Intracellular organelles mediate complex cellular functions that often require ion transport across their membranes. Melanosomes are organelles responsible for the synthesis of the major mammalian pigment melanin. Defects in melanin synthesis result in pigmentation defects, visual deficits, and increased susceptibility to skin and eye cancers. Although genes encoding putative melanosomal ion transporters have been identified as key regulators of melanin synthesis, melanosome ion transport and its contribution to pigmentation remain poorly understood. Here we identify two-pore channel 2 (TPC2) as the first reported melanosomal cation conductance by directly patch-clamping skin and eye melanosomes. TPC2 has been implicated in human pigmentation and melanoma, but the molecular mechanism mediating this function was entirely unknown. We demonstrate that the vesicular signaling lipid phosphatidylinositol bisphosphate PI(3,5)P2 modulates TPC2 activity to control melanosomal membrane potential, pH, and regulate pigmentation.

Metazoan development depends on the accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell-fate determination is less well understood. Here we identify the ubiquitin ligase CUL3 in complex with its vertebrate-specific substrate adaptor KBTBD8 (CUL3(KBTBD8)) as an essential regulator of human and Xenopus tropicalis neural crest specification. CUL3(KBTBD8) monoubiquitylates NOLC1 and its paralogue TCOF1, the mutation of which underlies the neurocristopathy Treacher Collins syndrome. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favour of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell-fate determination.

Previously, we identified ISRIB as a potent inhibitor of the integrated stress response (ISR) and showed that ISRIB makes cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents (Sidrauski et al., 2013). Here, we show by genome-wide in vivo ribosome profiling that translation of a restricted subset of mRNAs is induced upon ISR activation. ISRIB substantially reversed the translational effects elicited by phosphorylation of eIF2α and induced no major changes in translation or mRNA levels in unstressed cells. eIF2α phosphorylation-induced stress granule (SG) formation was blocked by ISRIB. Strikingly, ISRIB addition to stressed cells with pre-formed SGs induced their rapid disassembly, liberating mRNAs into the actively translating pool. Restoration of mRNA translation and modulation of SG dynamics may be an effective treatment of neurodegenerative diseases characterized by eIF2α phosphorylation, SG formation, and cognitive loss.

Brain derived peptides function as signaling molecules in the brain and regulate various physiological and behavioral processes. The low abundance and atypical fragmentation of these brain derived peptides makes detection using traditional proteomic methods challenging. In this study, we introduce and validate a new methodology for the discovery of novel peptides derived from mammalian brain. This methodology combines ribosome profiling and mass spectrometry-based peptidomics. Using this framework, we have identified a novel peptide in mouse whole brain whose expression is highest in the basal ganglia, hypothalamus and amygdala. Although its functional role is unknown, it has been previously detected in peripheral tissue as a component of the mRNA decapping complex. Continued discovery and studies of novel regulating peptides in mammalian brain may also provide insight into brain disorders.

Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be noncoding, including 5' UTRs and long noncoding RNAs (lncRNAs). Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs). Here, we show hallmarks of translation in these footprints: copurification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including the induction of immune responses following human cytomegalovirus (HCMV) infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts at understanding how cells manage and exploit its consequences.

Intracellular proteins with long lifespans have recently been linked to age-dependent defects, ranging from decreased fertility to the functional decline of neurons. Why long-lived proteins exist in metabolically active cellular environments and how they are maintained over time remains poorly understood. Here, we provide a system-wide identification of proteins with exceptional lifespans in the rat brain. These proteins are inefficiently replenished despite being translated robustly throughout adulthood. Using nucleoporins as a paradigm for long-term protein persistence, we found that nuclear pore complexes (NPCs) are maintained over a cell's life through slow but finite exchange of even its most stable subcomplexes. This maintenance is limited, however, as some nucleoporin levels decrease during aging, providing a rationale for the previously observed age-dependent deterioration of NPC function. Our identification of a long-lived proteome reveals cellular components that are at increased risk for damage accumulation, linking long-term protein persistence to the cellular aging process. PAPERCLIP:

Short hairpin RNA libraries are limited by low efficacy of many shRNAs and by off-target effects, which give rise to false negatives and false positives, respectively. Here we present a strategy for rapidly creating expanded shRNA pools (approximately 30 shRNAs per gene) that are analyzed by deep sequencing (EXPAND). This approach enables identification of multiple effective target-specific shRNAs from a complex pool, allowing a rigorous statistical evaluation of true hits.

Translation initiation at alternative start sites can dynamically control the synthesis of two or more functionally distinct protein isoforms from a single mRNA. Alternate isoforms of the hematopoietic transcription factor CCAAT-enhancer binding protein α (C/EBP α ) produced from different start sites exert opposing effects during myeloid cell development. This alternative initiation depends on sequence features of the CEBPA transcript, including a regulatory upstream open reading frame (uORF), but the molecular basis is not fully understood. Here we identify trans -acting factors that affect C/EBP α isoform choice using a sensitive and quantitative two-color fluorescence reporter coupled with CRISPRi screening. Our screen uncovered a role for the ribosome rescue factor PELOTA (PELO) in promoting expression of the longer C/EBP α isoform, by directly removing inhibitory unrecycled ribosomes and through indirect effects mediated by the mechanistic target of rapamycin (mTOR) kinase. Our work provides further mechanistic insights into coupling between ribosome recycling and translation reinitiation in regulation of a key transcription factor, with implications for normal hematopoiesis and leukemiagenesis.

Members of the Puf family of RNA-binding proteins typically associate via their Pumilio homology domain with specific short motifs in the 3'-UTR of an mRNA and thereby influence the stability, localization and/or efficiency of translation of the bound transcript. In our prior unbiased proteome-wide screen for targets of the TORC2-stimulated protein kinase Ypk1, we identified the paralogs Puf1/Jsn1 and Puf2 as high-confidence substrates. Earlier work by others had demonstrated that Puf1 and Puf2 exhibit a marked preference for interaction with mRNAs encoding plasma membrane-associated proteins, consistent with our previous studies documenting that a primary physiological role of TORC2-Ypk1 signaling is maintenance of plasma membrane homeostasis. Here, we show, first, that both Puf1 and Puf2 are authentic Ypk1 substrates both in vitro and in vivo. Fluorescently tagged Puf1 localizes constitutively in cortical puncta closely apposed to the plasma membrane, whereas Puf2 does so in the absence of its Ypk1 phosphorylation, but is dispersed in the cytosol when phosphorylated. We further demonstrate that Ypk1-mediated phosphorylation of Puf1 and Puf2 upregulates production of the protein products of the transcripts to which they bind, with a concomitant increase in the level of the cognate mRNAs. Thus, Ypk1 phosphorylation relieves Puf1- and Puf2-mediated post-transcriptional repression mainly by counteracting their negative effect on transcript stability. Using a heterologous protein-RNA tethering and fluorescent protein reporter assay, the consequence of Ypk1 phosphorylation in vivo was recapitulated for full-length Puf1 and even for N-terminal fragments (residues 1-340 and 143-295) corresponding to the region upstream of its dimerization domain (an RNA-recognition motif fold) encompassing its two Ypk1 phosphorylation sites (both also conserved in Puf2). This latter result suggests that alleviation of Puf1-imposed transcript destabilization does not obligatorily require dissociation of Ypk1-phosphorylated Puf1 from a transcript. Our findings add new insight about how the TORC2-Ypk1 signaling axis regulates the content of plasma membrane-associated proteins to promote maintenance of the integrity of the cell envelope.

No abstract available

Jellyfish and sea anemones fire single-use, venom-covered barbs to immobilize prey or predators. We previously showed that the anemone Nematostella vectensis uses a specialized voltage-gated calcium (CaV) channel to trigger stinging in response to synergistic prey-derived chemicals and touch (Weir et al., 2020). Here, we use experiments and theory to find that stinging behavior is suited to distinct ecological niches. We find that the burrowing anemone Nematostella uses uniquely strong CaV inactivation for precise control of predatory stinging. In contrast, the related anemone Exaiptasia diaphana inhabits exposed environments to support photosynthetic endosymbionts. Consistent with its niche, Exaiptasia indiscriminately stings for defense and expresses a CaV splice variant that confers weak inactivation. Chimeric analyses reveal that CaVβ subunit adaptations regulate inactivation, suggesting an evolutionary tuning mechanism for stinging behavior. These findings demonstrate how functional specialization of ion channel structure contributes to distinct organismal behavior.

Numerous proteins regulate gene expression by modulating mRNA translation and decay. To uncover the full scope of these post-transcriptional regulators, we conducted an unbiased survey that quantifies regulatory activity across the budding yeast proteome and delineates the protein domains responsible for these effects. Our approach couples a tethered function assay with quantitative single-cell fluorescence measurements to analyze ~50,000 protein fragments and determine their effects on a tethered mRNA. We characterize hundreds of strong regulators, which are enriched for canonical and unconventional mRNA-binding proteins. Regulatory activity typically maps outside the RNA-binding domains themselves, highlighting a modular architecture that separates mRNA targeting from post-transcriptional regulation. Activity often aligns with intrinsically disordered regions that can interact with other proteins, even in core mRNA translation and degradation factors. Our results thus reveal networks of interacting proteins that control mRNA fate and illuminate the molecular basis for post-transcriptional gene regulation.

Jellyfish and sea anemones fire single-use, venom-covered barbs to immobilize prey or predators. We previously showed that the anemone Nematostella vectensis uses a specialized voltage-gated calcium (CaV) channel to trigger stinging in response to synergistic prey-derived chemicals and touch (Weir et al., 2020). Here we use experiments and theory to find that stinging behavior is suited to distinct ecological niches. We find that the burrowing anemone Nematostella uses uniquely strong CaV inactivation for precise control of predatory stinging. In contrast, the related anemone Exaiptasia diaphana inhabits exposed environments to support photosynthetic endosymbionts. Consistent with its niche, Exaiptasia indiscriminately stings for defense and expresses a CaV splice variant that confers weak inactivation. Chimeric analyses reveal that CaVβ subunit adaptations regulate inactivation, suggesting an evolutionary tuning mechanism for stinging behavior. These findings demonstrate how functional specialization of ion channel structure contributes to distinct organismal behavior.

Voltage-gated sodium (Nav) channels initiate action potentials in most neurons, including primary afferent nerve fibres of the pain pathway. Local anaesthetics block pain through non-specific actions at all Nav channels, but the discovery of selective modulators would facilitate the analysis of individual subtypes of these channels and their contributions to chemical, mechanical, or thermal pain. Here we identify and characterize spider (Heteroscodra maculata) toxins that selectively activate the Nav1.1 subtype, the role of which in nociception and pain has not been elucidated. We use these probes to show that Nav1.1-expressing fibres are modality-specific nociceptors: their activation elicits robust pain behaviours without neurogenic inflammation and produces profound hypersensitivity to mechanical, but not thermal, stimuli. In the gut, high-threshold mechanosensitive fibres also express Nav1.1 and show enhanced toxin sensitivity in a mouse model of irritable bowel syndrome. Together, these findings establish an unexpected role for Nav1.1 channels in regulating the excitability of sensory nerve fibres that mediate mechanical pain.

Animals sense changes in ambient temperature irrespective of whether core body temperature is internally maintained (homeotherms) or subject to environmental variation (poikilotherms). Here we show that a cold-sensitive ion channel, TRPM8, displays dramatically different thermal activation ranges in frogs versus mammals or birds, consistent with variations in these species' cutaneous and core body temperatures. Thus, somatosensory receptors are not static through evolution, but show functional diversity reflecting the characteristics of an organism's ecological niche.

Vampire bats (Desmodus rotundus) are obligate blood feeders that have evolved specialized systems to suit their sanguinary lifestyle. Chief among such adaptations is the ability to detect infrared radiation as a means of locating hotspots on warm-blooded prey. Among vertebrates, only vampire bats, boas, pythons and pit vipers are capable of detecting infrared radiation. In each case, infrared signals are detected by trigeminal nerve fibres that innervate specialized pit organs on the animal's face. Thus, vampire bats and snakes have taken thermosensation to the extreme by developing specialized systems for detecting infrared radiation. As such, these creatures provide a window into the molecular and genetic mechanisms underlying evolutionary tuning of thermoreceptors in a species-specific or cell-type-specific manner. Previously, we have shown that snakes co-opt a non-heat-sensitive channel, vertebrate TRPA1 (transient receptor potential cation channel A1), to produce an infrared detector. Here we show that vampire bats tune a channel that is already heat-sensitive, TRPV1, by lowering its thermal activation threshold to about 30 °C. This is achieved through alternative splicing of TRPV1 transcripts to produce a channel with a truncated carboxy-terminal cytoplasmic domain. These splicing events occur exclusively in trigeminal ganglia, and not in dorsal root ganglia, thereby maintaining a role for TRPV1 as a detector of noxious heat in somatic afferents. This reflects a unique organization of the bat Trpv1 gene that we show to be characteristic of Laurasiatheria mammals (cows, dogs and moles), supporting a close phylogenetic relationship with bats. These findings reveal a novel molecular mechanism for physiological tuning of thermosensory nerve fibres.

While short exposure to solar ultraviolet radiation (UVR) can elicit increased skin pigmentation, a protective response mediated by epidermal melanocytes, chronic exposure can lead to skin cancer and photoaging. However, the molecular mechanisms that allow human skin to detect and respond to UVR remain incompletely understood. UVR stimulates a retinal-dependent signaling cascade in human melanocytes that requires GTP hydrolysis and phospholipase C β (PLCβ) activity. This pathway involves the activation of transient receptor potential A1 (TRPA1) ion channels, an increase in intracellular Ca(2+), and an increase in cellular melanin content. Here, we investigated the identity of the G protein and downstream elements of the signaling cascade and found that UVR phototransduction is Gαq/11 dependent. Activation of Gαq/11/PLCβ signaling leads to hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) to generate diacylglycerol (DAG) and inositol 1, 4, 5-trisphosphate (IP3). We found that PIP2 regulated TRPA1-mediated photocurrents, and IP3 stimulated intracellular Ca(2+) release. The UVR-elicited Ca(2+) response appears to involve both IP3-mediated release from intracellular stores and Ca(2+) influx through TRPA1 channels, showing the fast rising phase of the former and the slow decay of the latter. We propose that melanocytes use a UVR phototransduction mechanism that involves the activation of a Gαq/11-dependent phosphoinositide cascade, and resembles light phototransduction cascades of the eye.

CRISPR/Cas9-mediated transcriptional interference (CRISPRi) enables programmable gene knock-down, yielding loss-of-function phenotypes for nearly any gene. Effective, inducible CRISPRi has been demonstrated in budding yeast, and genome-scale guide libraries enable systematic, genome-wide genetic analysis.

Protein synthesis is a crucial but metabolically costly biological process that must be tightly coordinated with cellular needs and nutrient availability. In response to environmental stress, translation initiation is modulated to control protein output while meeting new demands. The cap-binding protein eIF4E-the earliest contact between mRNAs and the translation machinery-serves as one point of control, but its contributions to mRNA-specific translation regulation remain poorly understood. To survey eIF4E-dependent translational control, we acutely depleted eIF4E and determined how this impacts protein synthesis. Despite its essentiality, eIF4E depletion had surprisingly modest effects on cell growth and protein synthesis. Analysis of transcript-level changes revealed that long-lived transcripts were downregulated, likely reflecting accelerated turnover. Paradoxically, eIF4E depletion led to simultaneous upregulation of genes involved in catabolism of aromatic amino acids, which arose as secondary effects of reduced protein biosynthesis on amino acid pools, and genes involved in the biosynthesis of amino acids. These futile cycles of amino acid synthesis and degradation were driven, in part, by translational activation of GCN4, a transcription factor typically induced by amino acid starvation. Furthermore, we identified a novel regulatory mechanism governing translation of PCL5, a negative regulator of Gcn4, that provides a consistent protein-to-mRNA ratio under varied translation environments. This translational control was partial dependent on a uniquely long poly-(A) tract in the PCL5 5' UTR and on poly-(A) binding protein. Collectively, these results highlight how eIF4E connects translation to amino acid homeostasis and stress responses and uncovers new mechanisms underlying how cells tightly control protein synthesis during environmental challenges.

Nonstructural protein 1 (nsp1) is a coronavirus (CoV) virulence factor that restricts cellular gene expression by inhibiting translation through blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We perform a detailed structure-guided mutational analysis of severe acute respiratory syndrome (SARS)-CoV-2 nsp1, revealing insights into how it coordinates these activities against host but not viral mRNA. We find that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, both enhancing its restriction of host gene expression and enabling mRNA containing the SARS-CoV-2 leader sequence to escape translational repression. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in SARS-CoV-2 nsp1 could attenuate the virus.