RNA-based applications requiring high quality, non-degraded RNA are a foundational element of many research studies. As such, it is paramount that the integrity of experimental RNA is validated prior to cDNA synthesis or other downstream applications. In the absence of expensive equipment such as microfluidic electrophoretic devices, and as an alternative to the costly and time-consuming standard formaldehyde gel, RNA quality can be quickly analyzed by adding small amounts of commercial bleach to TAE buffer-based agarose gels prior to electrophoresis. In the presence of low concentrations of bleach, the secondary structure of RNA is denatured and potential contaminating RNases are destroyed. Because of this, the ‘bleach gel’ is a functional approach that addresses the need for an inexpensive and safe way to evaluate RNA integrity and will improve the ability of researchers to rapidly analyze RNA quality.

SignificanceThe molecular mechanism for sealing newly formed nuclear envelopes was unclear until the recent discovery that endosomal sorting complexes required for transport III (ESCRT-III) proteins mediate this process. Cmp7p (CHMP7), in particular, was identified as an early acting factor that recruits other ESCRT-III proteins to the nuclear envelope. A fundamental aspect of the varied roles of ESCRT factors is their recruitment by site-specific adaptors, yet the central question of how the ESCRT machinery is targeted to nuclear membranes has remained outstanding. Our study identifies the inner nuclear membrane protein LEM2 as a key, conserved factor that recruits CHMP7 and downstream ESCRT-III proteins to breaches in the nuclear envelope.

Summary Paragraph During cell division, remodeling of the nuclear envelope (NE) enables chromosome segregation by the mitotic spindle 1 . The reformation of sealed nuclei requires Endosomal Sorting Complexes Required for Transport (ESCRTs) and LEM2, a transmembrane ESCRT adapter 2 – 4 . Here, we show how LEM2’s ability to condense on microtubules governs ESCRT activation and coordinated spindle disassembly. The LEM motif of LEM2 binds barrier-to-autointegration factor (BAF), conferring affinity for chromatin 5 , 6 , while an adjacent low complexity domain (LCD) confers the ability to phase separate. A proline-arginine-rich sequence within the LCD binds microtubules, targeting LEM2 condensation to spindle microtubules traversing the nascent NE. Furthermore, LEM2’s winged-helix (WH) domain activates the ESCRT-II/ESCRT-III hybrid protein, CHMP7, to form co-oligomeric rings. Disrupting these events in cells prevented the recruitment of downstream ESCRTs, compromised spindle disassembly, and led to nuclear integrity defects and DNA damage. We propose that during nuclear reassembly, LEM2 condenses into a liquid-like phase and coassembles with CHMP7 to form a macromolecular O-ring seal at the confluence between membranes, chromatin, and the spindle. The properties of LEM2 described here, and the homologous architectures of related inner nuclear membrane proteins 7 , 8 , suggest that phase separation may contribute to other critical envelope functions, including interphase repair 8 – 13 and chromatin organization 14 – 17 .

ESCRT-III proteins have been implicated in sealing the nuclear envelope in mammals, spindle pole body dynamics in fission yeast, and surveillance of defective nuclear pore complexes in budding yeast. Here, we report that Lem2p (LEM2), a member of the LEM (Lap2-Emerin-Man1) family of inner nuclear membrane proteins, and the ESCRT-II/ESCRT-III hybrid protein Cmp7p (CHMP7), work together to recruit additional ESCRT-III proteins to holes in the nuclear membrane. In S. pombe, deletion of the ATPase vps4 leads to severe defects in nuclear morphology and integrity. These phenotypes are suppressed by loss-of-function mutations that arise spontaneously in lem2 or cmp7, implying that these proteins may function upstream in the same pathway. Building on these genetic interactions, we explored the role of LEM2 during nuclear envelope reformation in human cells. We found that CHMP7 and LEM2 enrich at the same region of the chromatin disc periphery during this window of cell division, and that CHMP7 can bind directly to the C-terminal domain of LEM2 in vitro. We further found that, during nuclear envelope formation, recruitment of the ESCRT factors CHMP7, CHMP2A and IST1/CHMP8 all depend on LEM2 in human cells. We conclude that Lem2p/LEM2 is a conserved nuclear sitespecific adaptor that recruits Cmp7p/CHMP7 and downstream ESCRT factors to the nuclear envelope.. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/049312 doi: bioRxiv preprint first posted online Apr. 19, 2016; 3 SignificanceThe molecular mechanism for sealing newly formed nuclear envelopes was unclear until the recent discovery that ESCRT-III proteins mediate this process. CHMP7, in particular, was identified as an early-acting factor that recruits other ESCRT-III proteins to the nuclear envelope.A fundamental aspect of the varied roles of ESCRT factors is their recruitment by site-specific adaptors, yet the central question of how the ESCRT machinery is targeted to nuclear membranes has remained outstanding. Our study identifies the inner nuclear membrane protein LEM2 as a key, conserved factor that recruits CHMP7 and downstream ESCRT-III proteins to breaches in the nuclear envelope.

Chronic inflammation has been recognized as a risk factor for the development and maintenance of malignant disease. Cytokines such as interleukin-6 (IL-6), oncostatin M (OSM), and interleukin-1 beta (IL-1β) promote the development of both acute and chronic inflammation while promoting in vitro metrics of breast cancer metastasis. However, anti-IL-6 and anti-IL-1β therapeutics have not yielded significant results against solid tumors in clinical trials. Here we show that these three cytokines are interrelated in expression. Using the Curtis TCGA™ dataset, we have determined that there is a correlation between expression levels of OSM, IL-6, and IL-1β and reduced breast cancer patient survival ( r = 0.6, p = 2.2 x 10 −23 ). Importantly, we confirm that OSM induces at least a 4-fold increase in IL-6 production from estrogen receptor-negative (ER−) breast cancer cells in a manner that is dependent on STAT3 signaling. Furthermore, OSM induces STAT3 phosphorylation and IL-1β promotes p65 phosphorylation to synergistically induce IL-6 secretion in ER− MDA-MB-231 and to a lesser extent in ER+ MCF7 human breast cancer cells. Induction may be reduced in the ER+ MCF7 cells due to a previously known suppressive interaction between ER and STAT3. Interestingly, we show in MCF7 cells that ER’s interaction with STAT3 is reduced by 50% through both OSM and IL-1β treatment, suggesting a role for ER in mitigating STAT3-mediated inflammatory cascades. Here, we provide a rationale for a breast cancer treatment regime that simultaneously suppresses multiple targets, as these cytokines possess many overlapping functions that increase metastasis and worsen patient survival.

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Breast cancer cell-response to inflammatory cytokines such as interleukin-6 (IL-6) and oncostatin M (OSM) may affect the course of clinical disease in a cancer subtype-dependent manner. Furthermore, vascular endothelial growth factor A (VEGF) secretion induced by IL-6 and OSM may also be subtype-dependent. Utilizing datasets from Oncomine, we show that poor survival of invasive ductal carcinoma (IDC) breast cancer patients is correlated with both high VEGF expression and high cytokine or cytokine receptor expression in tumors. Importantly, epidermal growth factor receptor-negative (HER2-), but not HER2-positive (HER2+), patient survival is significantly lower with high tumor co-expression of VEGF and OSM, OSMRβ, IL-6, or IL-6Rα compared to low co-expression. Furthermore, assessment of HER2- breast cancer cells in vitro identified unique signaling differences regulating cytokine-induced VEGF secretion. The levels of VEGF secretion were analyzed by ELISA with siRNAs for hypoxia inducible factor 1 α (HIF1α) and signal transducer and activator of transcription 3 (STAT3). Specifically, we found that estrogen receptor-negative (ER-) MDA-MB-231 cells respond only to OSM through STAT3 signaling, while ER+ T47D cells respond to both OSM and IL-6, though to IL-6 to a lesser extent. Additionally, in the ER+ T47D cells, OSM signals through both STAT3 and HIF1α. These results highlight that the survival of breast cancer patients with high co-expression of VEGF and IL-6 family cytokines is dependent on breast cancer subtype. Thus, the heterogeneity of human breast cancer in relation to IL-6 family cytokines and VEGF may have important implications in clinical treatment options, disease progression, and ultimately patient prognosis.

At mitotic exit, microtubule arrays are dismantled in concert with the reformation of the nuclear envelope. We show how the inner nuclear membrane protein, LEM2, exploits liquid-liquid phase separation to direct microtubule remodeling and nuclear envelope sealing via the Endosomal Sorting Complexes Required for Transport (ESCRT) pathway. LEM2 tethers membrane to chromatin disks through direct binding between its LEM motif and the chromatin-associated barrier-to-autointegration factor (BAF). Concurrently, a low-complexity domain within LEM2 undergoes liquid-liquid phase separation to coat spindle microtubule bundles. Spatially restricted, LEM2’s winged helix (WH) domain activates the ESCRT-II/ESCRT-III hybrid protein, CHMP7. Together LEM2 and CHMP7 copolymerize around microtubule bundles to form a molecular “O-ring” that promotes nuclear compartmentalization and initiates downstream ESCRT factor recruitment. These results demonstrate how multivalent interactions of a transmembrane protein, including those that mediate phase separation, coordinate localized ESCRT polymerization, mitotic spindle disassembly, and membrane fusion. Defects in this pathway compromise spindle disassembly, nuclear integrity, and genome stability.