Establishing RNA-RNA interactions remodels lncRNA structure and promotes PRC2 activity.

Human Polycomb Repressive Complex 2 (PRC2) catalysis of histone H3 lysine 27 methylation at certain loci depends on long noncoding RNAs (lncRNAs). Yet, in apparent contradiction, RNA is a potent catalytic inhibitor of PRC2. Here, we show that intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its targets can relieve RNA inhibition of PRC2. RNA bridging is promoted by heterogeneous nuclear ribonucleoprotein B1, which uses multiple protein domains to bind HOTAIR regions via multivalent protein-RNA interactions. Chemical probing demonstrates that establishing RNA-RNA interactions changes HOTAIR structure. Genome-wide HOTAIR/PRC2 activity occurs at genes whose transcripts can make favorable RNA-RNA interactions with HOTAIR. We demonstrate that RNA-RNA matches of HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity after B1 dissociation. Our work highlights an intrinsic switch that allows PRC2 activity in specific RNA contexts, which could explain how many lncRNAs work with PRC2.

RNA matchmaking in chromatin regulation.

Beyond being the product of gene expression, RNA can also influence the regulation of chromatin. The majority of the human genome has the capacity to be transcribed and the majority of the non-protein-coding transcripts made by RNA Polymerase II are enriched in the nucleus. Many chromatin regulators can bind to these ncRNAs in the nucleus; in some cases, there are clear examples of direct RNA-mediated chromatin regulation mechanisms stemming from these interactions, while others have yet to be determined. Recent studies have highlighted examples of chromatin regulation via RNA matchmaking, a term we use broadly here to describe intermolecular base-pairing interactions between one RNA molecule and an RNA or DNA match. This review provides examples of RNA matchmaking that regulates chromatin processes and summarizes the technical approaches used to capture these events.

Selinexor-induced thrombocytopenia results from inhibition of thrombopoietin signaling in early megakaryopoiesis.

Selinexor is the first oral selective inhibitor of nuclear export compound tested for cancer treatment. Selinexor has demonstrated a safety therapy profile with broad antitumor activity against solid and hematological malignancies in phases 2 and 3 clinical trials (#NCT03071276, #NCT02343042, #NCT02227251, #NCT03110562, and #NCT02606461). Although selinexor shows promising efficacy, its primary adverse effect is high-grade thrombocytopenia. Therefore, we aimed to identify the mechanism of selinexor-induced thrombocytopenia to relieve it and improve its clinical management. We determined that selinexor causes thrombocytopenia by blocking thrombopoietin (TPO) signaling and therefore differentiation of stem cells into megakaryocytes. We then used both in vitro and in vivo models and patient samples to show that selinexor-induced thrombocytopenia is indeed reversible when TPO agonists are administered in the absence of selinexor (drug holiday). In sum, these data reveal (1) the mechanism of selinexor-induced thrombocytopenia, (2) an effective way to reverse the dose-limiting thrombocytopenia, and (3) a novel role for XPO1 in megakaryopoiesis. The improved selinexor dosing regimen described herein is crucial to help reduce thrombocytopenia in selinexor patients, allowing them to continue their course of chemotherapy and have the best chance of survival. This trial was registered at www.clinicaltrials.gov as #NCT01607905.

Synthesis and dephosphorylation of MARCKS in the late stages of megakaryocyte maturation drive proplatelet formation.

Platelets are essential for hemostasis, and thrombocytopenia is a major clinical problem. Megakaryocytes (MKs) generate platelets by extending long processes, proplatelets, into sinusoidal blood vessels. However, very little is known about what regulates proplatelet formation. To uncover which proteins were dynamically changing during this process, we compared the proteome and transcriptome of round vs proplatelet-producing MKs by 2D difference gel electrophoresis (DIGE) and polysome profiling, respectively. Our data revealed a significant increase in a poorly-characterized MK protein, myristoylated alanine-rich C-kinase substrate (MARCKS), which was upregulated 3.4- and 5.7-fold in proplatelet-producing MKs in 2D DIGE and polysome profiling analyses, respectively. MARCKS is a protein kinase C (PKC) substrate that binds PIP2. In MKs, it localized to both the plasma and demarcation membranes. MARCKS inhibition by peptide significantly decreased proplatelet formation 53%. To examine the role of MARCKS in the PKC pathway, we treated MKs with polymethacrylate (PMA), which markedly increased MARCKS phosphorylation while significantly inhibiting proplatelet formation 84%, suggesting that MARCKS phosphorylation reduces proplatelet formation. We hypothesized that MARCKS phosphorylation promotes Arp2/3 phosphorylation, which subsequently downregulates proplatelet formation; both MARCKS and Arp2 were dephosphorylated in MKs making proplatelets, and Arp2 inhibition enhanced proplatelet formation. Finally, we used MARCKS knockout (KO) mice to probe the direct role of MARCKS in proplatelet formation; MARCKS KO MKs displayed significantly decreased proplatelet levels. MARCKS expression and signaling in primary MKs is a novel finding. We propose that MARCKS acts as a "molecular switch," binding to and regulating PIP2 signaling to regulate processes like proplatelet extension (microtubule-driven) vs proplatelet branching (Arp2/3 and actin polymerization-driven).

CCL5 derived from platelets increases megakaryocyte proplatelet formation.

In times of physiological stress, platelet count can transiently rise. What initiates this reactive thrombocytosis is poorly understood. Intriguingly, we found that treating megakaryocytes (MKs) with the releasate from activated platelets increased proplatelet production by 47%. Platelets store inflammatory cytokines, including the chemokine ligand 5 (CCL5, RANTES); after TRAP activation, platelets release over 25 ng/mL CCL5. We hypothesized that CCL5 could regulate platelet production by binding to its receptor, CCR5, on MKs. Maraviroc (CCR5 antagonist) or CCL5 immunodepletion diminished 95% and 70% of the effect of platelet releasate, respectively, suggesting CCL5 derived from platelets is sufficient to drive increased platelet production through MK CCR5. MKs cultured with recombinant CCL5 increased proplatelet production by 50% and had significantly higher ploidy. Pretreating the MK cultures with maraviroc prior to exposure to CCL5 reversed the augmented proplatelet formation and ploidy, suggesting that CCL5 increases MK ploidy and proplatelet formation in a CCR5-dependent manner. Interrogation of the Akt signaling pathway suggested that CCL5/CCR5 may influence proplatelet production by suppressing apoptosis. In an in vivo murine acute colitis model, platelet count significantly correlated with inflammation whereas maraviroc treatment abolished this correlation. We propose that CCL5 signaling through CCR5 may increase platelet counts during physiological stress.