For vertebrate olfactory signal transduction, a calcium-activated chloride conductance serves as a major amplification step. However, the molecular identity of the olfactory calcium-activated chloride channel (CaCC) is unknown. Here we report a proteomic screen for cilial membrane proteins of mouse olfactory sensory neurons (OSNs) that identified all the known olfactory transduction components as well as Anoctamin 2 (ANO2). Ano2 transcripts were expressed specifically in OSNs in the olfactory epithelium, and ANO2::EGFP fusion protein localized to the OSN cilia when expressed in vivo using an adenoviral vector. Patch-clamp analysis revealed that ANO2, when expressed in HEK-293 cells, forms a CaCC and exhibits channel properties closely resembling the native olfactory CaCC. Considering these findings together, we propose that ANO2 constitutes the olfactory calciumactivated chloride channel.Anoctamin ͉ cilia ͉ olfaction ͉ signal transduction ͉ TMEM16B

SUMMARY Nonsense-mediated decay (NMD) degrades both normal and aberrant transcripts harboring stop codons in particular contexts. Mutations that perturb NMD cause neurological disorders in humans, suggesting that NMD has roles in the brain. Here, we identify a brain-specific microRNA—miR-128—that represses NMD and thereby controls batteries of transcripts in neural cells. miR-128 represses NMD by targeting the RNA helicase UPF1 and the exon-junction complex core component MLN51. The ability of miR-128 to regulate NMD is a conserved response occuring in frogs, chickens, and mammals. miR-128 levels are dramatically increased in differentiating neuronal cells and during brain development, leading to repressed NMD and upregulation of mRNAs normally targeted for decay by NMD; overrepresented are those encoding proteins controlling neuron development and function. Together, these results suggest the existence of a conserved RNA circuit linking the microRNA and NMD pathways that induces cell type-specific transcripts during development.

SUMMARY Nonsense-mediated mRNA decay (NMD) is a conserved RNA decay pathway that degrades aberrant mRNAs and directly regulates many normal mRNAs. This dual role for NMD raises the possibility that its magnitude is buffered to prevent the potentially catastrophic alterations in gene expression that would otherwise occur if NMD were perturbed by environmental or genetic insults. In support of this, here we report the existence of a negative feedback regulatory network that directly acts on seven NMD factors. Feedback regulation is conferred by different branches of the NMD pathway in a cell type-specific and developmentally regulated manner. We identify feedback-regulated NMD factors that are rate limiting for NMD and demonstrate that reversal of feedback regulation in response to NMD perturbation is crucial for maintaining NMD. Together, our results suggest the existence of an intricate feedback network that maintains both RNA surveillance and the homeostasis of normal gene expression in mammalian cells.

SUMMARY Gene duplication is a major evolutionary force driving adaptation and speciation, as it allows for the acquisition of new functions and can augment or diversify existing functions. Here, we report a gene duplication event that yielded another outcome – the generation of antagonistic functions. One product of this duplication event – UPF3B – is critical for the nonsense-mediated RNA decay (NMD) pathway, while its autosomal counterpart – UPF3A – encodes an enigmatic protein previously shown to have trace NMD activity. Using loss-of-function approaches in vitro and in vivo, we discovered that UPF3A acts primarily as a potent NMD inhibitor that stabilizes hundreds of transcripts. Evidence suggests that UPF3A acquired repressor activity through simple impairment of a critical domain, a rapid mechanism that may have been widely used in evolution. Mice conditionally lacking UPF3A exhibit “hyper” NMD and display defects in embryogenesis and gametogenesis, consistent with UPF3A serving as a molecular rheostat that directs developmental events.

SUMMARY The mechanisms dictating whether a cell proliferates or differentiates have undergone intense scrutiny but remain poorly understood. Here, we report that a central component in the nonsense-mediated RNA decay (NMD) pathway—UPF1—plays a key role in this decision by promoting the proliferative, undifferentiated cell state. UPF1 acts, in part, by destabilizing the NMD substrate encoding the TGFβ inhibitor, SMAD7, and stimulating TGFβ signaling. UPF1 also promotes the decay of mRNAs encoding many other proteins that oppose the proliferative, undifferentiated cell state. Neural differentiation is triggered when NMD is downregulated by neurally expressed microRNAs (miRNAs). This UPF1-miRNA circuitry is highly conserved and harbors negative feedback loops that act as a molecular switch. Our results suggest that the NMD RNA decay pathway collaborates with the TGF-β signaling pathway to lock-in the stem-like state, a cellular state that is stably reversed when neural differentiation signals that induce NMD-repressive miRNAs are received.

SummaryNonsense-mediated RNA decay (NMD) is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs) demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-β and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages.

Summary The developmental origins of most adult stem cells are poorly understood. Here, we report the identification of a transcription factor—RHOX10—that is critical for the initial establishment of spermatogonial stem cells (SSCs). Conditional loss of the entire 33-gene X-linked homeobox gene cluster that includes Rhox10 causes progressive spermatogenic decline, a phenotype indistinguishable from that caused by loss of only Rhox10. We demonstrate that this phenotype results from dramatically reduced SSC generation. Using a battery of approaches, including single cell-RNAseq (scRNAseq) analysis, we show that Rhox10 drives SSC generation by promoting Pro-spermatogonia differentiation. Rhox10 also regulates batteries of migration genes and promotes the migration of Pro-spermatogonia into the SSC niche. The identification of an X-linked homeobox gene that drives the initial generation of SSCs has implications for the evolution of X-linked gene clusters and sheds light on regulatory mechanisms influencing adult stem cell generation in general.

Nonsense-mediated RNA decay (NMD) is a highly conserved and selective RNA degradation pathway that acts on RNAs terminating their reading frames in specific contexts. NMD is regulated in a tissue-specific and developmentally controlled manner, raising the possibility that it influences developmental events. Indeed, loss or depletion of NMD factors have been shown to disrupt developmental events in organisms spanning the phylogenetic scale. In humans, mutations in the NMD factor gene, UPF3B, cause intellectual disability (ID) and are strongly associated with autism spectrum (ASD), attention deficit hyperactivity disorder (ADHD), and schizophrenia (SCZ). Here, we report the generation and characterization of mice harboring a null Upf3b allele. These Upf3b-null mice exhibit deficits in fear-conditioned learning, but not spatial learning. Upf3b-null mice also have a profound defect in prepulse inhibition (PPI), a measure of sensorimotor gating commonly deficient in individuals with SCZ and other brain disorders. Consistent with both their PPI and learning defects, cortical pyramidal neurons from Upf3b-null mice display deficient dendritic spine maturation in vivo. In addition, neural stem cells from Upf3b-null mice have impaired ability to undergo differentiation and require prolonged culture to give rise to functional neurons with electrical activity. RNA sequencing (RNAseq) analysis of the frontal cortex identified UPF3B-regulated RNAs, including direct NMD target transcripts encoding proteins with known functions in neural differentiation, maturation, and disease. We suggest Upf3b-null mice serve as a novel model system to decipher cellular and molecular defects underlying ID and neuro-developmental disorders.