. Here, we report that fibroblast-secreted exosomes promote breast cancer cell (BCC) protrusive activity and motility via Wnt-planar cell polarity (PCP) signaling. We show that exosome-stimulated BCC protrusions display mutually exclusive localization of the core PCP complexes, Fzd-Dvl and Vangl-Pk. In orthotopic mouse models of breast cancer, coinjection of BCCs with fibroblasts dramatically enhances metastasis that is dependent on PCP signaling in BCCs and the exosome component, Cd81 in fibroblasts. Moreover, we demonstrate that trafficking in BCCs promotes tethering of autocrine Wnt11 to fibroblast-derived exosomes. This work reveals an intercellular communication pathway whereby fibroblast exosomes mobilize autocrine Wnt-PCP signaling to drive BCC invasive behavior.

An alternative splicing switch regulates embryonic stem cell pluripotency and reprogramming.

Alternative splicing (AS) is a key process underlying the expansion of proteomic diversity and the regulation of gene expression. Here, we identify an evolutionarily conserved embryonic stem cell (ESC)-specific AS event that changes the DNA-binding preference of the forkhead family transcription factor FOXP1. We show that the ESC-specific isoform of FOXP1 stimulates the expression of transcription factor genes required for pluripotency, including OCT4, NANOG, NR5A2, and GDF3, while concomitantly repressing genes required for ESC differentiation. This isoform also promotes the maintenance of ESC pluripotency and contributes to efficient reprogramming of somatic cells into induced pluripotent stem cells. These results reveal a pivotal role for an AS event in the regulation of pluripotency through the control of critical ESC-specific transcriptional programs.

Single-cell transcriptomes of the regenerating intestine reveal a revival stem cell.

The turnover of the intestinal epithelium is driven by multipotent LGR5+ crypt-base columnar cells (CBCs) located at the bottom of crypt zones1. However, CBCs are lost following injury, such as irradiation2, but the intestinal epithelium is nevertheless able to recover3. Thus, a second population of quiescent '+4' cells, or reserve stem cells (RSCs), has previously been proposed to regenerate the damaged intestine4-7. Although CBCs and RSCs were thought to be mutually exclusive4,8, subsequent studies have found that LGR5+ CBCs express RSC markers9 and that RSCs were dispensable-whereas LGR5+ cells were essential-for repair of the damaged intestine3. In addition, progenitors of absorptive enterocytes10, secretory cells11-15 and slow cycling LGR5+ cells16 have been shown to contribute to regeneration whereas the transcriptional regulator YAP1, which is important for intestinal regeneration, was suggested to induce a pro-survival phenotype in LGR5+ cells17. Thus, whether cellular plasticity or distinct cell populations are critical for intestinal regeneration remains unknown. Here we applied single-cell RNA sequencing to profile the regenerating mouse intestine and identified a distinct, damage-induced quiescent cell type that we term the revival stem cell (revSC). revSCs are marked by high clusterin expression and are extremely rare under homoeostatic conditions, yet give rise-in a temporal hierarchy-to all the major cell types of the intestine, including LGR5+ CBCs. After intestinal damage by irradiation, targeted ablation of LGR5+ CBCs, or treatment with dextran sodium sulfate, revSCs undergo a YAP1-dependent transient expansion, reconstitute the LGR5+ CBC compartment and are required to regenerate a functional intestine. These studies thus define a unique stem cell that is mobilized by damage to revive the homoeostatic stem cell compartment and regenerate the intestinal epithelium.

Multilayered Control of Alternative Splicing Regulatory Networks by Transcription Factors.

Networks of coordinated alternative splicing (AS) events play critical roles in development and disease. However, a comprehensive knowledge of the factors that regulate these networks is lacking. We describe a high-throughput system for systematically linking trans-acting factors to endogenous RNA regulatory events. Using this system, we identify hundreds of factors associated with diverse regulatory layers that positively or negatively control AS events linked to cell fate. Remarkably, more than one-third of the regulators are transcription factors. Further analyses of the zinc finger protein Zfp871 and BTB/POZ domain transcription factor Nacc1, which regulate neural and stem cell AS programs, respectively, reveal roles in controlling the expression of specific splicing regulators. Surprisingly, these proteins also appear to regulate target AS programs via binding RNA. Our results thus uncover a large "missing cache" of splicing regulators among annotated transcription factors, some of which dually regulate AS through direct and indirect mechanisms.

YAP establishes epiblast responsiveness to inductive signals for germ cell fate.

The germ cell lineage in mammals is induced by the stimulation of pluripotent epiblast cells by signaling molecules. Previous studies have suggested that the germ cell differentiation competence or responsiveness of epiblast cells to signaling molecules is established and maintained in epiblast cells of a specific differentiation state. However, the molecular mechanism underlying this process has not been well defined. Here, using the differentiation model of mouse epiblast stem cells (EpiSCs), we have shown that two defined EpiSC lines have robust germ cell differentiation competence. However, another defined EpiSC line has no competence. By evaluating the molecular basis of EpiSCs with distinct germ cell differentiation competence, we identified YAP, an intracellular mediator of the Hippo signaling pathway, as crucial for the establishment of germ cell induction. Strikingly, deletion of YAP severely affected responsiveness to inductive stimuli, leading to a defect in WNT target activation and germ cell differentiation. In conclusion, we propose that the Hippo/YAP signaling pathway creates a potential for germ cell fate induction via mesodermal WNT signaling in pluripotent epiblast cells.

The secret life of Smad4.

Smad4 is an important mediator of signaling by TGFbeta family members that is presumed to be controlled by the phosphorylation status of its partners, the receptor-regulated R-Smads. In this issue, Dupont et al. (2009) now reveal that Smad4 itself is subject to cycles of ubiquitination and deubiquitination that regulate its interactions with the R-Smads.

Regulation of planar cell polarity by Smurf ubiquitin ligases.

Planar cell polarity (PCP) is critical for morphogenesis in metazoans. PCP in vertebrates regulates stereocilia alignment in neurosensory cells of the cochlea and closure of the neural tube through convergence and extension movements (CE). Noncanonical Wnt morphogens regulate PCP and CE in vertebrates, but the molecular mechanisms remain unclear. Smurfs are ubiquitin ligases that regulate signaling, cell polarity and motility through spatiotemporally restricted ubiquitination of diverse substrates. Here, we report an unexpected role for Smurfs in controlling PCP and CE. Mice mutant for Smurf1 and Smurf2 display PCP defects in the cochlea and CE defects that include a failure to close the neural tube. Further, we show that Smurfs engage in a noncanonical Wnt signaling pathway that targets the core PCP protein Prickle1 for ubiquitin-mediated degradation. Our work thus uncovers ubiquitin ligases in a mechanistic link between noncanonical Wnt signaling and PCP/CE.

Regulation of homeostasis and regeneration in the adult intestinal epithelium by the TGF-ß superfamily.

The delicate balance between the homeostatic maintenance and regenerative capacity of the intestine makes this a fascinating tissue of study. The intestinal epithelium undergoes continuous homeostatic renewal but is also exposed to a diverse array of stresses that can range from physiological processes such as digestion to exposure to infectious agents, drugs, radiation therapy, and inflammatory stimuli. The intestinal epithelium has thus evolved to efficiently maintain and reinstate proper barrier function that is essential for intestinal integrity and function. Factors governing homeostatic epithelial turnover are well described; however, the dynamic regenerative mechanisms that occur following injury are the subject of intense ongoing investigations. The TGF-ß superfamily is a key regulator of both homeostatic renewal and regenerative processes of the intestine. Here, we review the roles of TGF-ß and BMP on the adult intestinal epithelium during self-renewal and injury to provide a framework for understanding how this major family of morphogens can tip the scale between intestinal health and disease.

Myc and SAGA rewire an alternative splicing network during early somatic cell reprogramming.

Embryonic stem cells are maintained in a self-renewing and pluripotent state by multiple regulatory pathways. Pluripotent-specific transcriptional networks are sequentially reactivated as somatic cells reprogram to achieve pluripotency. How epigenetic regulators modulate this process and contribute to somatic cell reprogramming is not clear. Here we performed a functional RNAi screen to identify the earliest epigenetic regulators required for reprogramming. We identified components of the SAGA histone acetyltransferase complex, in particular Gcn5, as critical regulators of reprogramming initiation. Furthermore, we showed in mouse pluripotent stem cells that Gcn5 strongly associates with Myc and that, upon initiation of somatic reprogramming, Gcn5 and Myc form a positive feed-forward loop that activates a distinct alternative splicing network and the early acquisition of pluripotency-associated splicing events. These studies expose a Myc-SAGA pathway that drives expression of an essential alternative splicing regulatory network during somatic cell reprogramming.

An endogenous PI3K interactome promoting astrocyte-mediated neuroprotection identifies a novel association with RNA-binding protein ZC3H14.

Astrocytes can support neuronal survival through a range of secreted signals that protect against neurotoxicity, oxidative stress, and apoptotic cascades. Thus, analyzing the effects of the astrocyte secretome may provide valuable insight into these neuroprotective mechanisms. Previously, we characterized a potent neuroprotective activity mediated by retinal astrocyte conditioned media (ACM) on retinal and cortical neurons in metabolic stress models. However, the molecular mechanism underlying this complex activity in neuronal cells has remained unclear. Here, a chemical genetics screen of kinase inhibitors revealed phosphoinositide 3-kinase (PI3K) as a central player transducing ACM-mediated neuroprotection. To identify additional proteins contributing to the protective cascade, endogenous PI3K was immunoprecipitated from neuronal cells exposed to ACM or control media, followed by MS/MS proteomic analyses. These data pointed toward a relatively small number of proteins that coimmunoprecipitated with PI3K, and surprisingly only five were regulated by the ACM signal. These hits included expected PI3K interactors, such as the platelet-derived growth factor receptor A (PDGFRA), as well as novel RNA-binding protein interactors ZC3H14 (zinc finger CCCH-type containing 14) and THOC1 (THO complex protein 1). In particular, ZC3H14 has recently emerged as an important RNA-binding protein with multiple roles in posttranscriptional regulation. In validation studies, we show that PI3K recruitment of ZC3H14 is necessary for PDGF-induced neuroprotection and that this interaction is present in primary retinal ganglion cells. Thus, we identified a novel non-cell autonomous neuroprotective signaling cascade mediated through PI3K that requires recruitment of ZC3H14 and may present a promising strategy to promote astrocyte-secreted prosurvival signals.

The hippo pathway: A master regulator of liver metabolism, regeneration, and disease.

The liver is the only visceral organ in the body with a tremendous capacity to regenerate in response to insults that induce inflammation, cell death, and injury. Liver regeneration is a complicated process involving a well-orchestrated activation of non-parenchymal cells in the injured area and proliferation of undamaged hepatocytes. Furthermore, the liver has a Hepatostat, defined as adjustment of its volume to that required for homeostasis. Understanding the mechanisms that control different steps of liver regeneration is critical to informing therapies for liver repair, to help patients with liver disease. The Hippo signaling pathway is well known for playing an essential role in the control and regulation of liver size, regeneration, stem cell self-renewal, and liver cancer. Thus, the Hippo pathway regulates dynamic cell fates in liver, and in absence of its downstream effectors YAP and TAZ, liver regeneration is severely impaired, and the proliferative expansion of liver cells blocked. We will mainly review upstream mechanisms activating the Hippo signaling pathway following partial hepatectomy in mouse model and patients, its roles during different steps of liver regeneration, metabolism, and cancer. We will also discuss how targeting the Hippo signaling cascade might improve liver regeneration and suppress liver tumorigenesis.

Comparison of SARS-CoV-2 indirect and direct RT-qPCR detection methods.

BACKGROUND: Sensitive, rapid, and accessible diagnostics continue to be critical to track the COVID-19 pandemic caused by the SARS-CoV-2 virus. RT-qPCR is the gold standard test, and comparison of methodologies and reagents, utilizing patient samples, is important to establish reliable diagnostic pipelines. METHODS: Here, we assessed indirect methods that require RNA extraction with direct RT-qPCR on patient samples. Four different RNA extraction kits (Qiagen, Invitrogen, BGI and Norgen Biotek) were compared. For detection, we assessed two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). We also tested and optimized direct, extraction-free detection using these RT-qPCR systems and performed a cost analysis of the different methods evaluated here. RESULTS: Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system provided overall superior performance (lower detection limit, lower Ct values and higher sensitivity), generating comparable results to original clinical diagnostic data, and identifying samples ranging from 65 copies to 2.1 × 105 copies of viral genome/µl. However, the BGI detection system is more expensive than other options tested here. With direct RT-qPCR, simply adding an RNase inhibitor greatly improved detection, without the need for any other treatments (e.g. lysis buffers or boiling). The best direct methods detected ~ 10 fold less virus than indirect methods, but this simplified approach reduced sample handling, as well as assay time and cost. CONCLUSIONS: With extracted RNA, the BGI RT-qPCR detection system exhibited superior performance over the Norgen system, matching initial clinical diagnosis with the Seegene Allplex assay. The BGI system was also suitable for direct, extraction-free analysis, providing 78.4% sensitivity. The Norgen system, however, still accurately detected samples with a clinical Ct < 33 from extracted RNA, provided significant cost savings, and was superior to SYBR green assays that exhibited reduced specificity.

Yap-dependent reprogramming of Lgr5(+) stem cells drives intestinal regeneration and cancer.

The gut epithelium has remarkable self-renewal capacity that under homeostatic conditions is driven by Wnt signalling in Lgr5(+) intestinal stem cells (ISCs). However, the mechanisms underlying ISC regeneration after injury remain poorly understood. The Hippo signalling pathway mediates tissue growth and is important for regeneration. Here we demonstrate in mice that Yap, a downstream transcriptional effector of Hippo, is critical for recovery of intestinal epithelium after exposure to ionizing radiation. Yap transiently reprograms Lgr5(+) ISCs by suppressing Wnt signalling and excessive Paneth cell differentiation, while promoting cell survival and inducing a regenerative program that includes Egf pathway activation. Accordingly, growth of Yap-deficient organoids is rescued by the Egfr ligand epiregulin, and we find that non-cell-autonomous production of stromal epiregulin may compensate for Yap loss in vivo. Consistent with key roles for regenerative signalling in tumorigenesis, we further demonstrate that Yap inactivation abolishes adenomas in the Apc(Min) mouse model of colon cancer, and that Yap-driven expansion of Apc(-/-) organoids requires the Egfr module of the Yap regenerative program. Finally, we show that in vivo Yap is required for progression of early Apc mutant tumour-initiating cells, suppresses their differentiation into Paneth cells, and induces a regenerative program and Egfr signalling. Our studies reveal that upon tissue injury, Yap reprograms Lgr5(+) ISCs by inhibiting the Wnt homeostatic program, while inducing a regenerative program that includes activation of Egfr signalling. Moreover, our findings reveal a key role for the Yap regenerative pathway in driving cancer initiation.

Tissue-specific alternative splicing remodels protein-protein interaction networks.

Alternative splicing plays a key role in the expansion of proteomic and regulatory complexity, yet the functions of the vast majority of differentially spliced exons are not known. In this study, we observe that brain and other tissue-regulated exons are significantly enriched in flexible regions of proteins that likely form conserved interaction surfaces. These proteins participate in significantly more interactions in protein-protein interaction (PPI) networks than other proteins. Using LUMIER, an automated PPI assay, we observe that approximately one-third of analyzed neural-regulated exons affect PPIs. Inclusion of these exons stimulated and repressed different partner interactions at comparable frequencies. This assay further revealed functions of individual exons, including a role for a neural-specific exon in promoting an interaction between Bridging Integrator 1 (Bin1)/Amphiphysin II and Dynamin 2 (Dnm2) that facilitates endocytosis. Collectively, our results provide evidence that regulated alternative exons frequently remodel interactions to establish tissue-dependent PPI networks.

MBNL proteins repress ES-cell-specific alternative splicing and reprogramming.

Previous investigations of the core gene regulatory circuitry that controls the pluripotency of embryonic stem (ES) cells have largely focused on the roles of transcription, chromatin and non-coding RNA regulators. Alternative splicing represents a widely acting mode of gene regulation, yet its role in regulating ES-cell pluripotency and differentiation is poorly understood. Here we identify the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between ES cells and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ES-cell-like alternative splicing pattern for approximately half of these events, whereas overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns. Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in the forkhead family transcription factor FOXP1 that controls pluripotency. Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming.

The RNF146 and tankyrase pathway maintains the junctional Crumbs complex through regulation of angiomotin.

The Crumbs complex is an important determinant of epithelial apical-basal polarity that functions in regulation of tight junctions, resistance to epithelial-to-mesenchymal transitions and as a tumour suppressor. Although the functional role of the Crumbs complex is being elucidated, its regulation is poorly understood. Here, we show that suppression of RNF146, an E3 ubiquitin ligase that recognizes ADP-ribosylated substrates, and tankyrase, a poly(ADP-ribose) polymerase, disrupts the junctional Crumbs complex and disturbs the function of tight junctions. We show that RNF146 binds a number of polarity-associated proteins, in particular members of the angiomotin (AMOT) family. Accordingly, AMOT proteins are ADP-ribosylated by TNKS2, which drives ubiquitylation by RNF146 and subsequent degradation. Ablation of RNF146 or tankyrase, as well as overexpression of AMOT, led to the relocation of PALS1 (a Crumbs complex component) from the apical membrane to internal puncta, a phenotype that is rescued by AMOTL2 knockdown. We thus reveal a new function of RNF146 and tankyrase in stabilizing the Crumbs complex through downregulation of AMOT proteins at the apical membrane.