Phosphorylation-regulated binding of RNA polymerase II to fibrous polymers of low-complexity domains.

The low-complexity (LC) domains of the products of the fused in sarcoma (FUS), Ewings sarcoma (EWS), and TAF15 genes are translocated onto a variety of different DNA-binding domains and thereby assist in driving the formation of cancerous cells. In the context of the translocated fusion proteins, these LC sequences function as transcriptional activation domains. Here, we show that polymeric fibers formed from these LC domains directly bind the C-terminal domain (CTD) of RNA polymerase II in a manner reversible by phosphorylation of the iterated, heptad repeats of the CTD. Mutational analysis indicates that the degree of binding between the CTD and the LC domain polymers correlates with the strength of transcriptional activation. These studies offer a simple means of conceptualizing how RNA polymerase II is recruited to active genes in its unphosphorylated state and released for elongation following phosphorylation of the CTD.

The microtubule regulator ringer functions downstream from the RNA repair/splicing pathway to promote axon regeneration.

Promoting axon regeneration in the central and peripheral nervous system is of clinical importance in neural injury and neurodegenerative diseases. Both pro- and antiregeneration factors are being identified. We previously reported that the Rtca mediated RNA repair/splicing pathway restricts axon regeneration by inhibiting the nonconventional splicing of Xbp1 mRNA under cellular stress. However, the downstream effectors remain unknown. Here, through transcriptome profiling, we show that the tubulin polymerization-promoting protein (TPPP) ringmaker/ringer is dramatically increased in Rtca-deficient Drosophila sensory neurons, which is dependent on Xbp1. Ringer is expressed in sensory neurons before and after injury, and is cell-autonomously required for axon regeneration. While loss of ringer abolishes the regeneration enhancement in Rtca mutants, its overexpression is sufficient to promote regeneration both in the peripheral and central nervous system. Ringer maintains microtubule stability/dynamics with the microtubule-associated protein futsch/MAP1B, which is also required for axon regeneration. Furthermore, ringer lies downstream from and is negatively regulated by the microtubule-associated deacetylase HDAC6, which functions as a regeneration inhibitor. Taken together, our findings suggest that ringer acts as a hub for microtubule regulators that relays cellular status information, such as cellular stress, to the integrity of microtubules in order to instruct neuroregeneration.

Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies.

Cellular granules lacking boundary membranes harbor RNAs and their associated proteins and play diverse roles controlling the timing and location of protein synthesis. Formation of such granules was emulated by treatment of mouse brain extracts and human cell lysates with a biotinylated isoxazole (b-isox) chemical. Deep sequencing of the associated RNAs revealed an enrichment for mRNAs known to be recruited to neuronal granules used for dendritic transport and localized translation at synapses. Precipitated mRNAs contain extended 3' UTR sequences and an enrichment in binding sites for known granule-associated proteins. Hydrogels composed of the low complexity (LC) sequence domain of FUS recruited and retained the same mRNAs as were selectively precipitated by the b-isox chemical. Phosphorylation of the LC domain of FUS prevented hydrogel retention, offering a conceptual means of dynamic, signal-dependent control of RNA granule assembly.

Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels.

Eukaryotic cells contain assemblies of RNAs and proteins termed RNA granules. Many proteins within these bodies contain KH or RRM RNA-binding domains as well as low complexity (LC) sequences of unknown function. We discovered that exposure of cell or tissue lysates to a biotinylated isoxazole (b-isox) chemical precipitated hundreds of RNA-binding proteins with significant overlap to the constituents of RNA granules. The LC sequences within these proteins are both necessary and sufficient for b-isox-mediated aggregation, and these domains can undergo a concentration-dependent phase transition to a hydrogel-like state in the absence of the chemical. X-ray diffraction and EM studies revealed the hydrogels to be composed of uniformly polymerized amyloid-like fibers. Unlike pathogenic fibers, the LC sequence-based polymers described here are dynamic and accommodate heterotypic polymerization. These observations offer a framework for understanding the function of LC sequences as well as an organizing principle for cellular structures that are not membrane bound.

Implementation of a Suicide Risk Screening Clinical Pathway in a Children's Hospital: A Feasibility Study.

OBJECTIVES: Youth suicide is a pressing global concern. Prior research has developed evidence-driven clinical pathways to screen and identify suicide risk among pediatric patients in outpatient clinics, emergency departments (ED) and inpatient hospital units. However, the feasibility of implementing these pathways remains to be established. Here, we share the results of a hospital-wide "youth suicide risk screening pathway" implementation trial at an urban academic pediatric hospital to address this gap. METHODS: A 3-tier "youth suicide risk screening pathway" using The Ask Suicide-Screening Questions (ASQ) was implemented for patients aged 10 to 26 years who received care at an urban academic pediatric hospital's emergency department or inpatient units. We retrospectively reviewed implementation outcomes of this pathway from January 1 to August 31, 2019. The feasibility of this implementation was measured by assessing the pathway's degree of execution, fidelity, resource utilization, and acceptability. RESULTS: Of 4108 eligible patient encounters, 3424 (83%) completed the screen. Forty-eight (1%) screened acute positive, 263 (8%) screened nonacute positive and 3113 (91%) screened negative. Patients reporting positive suicide risk were more likely to be older and female, although more males required specialty mental health evaluations. Pathway fidelity was 83% among all positive screens and 94% among acute positive screens. The clinical pathway implementation required 16 hours of provider training time and was associated with slightly longer length of stay for inpatients that screened positive (4 vs 3 days). Sixty-five percent of nurses and 78% of social work providers surveyed supported participation in this effort. CONCLUSIONS: It is feasible to implement a youth suicide risk screening pathway without overburdening the system at an urban academic pediatric hospital.

Chemically induced vesiculation as a platform for studying TMEM16F activity.

Calcium-activated phospholipid scramblase mediates the energy-independent bidirectional translocation of lipids across the bilayer, leading to transient or, in the case of apoptotic scrambling, sustained collapse of membrane asymmetry. Cells lacking TMEM16F-dependent lipid scrambling activity are deficient in generation of extracellular vesicles (EVs) that shed from the plasma membrane in a Ca2+-dependent manner, namely microvesicles. We have adapted chemical induction of giant plasma membrane vesicles (GPMVs), which require both TMEM16F-dependent phospholipid scrambling and calcium influx, as a kinetic assay to investigate the mechanism of TMEM16F activity. Using the GPMV assay, we identify and characterize both inactivating and activating mutants that elucidate the mechanism for TMEM16F activation and facilitate further investigation of TMEM16F-mediated lipid translocation and its role in extracellular vesiculation.

Phosphatidylinositol-(4, 5)-bisphosphate regulates calcium gating of small-conductance cation channel TMEM16F.

TMEM16F, which is activated by elevation of intracellular calcium to trigger phospholipid scrambling and the collapse of lipid bilayer asymmetry to mediate important cellular functions such as blood coagulation, also generates a small-conductance calcium-activated cation current. How TMEM16F activation may be regulated is an open question. By recording TMEM16F Ca2+-activated current, we found that the TMEM16F Ca2+-response is desensitized by a brief exposure to high intracellular Ca2+, which is associated with depletion of phosphatidylinositol-(4, 5)-bisphosphate (PIP2) from the inner leaflet of the membrane. Application of artificial or natural PIP2 restores TMEM16F channel activity. PIP2 modulation of TMEM16F requires the presence of several positively charged amino acids in its cytoplasmic N-terminal domain. TMEM16F interaction with PIP2 works synergistically with membrane depolarization to facilitate Ca2+-gating of TMEM16F. Our study reveals the dependence of TMEM16F activity on phosphoinositides and provides one mechanism for TMEM16F activation to be strictly regulated in the cell membrane.

Closing the gap: Solving complex medically relevant genes at scale.

Comprehending the mechanism behind human diseases with an established heritable component represents the forefront of personalized medicine. Nevertheless, numerous medically important genes are inaccurately represented in short-read sequencing data analysis due to their complexity and repetitiveness or the so-called 'dark regions' of the human genome. The advent of PacBio as a long-read platform has provided new insights, yet HiFi whole-genome sequencing (WGS) cost remains frequently prohibitive. We introduce a targeted sequencing and analysis framework, Twist Alliance Dark Genes Panel (TADGP), designed to offer phased variants across 389 medically important yet complex autosomal genes. We highlight TADGP accuracy across eleven control samples and compare it to WGS. This demonstrates that TADGP achieves variant calling accuracy comparable to HiFi-WGS data, but at a fraction of the cost. Thus, enabling scalability and broad applicability for studying rare diseases or complementing previously sequenced samples to gain insights into these complex genes. TADGP revealed several candidate variants across all cases and provided insight into LPA diversity when tested on samples from rare disease and cardiovascular disease cohorts. In both cohorts, we identified novel variants affecting individual disease-associated genes (e.g., IKZF1, KCNE1). Nevertheless, the annotation of the variants across these 389 medically important genes remains challenging due to their underrepresentation in ClinVar and gnomAD. Consequently, we also offer an annotation resource to enhance the evaluation and prioritization of these variants. Overall, we can demonstrate that TADGP offers a cost-efficient and scalable approach to routinely assess the dark regions of the human genome with clinical relevance.

Identification of a drug binding pocket in TMEM16F calcium-activated ion channel and lipid scramblase.

The dual functions of TMEM16F as Ca2+-activated ion channel and lipid scramblase raise intriguing questions regarding their molecular basis. Intrigued by the ability of the FDA-approved drug niclosamide to inhibit TMEM16F-dependent syncytia formation induced by SARS-CoV-2, we examined cryo-EM structures of TMEM16F with or without bound niclosamide or 1PBC, a known blocker of TMEM16A Ca2+-activated Cl- channel. Here, we report evidence for a lipid scrambling pathway along a groove harboring a lipid trail outside the ion permeation pore. This groove contains the binding pocket for niclosamide and 1PBC. Mutations of two residues in this groove specifically affect lipid scrambling. Whereas mutations of some residues in the binding pocket of niclosamide and 1PBC reduce their inhibition of TMEM16F-mediated Ca2+ influx and PS exposure, other mutations preferentially affect the ability of niclosamide and/or 1PBC to inhibit TMEM16F-mediated PS exposure, providing further support for separate pathways for ion permeation and lipid scrambling.

A Case Report of Diffuse Alveolar Hemorrhage Coexisting With Immunoglobulin A (IgA) Nephropathy.

Immunoglobulin A (IgA) nephropathy is the most common cause of primary glomerulonephritis worldwide. IgA vasculitis (formerly known as Henoch-Schonlein purpura) typically presents with IgA nephropathy on renal biopsy in addition to extrarenal symptoms like purpura, abdominal pain, and arthritis. Diffuse alveolar hemorrhage (DAH) is the most common pulmonary complication, but this is rarely seen. In this case report, we describe a 35-year-old male with chronic untreated hepatitis B infection who presented with pulmonary-renal syndrome. He was found to have clinical findings of DAH and concomitant IgA nephropathy on renal biopsy, without having any other typical manifestations of IgA vasculitis. This shows that IgA nephropathy should be considered in the differential diagnosis of DAH and emphasizes the importance of a renal biopsy in patients presenting with pulmonary-renal syndrome.

Identification of a conserved drug binding pocket in TMEM16 proteins.

The TMEM16 family of calcium-activated membrane proteins includes ten mammalian paralogs (TMEM16A-K) playing distinct physiological roles with some implicated in cancer and airway diseases. Their modulators with therapeutic potential include 1PBC, a potent inhibitor with anti-tumoral properties, and the FDA-approved drug niclosamide that targets TMEM16F to inhibit syncytia formation induced by SARS-CoV-2 infection. Here, we report cryo-EM structures of TMEM16F associated with 1PBC and niclosamide, revealing that both molecules bind the same drug binding pocket. We functionally and computationally validate this binding pocket in TMEM16A as well as TMEM16F, thereby showing that drug modulation also involves residues that are not conserved between TMEM16A and TMEM16F. This study establishes a much-needed structural framework for the development of more potent and more specific drug molecules targeting TMEM16 proteins.

Dynamic change of electrostatic field in TMEM16F permeation pathway shifts its ion selectivity.

TMEM16F is activated by elevated intracellular Ca2+, and functions as a small-conductance ion channel and as a phospholipid scramblase. In contrast to its paralogs, the TMEM16A/B calcium-activated chloride channels, mouse TMEM16F has been reported as a cation-, anion-, or non-selective ion channel, without a definite conclusion. Starting with the Q559K mutant that shows no current rundown and less outward rectification in excised patch, we found that the channel shifted its ion selectivity in response to the change of intracellular Ca2+ concentration, with an increased permeability ratio of Cl- to Na+ (PCl-/PNa+) at a higher Ca2+ level. The gradual shift of relative ion permeability did not correlate with the channel activation state. Instead, it was indicative of an alteration of electrostatic field in the permeation pathway. The dynamic change of ion selectivity suggests a charge-screening mechanism for TMEM16F ion conduction, and it provides hints to further studies of TMEM16F physiological functions.

Cryo-EM Studies of TMEM16F Calcium-Activated Ion Channel Suggest Features Important for Lipid Scrambling.

As a Ca2+-activated lipid scramblase and ion channel that mediates Ca2+ influx, TMEM16F relies on both functions to facilitate extracellular vesicle generation, blood coagulation, and bone formation. How a bona fide ion channel scrambles lipids remains elusive. Our structural analyses revealed the coexistence of an intact channel pore and PIP2-dependent protein conformation changes leading to membrane distortion. Correlated to the extent of membrane distortion, many tightly bound lipids are slanted. Structure-based mutagenesis studies further reveal that neutralization of some lipid-binding residues or those near membrane distortion specifically alters the onset of lipid scrambling, but not Ca2+ influx, thus identifying features outside of channel pore that are important for lipid scrambling. Together, our studies demonstrate that membrane distortion does not require open hydrophilic grooves facing the membrane interior and provide further evidence to suggest separate pathways for lipid scrambling and ion permeation.

A web based resource characterizing the zebrafish developmental profile of over 16,000 transcripts.

Using a spotted 65-mer oligonucleotide microarray, we have characterized the developmental expression profile from mid-gastrulation (75% epiboly) to 5 days post-fertilization (dpf) for >16,000 unique transcripts in the zebrafish genome. Microarray profiling data sets are often immense, and one challenge is validating the results and prioritizing genes for further study. The purpose of the current study was to address such issues, as well as to generate a publicly available resource for investigators to examine the developmental expression profile of any of the over 16,000 zebrafish genes on the array. On the chips, there are 16,459 printed spots corresponding to 16,288 unique transcripts and 172 beta-actin (AF025305) spots spatially distributed throughout the chip as a positive control. We have collected 55 microarray gene expression profiling results from various zebrafish laboratories and created a Perl/CGI-based software tool (http://serine.umdnj.edu/approximately ouyangmi/cgi-bin/zebrafish/profile.htm) for researchers to look for the expression patterns of their gene of interest. Users can search for their genes of interest by entering the accession numbers or the nucleotide sequences and the expression profiling will be reported in the form of expression intensities versus time-course graphical displays. In order to validate this web tool, we compared 74 genes' expression results between our web tool and the in situ hybridization results from Thisse et al. [Thisse, B., Heyer, V., Lux, A., Alunni, A., Degrave, A., Seiliez, I., Kirchner, J., Parkhill, J.-P., Thisse, C., 2004. Spatial and temporal expression of the zebrafish genome by large-scale in situ hybridization screening. Meth. Cell. Biol. 77, 505-519] as well as those reported by Mathavan et al. [Mathavan, S., Lee, S.G., mark, A., Miller, L.D., Murthy, K.R., Tong, Y., Wu, Y.L., Lam, S.H., Yang, H., Ruan, Y., Korzh, V., Gong, Z., Liu, E.T., Lufkin, T., 2005. Transcriptome analysis of zebrafish embryogenesis using microarrays. PLoS Genet. 1, 260-276]. The comparison indicates that our microarray-derived expression patterns are 80% and 75% in agreement with the in situ database (Thisse et al., 2004) and previously published microarray data (Mathavan et al., 2005), respectively. Those genes that conflict between our web tool and the in situ database either have high sequence similarity with other genes or the in situ probes are not reliable. Among those genes that disagree between our web tool and those reported by Mathavan et al. (2005), 93% of the genes are in agreement between our web tool and the in situ database, indicating our web tool results are quite reliable. Thus, this resource provides a user-friendly web based platform for researchers to determine the developmental profile of their gene of interest and to prioritize genes identified in microarray analyses by their developmental expression profile.

Identification of direct T-box target genes in the developing zebrafish mesoderm.

The zebrafish genes spadetail (spt) and no tail (ntl) encode T-box transcription factors that are important for early mesoderm development. Although much has been done to characterize these genes, the identity and location of target regulatory elements remain largely unknown. Here, we survey the genome for downstream target genes of the Spt and Ntl T-box transcription factors. We find evidence for extensive additive interactions towards gene activation and limited evidence for combinatorial and antagonistic interactions between the two factors. Using in vitro binding selection assays to define Spt- and Ntl-binding motifs, we searched for target regulatory sequence via a combination of binding motif searches and comparative genomics. We identified regulatory elements for tbx6 and deltaD, and, using chromatin immunoprecipitation, in vitro DNA binding assays and transgenic methods, we provide evidence that both are directly regulated by T-box transcription factors. We also find that deltaD is directly activated by T-box factors in the tail bud, where it has been implicated in starting the segmentation clock, suggesting that spt and ntl act upstream of this process.

The neuronal PAS domain protein 3 transcription factor controls FGF-mediated adult hippocampal neurogenesis in mice.

The neuronal PAS domain protein 3 (NPAS3) gene encoding a brain-enriched transcription factor was recently found to be disrupted in a family suffering from schizophrenia. Mice harboring compound disruptions in the NPAS3 and related NPAS1 genes manifest behavioral and neuroanatomical abnormalities reminiscent of schizophrenia. Herein we demonstrate that Npas3-/- mice are deficient in expression of hippocampal FGF receptor subtype 1 mRNA, most notably in the dentate gyrus. In vivo BrdUrd-labeling shows that basal neural precursor cell proliferation in the dentate gyrus of Npas3-/- mice is reduced by 84% relative to wild-type littermates. We propose that a deficiency in adult neurogenesis may cause the behavioral and neuroanatomical abnormalities seen in Npas3-/- mice, and we speculate that impaired neurogenesis may be involved in the pathophysiology of schizophrenia.

Organizational effects of testosterone, estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis.

In adulthood, male rats express higher levels of arginine vasopressin (AVP) mRNA in the bed nucleus of the stria terminalis (BST) than do female rats. We tested whether this sex difference is primarily due to differences in neonatal levels of testosterone. Male and female rats were gonadectomized on the day of birth and treated with testosterone propionate (TP) or vehicle on postnatal days 1, 3, and 5 (P1, P3, and P5). Three months later, all rats were implanted with testosterone-filled capsules. Two weeks later, brains were processed for in situ hybridization to detect AVP mRNA. We found that neonatal TP treatment significantly increased the number of vasopressinergic cells in the BST over control injections. We then sought to determine the effects of testosterone metabolites, estradiol and dihydrotestosterone, given alone or in combination, on AVP expression in the BST. Rat pups were treated as described above, except that instead of testosterone, estradiol benzoate (EB), dihydrotestosterone propionate (DHTP), a combination of EB and DHTP (EB+DHTP), or vehicle was injected neonatally. Neonatal treatment with either EB or EB+DHTP increased the number of vasopressinergic cells in the BST over that of DHTP or oil treatment. However, treatment with DHTP also significantly increased the number of vasopressinergic cells over that of oil treatment. Hence, in addition to bolstering evidence that estradiol is the more potent metabolite of testosterone in causing sexual differentiation of the brain, these data provide the first example of a masculinizing effect of a nonaromatizable androgen on a sexually dimorphic neuropeptide system.