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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.

Highlights d Increased lymphatic specification in zebrafish mutants for splicing factor Nova2 d Vascular Nova2 regulates splicing of pre-mRNAs encoding signaling molecules d Nova2-regulated splicing limits MAPK/ERK signaling in developing vessels d Nova2 limits the Vegfc-Flt4-Mapk-Erk-Prox1 axis to restrict lymphatic specification

The Estuarine stonefish (Synanceia horrida) is recognised as one of the most venomous fish species in the world but the overall venom composition has yet to be investigated using indepth transcriptomic and proteomic methods. To date, known venom components are restricted to a hyaluronidase and a large, pore-forming toxin known as Stonustoxin (SNTX). Transcriptomic sequencing of the venom gland resulted in over 170,000 contigs with only 0.4% that were homologous to putative venom proteins. Integration of the transcriptomic data with proteomic data from the S. horrida venom confirmed the hyaluronidase and SNTX to be present, together with several other protein families including major contributions from Ctype lectins. Other protein families observed included peroxiredoxin and several minor protein families such as Golgi-associated plant pathogenesis related proteins, tissue pathway factor inhibitors, and Kazal-type serine protease inhibitors that, although not putative venom proteins, may contribute to the venom's adverse effects. Biological Significance: Proteomic analysis of milked Synanceia horrida venom, paired with transcriptomic analysis of the venom gland tissue revealed for the first time the composition of one of the world's most dangerous fish venoms. The results demonstrate that the venom is relatively less complex compared to other well-studied venomous animals with a number of unique proteins not previously found in animal venoms.

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The lymphatic vasculature develops primarily from pre-existing veins. A pool of lymphatic endothelial cells (LECs) first sprout from cardinal veins followed by migration and proliferation to colonise embryonic tissues. While much is known about the molecular regulation of LEC fate and sprouting during early lymphangiogenesis, we know far less about the instructive and permissive signals that support LEC migration through the embryo. Using a forward genetic screen, we identified mbtps1 and sec23a, components of the COP-II protein secretory pathway, as essential for developmental lymphangiogenesis. In both mutants, LECs initially depart the cardinal vein but then fail in their ongoing migration. A key cargo that failed to be secreted in both mutants was a type II collagen (Col2a1). Col2a1 is normally secreted by notochord sheath cells alongside which LECs migrate. col2a1a mutants displayed defects in the migratory behaviour of LECs and failed lymphangiogenesis. These studies thus identify Col2a1 as a key cargo secreted by notochord sheath cells and required for the migration of LECs. These findings combine with our current understanding to suggest that successive cell-to-cell and cell-matrix interactions regulate the migration of LECs through the embryonic environment during development.

MicroRNAs (miRNAs) are highly conserved ∼22 nt small noncoding RNAs that bind partially complementary sequences in target transcripts. MicroRNAs regulate both translation and transcript stability, and play important roles in development, cellular homeostasis, and disease. There are limited approaches available to agnostically identify microRNA targets transcriptome-wide, and methods using miRNA mimics, which in principle identify direct miRNA:transcript pairs, have low sensitivity and specificity. Here, we describe a novel method to identify microRNA targets using miR-29b mimics containing 3-cyanovinylcarbazole (K), a photolabile nucleoside analog. We demonstrate that biotin-tagged, K-containing miR-29b (K-miR-29b) mimics are nontoxic in cell culture, associate with endogenous mammalian Argonaute2, are sensitive for known targets and recapitulate endogenous transcript destabilization. Partnering K-miR-29b with ultra-low-input RNA sequencing, we recover ∼40% of known miR-29b targets and find conservation of the focal adhesion and apoptotic target pathways in mouse and human. We also identify hundreds of novel targets, including, , and, with a validation rate of 71% for a subset of 73 novel target transcripts interrogated using a high-throughput luciferase assay. Consistent with previous reports, we show that both endogenous miR-29b and K-miR-29b are trafficked to the nucleus, but find no evidence of nuclear-specific miR-29b transcript binding. This may indicate that miR-29b nuclear sequestration is a regulatory mechanism in itself. We suggest thatK-containing small RNA mimics may find applicability in other experimental models.

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