RNA binding protein SYNCRIP maintains proteostasis and self-renewal of hematopoietic stem and progenitor cells.

Tissue homeostasis is maintained after stress by engaging and activating the hematopoietic stem and progenitor compartments in the blood. Hematopoietic stem cells (HSCs) are essential for long-term repopulation after secondary transplantation. Here, using a conditional knockout mouse model, we revealed that the RNA-binding protein SYNCRIP is required for maintenance of blood homeostasis especially after regenerative stress due to defects in HSCs and progenitors. Mechanistically, we find that SYNCRIP loss results in a failure to maintain proteome homeostasis that is essential for HSC maintenance. SYNCRIP depletion results in increased protein synthesis, a dysregulated epichaperome, an accumulation of misfolded proteins and induces endoplasmic reticulum stress. Additionally, we find that SYNCRIP is required for translation of CDC42 RHO-GTPase, and loss of SYNCRIP results in defects in polarity, asymmetric segregation, and dilution of unfolded proteins. Forced expression of CDC42 recovers polarity and in vitro replating activities of HSCs. Taken together, we uncovered a post-transcriptional regulatory program that safeguards HSC self-renewal capacity and blood homeostasis.

BTG1 mutation yields supercompetitive B cells primed for malignant transformation.

Multicellular life requires altruistic cooperation between cells. The adaptive immune system is a notable exception, wherein germinal center B cells compete vigorously for limiting positive selection signals. Studying primary human lymphomas and developing new mouse models, we found that mutations affecting BTG1 disrupt a critical immune gatekeeper mechanism that strictly limits B cell fitness during antibody affinity maturation. This mechanism converted germinal center B cells into supercompetitors that rapidly outstrip their normal counterparts. This effect was conferred by a small shift in MYC protein induction kinetics but resulted in aggressive invasive lymphomas, which in humans are linked to dire clinical outcomes. Our findings reveal a delicate evolutionary trade-off between natural selection of B cells to provide immunity and potentially dangerous features that recall the more competitive nature of unicellular organisms.

Transcriptional control of CBX5 by the RNA binding proteins RBMX and RBMXL1 maintains chromatin state in myeloid leukemia.

RNA binding proteins (RBPs) are key arbiters of post-transcriptional regulation and are found to be found dysregulated in hematological malignancies. Here, we identify the RBP RBMX and its retrogene RBMXL1 to be required for murine and human myeloid leukemogenesis. RBMX/L1 are overexpressed in acute myeloid leukemia (AML) primary patients compared to healthy individuals, and RBMX/L1 loss delayed leukemia development. RBMX/L1 loss lead to significant changes in chromatin accessibility, as well as chromosomal breaks and gaps. We found that RBMX/L1 directly bind to mRNAs, affect transcription of multiple loci, including CBX5 (HP1α), and control the nascent transcription of the CBX5 locus. Forced CBX5 expression rescued the RBMX/L1 depletion effects on cell growth and apoptosis. Overall, we determine that RBMX/L1 control leukemia cell survival by regulating chromatin state through their downstream target CBX5. These findings identify a mechanism for RBPs directly promoting transcription and suggest RBMX/L1, as well as CBX5, as potential therapeutic targets in myeloid malignancies.

N6-Methyladenosine on mRNA facilitates a phase-separated nuclear body that suppresses myeloid leukemic differentiation.

N6-Methyladenosine (m6A) on mRNAs mediates different biological processes and its dysregulation contributes to tumorigenesis. How m6A dictates its diverse molecular and cellular effects in leukemias remains unknown. We found that YTHDC1 is the essential m6A reader in myeloid leukemia from a genome-wide CRISPR screen and that m6A is required for YTHDC1 to undergo liquid-liquid phase separation and form nuclear YTHDC1-m6A condensates (nYACs). The number of nYACs increases in acute myeloid leukemia (AML) cells compared with normal hematopoietic stem and progenitor cells. AML cells require the nYACs to maintain cell survival and the undifferentiated state that is critical for leukemia maintenance. Furthermore, nYACs enable YTHDC1 to protect m6A-mRNAs from the PAXT complex and exosome-associated RNA degradation. Collectively, m6A is required for the formation of a nuclear body mediated by phase separation that maintains mRNA stability and control cancer cell survival and differentiation.

HyperTRIBE uncovers increased MUSASHI-2 RNA binding activity and differential regulation in leukemic stem cells.

The cell-context dependency for RNA binding proteins (RBPs) mediated control of stem cell fate remains to be defined. Here we adapt the HyperTRIBE method using an RBP fused to a Drosophila RNA editing enzyme (ADAR) to globally map the mRNA targets of the RBP MSI2 in mammalian adult normal and malignant stem cells. We reveal a unique MUSASHI-2 (MSI2) mRNA binding network in hematopoietic stem cells that changes during transition to multipotent progenitors. Additionally, we discover a significant increase in RNA binding activity of MSI2 in leukemic stem cells compared with normal hematopoietic stem and progenitor cells, resulting in selective regulation of MSI2's oncogenic targets. This provides a basis for MSI2 increased dependency in leukemia cells compared to normal cells. Moreover, our study provides a way to measure RBP function in rare cells and suggests that RBPs can achieve differential binding activity during cell state transition independent of gene expression.

m6A RNA Methylation Maintains Hematopoietic Stem Cell Identity and Symmetric Commitment.

Stem cells balance cellular fates through asymmetric and symmetric divisions in order to self-renew or to generate downstream progenitors. Symmetric commitment divisions in stem cells are required for rapid regeneration during tissue damage and stress. The control of symmetric commitment remains poorly defined. Using single-cell RNA sequencing (scRNA-seq) in combination with transcriptomic profiling of HSPCs (hematopoietic stem and progenitor cells) from control and m6A methyltransferase Mettl3 conditional knockout mice, we found that m6A-deficient hematopoietic stem cells (HSCs) fail to symmetrically differentiate. Dividing HSCs are expanded and are blocked in an intermediate state that molecularly and functionally resembles multipotent progenitors. Mechanistically, RNA methylation controls Myc mRNA abundance in differentiating HSCs. We identified MYC as a marker for HSC asymmetric and symmetric commitment. Overall, our results indicate that RNA methylation controls symmetric commitment and cell identity of HSCs and may provide a general mechanism for how stem cells regulate differentiation fate choice.

A novel application of membrane distillation to facilitate nickel recovery from electroplating wastewater.

In many years, the nickel electroplating technique has been applied to coat nickel on other materials for their increased properties. Nickel electroplating has played a vital role in our modern society but also caused considerable environmental concerns due to the mass discharge of its wastewater (i.e. containing nickel and other heavy metals) to the environment. Thus, there is a growing need for treating nickel electroplating wastewater to protect the environment and, in tandem, recover nickel for beneficial use. This study explores a novel application of membrane distillation (MD) for the treatment of nickel electroplating wastewater for a dual purpose: facilitating the nickel recovery and obtaining fresh water. The experimental results demonstrate the technical capability of MD to pre-concentrate nickel in the wastewater (i.e. hence pave the way for subsequent nickel recovery via chemical precipitation or electrodeposition) and extract fresh water. At a low operating feed temperature of 60 °C, the MD process increased the nickel content in the wastewater by more than 100-fold from 0.31 to 33 g/L with only a 20% reduction in the process water flux and obtained pure fresh water. At such high concentration factors, the membrane surface was slightly fouled by inorganic precipitates; however, membrane pore wetting was not evident, confirmed by the purity of the obtained fresh water. The fouled membrane was effectively cleaned using a 3% HCl solution to restore its surface morphology. Finally, the preliminary thermal energy analysis of the combined MD-chemical precipitation/electrodeposition process reveals a considerable reduction in energy consumption of the nickel recovery process.

The N6-methyladenosine (m6A)-forming enzyme METTL3 controls myeloid differentiation of normal hematopoietic and leukemia cells.

N6-methyladenosine (m6A) is an abundant nucleotide modification in mRNA that is required for the differentiation of mouse embryonic stem cells. However, it remains unknown whether the m6A modification controls the differentiation of normal and/or malignant myeloid hematopoietic cells. Here we show that shRNA-mediated depletion of the m6A-forming enzyme METTL3 in human hematopoietic stem/progenitor cells (HSPCs) promotes cell differentiation, coupled with reduced cell proliferation. Conversely, overexpression of wild-type METTL3, but not of a catalytically inactive form of METTL3, inhibits cell differentiation and increases cell growth. METTL3 mRNA and protein are expressed more abundantly in acute myeloid leukemia (AML) cells than in healthy HSPCs or other types of tumor cells. Furthermore, METTL3 depletion in human myeloid leukemia cell lines induces cell differentiation and apoptosis and delays leukemia progression in recipient mice in vivo. Single-nucleotide-resolution mapping of m6A coupled with ribosome profiling reveals that m6A promotes the translation of c-MYC, BCL2 and PTEN mRNAs in the human acute myeloid leukemia MOLM-13 cell line. Moreover, loss of METTL3 leads to increased levels of phosphorylated AKT, which contributes to the differentiation-promoting effects of METTL3 depletion. Overall, these results provide a rationale for the therapeutic targeting of METTL3 in myeloid leukemia.

The chromatin remodelling factor ATRX suppresses R-loops in transcribed telomeric repeats.

ATRX is a chromatin remodelling factor found at a wide range of tandemly repeated sequences including telomeres (TTAGGG)n ATRX mutations are found in nearly all tumours that maintain their telomeres via the alternative lengthening of telomere (ALT) pathway, and ATRX is known to suppress this pathway. Here, we show that recruitment of ATRX to telomeric repeats depends on repeat number, orientation and, critically, on repeat transcription. Importantly, the transcribed telomeric repeats form RNA-DNA hybrids (R-loops) whose abundance correlates with the recruitment of ATRX Here, we show loss of ATRX is also associated with increased R-loop formation. Our data suggest that the presence of ATRX at telomeres may have a central role in suppressing deleterious DNA secondary structures that form at transcribed telomeric repeats, and this may account for the increased DNA damage, stalling of replication and homology-directed repair previously observed upon loss of ATRX function.

Female sexual partners of male people who inject drugs in vietnam have poor knowledge of their male partners' HIV status.

BACKGROUND: Vietnam's HIV epidemic is concentrated among male people who inject drugs (PWID), and their female sexual partners (SPs) may be at risk for infection. HIV prevention interventions for SPs were implemented in Hanoi, Dien Bien Province, and Ho Chi Minh City (HCMC), and data from linked surveys used to evaluate these interventions offered an unusual opportunity to assess knowledge of HIV status within couples. METHODS: Linked surveys (behavioral interviews and HIV testing) among 200 PWID-SP couples in Hanoi, 300 in Dien Bien, and 249 in HCMC. RESULTS: HIV prevalence among male PWID was 53% in Hanoi, 30% in Dien Bien, and 46% in HCMC, and lower among their SPs: 44%, 10%, and 37%, respectively. Comparison of SPs' beliefs regarding male PWID partners' HIV status with the PWIDs' actual test results revealed that 32% of SPs in Dien Bien and 44% in Hanoi and HCMC lacked correct knowledge of their male partners' status. This proportion was slightly lower (21%-33%) among SPs whose PWID partners reported having been previously tested and received HIV+ results. CONCLUSIONS: SP interventions reached HIV-negative women in serodiscordant relationships, and some improvements occurred in condom use and relationship characteristics. Nevertheless, our findings suggest that at least 11,000 SPs in Vietnam may be at high risk for HIV infection because of incorrect knowledge of their partners' HIV status. Interventions should be strengthened in HIV testing, disclosure, and treatment, as well as empowerment of SPs as individuals, within couples, and as communities.

MicroRNA-125b transforms myeloid cell lines by repressing multiple mRNA.

BACKGROUND: We previously described a t(2;11)(p21;q23) chromosomal translocation found in patients with myelodysplasia or acute myeloid leukemia that leads to over-expression of the microRNA miR-125b, and we showed that transplantation of mice with murine stem/progenitor cells overexpressing miR-125b is able to induce leukemia. In this study, we investigated the mechanism of myeloid transformation by miR-125b. DESIGN AND METHODS: To investigate the consequences of miR-125b over-expression on myeloid differentiation, apoptosis and proliferation, we used the NB4 and HL60 human promyelocytic cell lines and the 32Dclone3 murine promyelocytic cell line. To test whether miR-125b is able to transform myeloid cells, we used the non-tumorigenic and interleukin-3-dependent 32Dclone3 cell line over-expressing miR-125b, in xenograft experiments in nude mice and in conditions of interleukin-3 deprivation. To identify new miR-125b targets, we compared, by RNA-sequencing, the transcriptome of cell lines that do or do not over-express miR-125b. RESULTS: We showed that miR-125b over-expression blocks apoptosis and myeloid differentiation and enhances proliferation in both species. More importantly, we demonstrated that miR-125b is able to transform the 32Dclone3 cell line by conferring growth independence from interleukin-3; xenograft experiments showed that these cells form tumors in nude mice. Using RNA-sequencing and quantitative real-time polymerase chain reaction experiments, we identified multiple miR-125b targets. We demonstrated that ABTB1, an anti-proliferative factor, is a new direct target of miR-125b and we confirmed that CBFB, a transcription factor involved in hematopoiesis, is also targeted by miR-125b. MiR-125b controls apoptosis by down-regulating genes involved in the p53 pathway including BAK1 and TP53INP1. CONCLUSIONS: This study demonstrates that in a myeloid context, miR-125b is an oncomiR able to transform cell lines. miR-125b blocks myeloid differentiation in part by targeting CBFB, blocks apoptosis through down-regulation of multiple genes involved in the p53 pathway, and confers a proliferative advantage to human and mouse myeloid cell lines in part by targeting ABTB1.