Gregory J. Baillie

Significance: The heart initiates rhythmic contractions early during development. A unique zebrafish mutant with specific cardiac arrhythmia phenotypes was identified and mutation of the uncharacterizedtmem161bgene shown to be causative. Tmem161b knockout mice are perinatal lethal and isolated embryonic cardiomyocytes exhibit arrhythmic Ca²⁺oscillations, suggesting functional conservation. Protein localization studies show Tmem161b is expressed in excitable cells and at cardiomyocyte plasma membranes. Imaging of in vivo action potentials showstmem161bmutants have prolonged action potential repolarization. Patch-clamping of isolated cardiomyocytes confirms prolonged action potential repolarization and shows increased Ca²⁺and K⁺currents are likely the cause of altered action potential morphology. This suggests Tmem161b is a new conserved regulator of cardiac rhythm by inhibiting Ca²⁺and K⁺currents.

Background: During heart morphogenesis, the cardiac chambers undergo ballooning: a process involving regionalized elongation of cardiomyocytes. Cardiomyocyte shape changes require reorganization of the actin cytoskeleton; however, the genetic regulation of this process is not well understood.Results: From a forward genetic screen, we identified the zebrafish uq ^23ks mutant which manifests chamber ballooning defects. Whole‐genome sequencing‐mapping identified a truncating mutation in the gene, myo5b. myo5b encodes an atypical myosin required for endosome recycling and, consistent with this, increased vesicles were observed in myo5b mutant cardiomyocytes. Expression of RFP‐Rab11a (a recycling endosome marker) confirmed increased recycling endosomes in cardiomyocytes of myo5b mutants. To investigate potential cargo of MyoVb‐associated vesicles, we examined the adherens junction protein, N‐cadherin. N‐cadherin appeared mispatterned at cell junctions, and an increase in the number of intracellular particles was also apparent. Co‐localization with RFP‐Rab11a confirmed increased N‐cadherin‐positive recycling endosomes, demonstrating N‐cadherin trafficking is perturbed in myo5b mutants. Finally, phalloidin staining showed disorganized F‐actin in myo5b cardiomyocytes, suggesting the cytoskeleton fails to remodel, obstructing chamber ballooning.Conclusions: MyoVb is required for cardiomyocyte endosomal recycling and appropriate N‐cadherin localization during the onset of chamber ballooning. Cardiomyocytes lacking MyoVb are unable to reorganize their actin cytoskeleton, resulting in failed chamber ballooning. Developmental Dynamics 248:284–295, 2019.

The remodeling of specific calcium-permeable ion channels is a feature of some breast cancer subtypes. ORAI1 is a protein that forms a calcium-permeable ion channel responsible for store-operated calcium entry (SOCE) in a variety of cell types. ORAI3, a related isoform, is not a regulator of SOCE in most cell types. However, ORAI3 does control SOCE in many estrogen receptor-positive breast cancer cell lines, where it also controls proliferation. ORAI1 is a well-characterized regulator of the proliferation and migration of many basal breast cancer cells; however, the role of ORAI3 in these types of breast cancer cells remains unclear. Here, we sought to define ORAI1 and ORAI3 expression in breast cancer cell lines of different molecular subtypes and assess the potential role and regulation of ORAI3 in basal breast cancer cells. Our study demonstrates that elevated ORAI1 is a feature of basal-like breast cancers, while elevated ORAI3 is a feature of luminal breast cancers. Intriguingly, we found that ORAI3 is over-expressed in the mesenchymal subtype of triple-negative breast cancer. Given this, we assessed ORAI3 levels in the presence of two inducers of the mesenchymal phenotype, hypoxia and epidermal growth factor (EGF). Hypoxia induced ORAI3 levels in basal breast cancer cell lines through a pathway involving hypoxia-inducible factor-1 alpha (HIF1α. The silencing of ORAI3 attenuated hypoxia-associated phosphorylation of the EGF receptor (EGFR) and the expression of genes associated with cell migration and inflammatory/immune responses in the MDA-MB-468 model of basal breast cancer. Although elevated ORAI3 levels were not associated with survival; basal, estrogen receptor-negative and triple-negative breast cancers with high ORAI3 and low ORAI1 levels were associated with poorer clinical outcomes. This study defines ORAI3 as a potential fine-tuner for processes relevant to the progression of basal breast cancers.

The platypus (Ornithorhynchus anatinus) is an egg-laying monotreme mammal whose ancestors diverged approximately 166 million years ago from the evolutionary pathway that eventually gave rise to both marsupial and eutherian mammals. Consequently, its genome is an extraordinary amalgam of both ancestral reptilian and derived mammalian features. To gain insight into the evolution of mammalian pluripotency we have generated induced pluripotent stem cells (piPSCs) from the platypus. Deep sequencing of the piPSC transcriptome revealed that piPSCs robustly express the core eutherian pluripotency factors OCT4, SOX2 and NANOG. Given the more extensive role of SOX3 over SOX2 in avian pluripotency, our data indicate that between 315 million years and 166 million years ago primitive mammals replaced the role of SOX3 in the vertebrate pluripotency network with SOX2. DAX1/NR0B1 is not expressed in piPSCs and an analysis of the platypus DAX1 promoter revealed the absence of a proximal SOX2-binding DNA motif known to be critical for DAX1 expression in eutherian pluripotent stem cells, suggesting that the acquisition of SOX2 responsiveness by DAX1 has facilitated its recruitment into the pluripotency network of eutherians. Using the RNAseq data, we were also able to demonstrate that in both fibroblasts and piPSCs the expression ratio of X chromosomes to autosomes (X1-5 X1-5:AA) is approximately equal to 1 indicating that there is no upregulation of X-linked genes. Finally, the RNAseq data also allowed us to explore the process of X-linked gene inactivation in the platypus, where we determined that for any given gene there is no preference for silencing of the maternal or paternal allele; that is, within a population of cells the silencing of X-linked genes is not imprinted.

Summary Statement: The generation and transcriptome analysis of the first induced pluripotent stem cells from the platypus reveals SOX2 has been a key driver of the expanded pluripotency regulatory network in placental mammals. The mechanisms by which pluripotency has evolved remain unclear. To gain insight into the evolution of mammalian pluripotency we have generated induced pluripotent stem cells (piPSCs) from the platypus. Deep sequencing of the piPSC transcriptome revealed that piPSCs robustly express the core eutherian pluripotency factors OCT4, SOX2 and NANOG . Given the more extensive role of SOX3 over SOX2 in avian pluripotency, our data indicate that between 315 million years and 166 million years ago primitive mammals replaced the role of SOX3 in the vertebrate pluripotency network with SOX2. DAX1/NR0B1 is not expressed in piPSCs and an analysis of the platypus DAX1 promoter revealed the absence of a proximal SOX2-binding DNA motif known to be critical for DAX1 expression in eutherian pluripotent stem cells, suggesting that the acquisition of SOX2 responsiveness by DAX1 has facilitated its recruitment into the pluripotency network of eutherians. We further show that the expression ratio of X chromosomes to autosomes (X _1-5 X _1-5 :AA) is approximately equal to 1 indicating that there is no upregulation of X-linked genes and that there is no preference for silencing of maternal or paternal alleles (ie imprinting).

Transcription initiation RNAs (tiRNAs) are small, predominantly 18 nt, RNAs whose biogenesis is associated with nucleosomes adjacent to active transcription initiation sites. These loci usually contain modified histones associated with transcription initiation, including histone H3 trimethylated at lysine 4 (H3K4me3). To further characterize the relationship of tiRNAs and H3K4me3 marked nucleosomes, H3K4me3-targeted RNA:chromatin immunoprecipitations were performed in a murine macrophage cell line, and small RNA sequence libraries were constructed and subjected to deep sequencing. The H3K4me3 libraries exhibited a distinct profile of read lengths with a noticeable enrichment of sequences 17-26 nt in length, with a peak at ∼18nt that included tiRNAs. These RNAs show clear enrichment of sequences that map to genomic features known to be associated with transcription initiation, including CAGE transcription initiation sites (TSSs), sites of RNAPII occupancy, and H3K4me3 sites. The distribution of sequences that map in the vicinity of TSSs is consistent with previous descriptions of tiRNAs; viz . a major peak at approximately 40 nt downstream of the TSS, and a minor broader peak approximately 150-200 nt upstream of, and on the opposite strand to, the TSS. These results show that tiRNAs are physically associated with H3K4me3-marked chromatin. tiRNAs may be markers of RNAPII pausing and it remains a possibility that their association with H3K4me3 is part of an epigenetic signaling system.

A monthly journal publishing high-quality, peer-reviewed research on all topics related to RNA and its metabolism in all organisms

Millions of years of evolution have fine-tuned the ability of venom peptides to rapidly incapacitate both prey and potential predators. Toxicofera reptiles are characterized by serous-secreting mandibular or maxillary glands with heightened levels of protein expression. These glands are the core anatomical components of the toxicoferan venom system, which exists in myriad points along an evolutionary continuum. Neofunctionalisation of toxins is facilitated by positive selection at functional hotspots on the ancestral protein and venom proteins have undergone dynamic diversification in helodermatid and varanid lizards as well as advanced snakes. A spectacular point on the venom system continuum is the long-glanded blue coral snake (Calliophis bivirgatus), a specialist feeder that preys on fast moving, venomous snakes which have both a high likelihood of prey escape but also represent significant danger to the predator itself. The maxillary venom glands of C. bivirgatus extend one quarter of the snake’s body length and nestle within the rib cavity. Despite the snake’s notoriety its venom has remained largely unstudied. Here we show that the venom uniquely produces spastic paralysis, in contrast to the flaccid paralysis typically produced by neurotoxic snake venoms. The toxin responsible, which we have called calliotoxin (δ-elapitoxin-Cb1a), is a three-finger toxin (3FTx). Calliotoxin shifts the voltage-dependence of Na_V1.4 activation to more hyperpolarised potentials, inhibits inactivation, and produces large ramp currents, consistent with its profound effects on contractile force in an isolated skeletal muscle preparation. Voltage-gated sodium channels (Na_V) are a particularly attractive pharmacological target as they are involved in almost all physiological processes including action potential generation and conduction. Accordingly, venom peptides that interfere with Na_V function provide a key defensive and predatory advantage to a range of invertebrate venomous species including cone snails, scorpions, spiders, and anemones. Enhanced activation or delayed inactivation of sodium channels by toxins is associated with the extremely rapid onset of tetanic/excitatory paralysis in envenomed prey animals. A strong selection pressure exists for the evolution of such toxins where there is a high chance of prey escape. However, despite their prevalence in other venomous species, toxins causing delay of sodium channel inhibition have never previously been described in vertebrate venoms. Here we show that Na_V modulators, convergent with those of invertebrates, have evolved in the venom of the long-glanded coral snake. Calliotoxin represents a functionally novel class of 3FTx and a structurally novel class of Na_V toxins that will provide significant insights into the pharmacology and physiology of Na_V. The toxin represents a remarkable case of functional convergence between invertebrate and vertebrate venom systems in response to similar selection pressures. These results underscore the dynamic evolution of the Toxicofera reptile system and reinforces the value of using evolution as a roadmap for biodiscovery.

Here we report whole exome sequencing (WES) on a cohort of 71 patients with persistently unresolved white matter abnormalities with a suspected diagnosis of leukodystrophy or genetic leukoencephalopathy. WES analyses were performed on trio, or greater, family groups. Diagnostic pathogenic variants were identified in 35% (25 of 71) of patients. Potentially pathogenic variants were identified in clinically relevant genes in a further 7% (5 of 71) of cases, giving a total yield of clinical diagnoses in 42% of individuals. These findings provide evidence that WES can substantially decrease the number of unresolved white matter cases. Ann Neurol 2016;79:1031–1037