Genome-wide association studies (GWASs) have linked genes to various pathological traits. However, the potential contribution of regulatory noncoding RNAs, such as microRNAs (miRNAs), to a genetic predisposition to pathological conditions has remained unclear. We leveraged GWAS meta-analysis data from >188,000 individuals to identify 69 miRNAs in physical proximity to single-nucleotide polymorphisms (SNPs) associated with abnormal levels of circulating lipids. Several of these miRNAs (miR-128-1, miR-148a, miR-130b, and miR-301b) control the expression of key proteins involved in cholesterol-lipoprotein trafficking, such as the low-density lipoprotein (LDL) receptor (LDLR) and the ATP-binding cassette A1 (ABCA1) cholesterol transporter. Consistent with human liver expression data and genetic links to abnormal blood lipid levels, overexpression and antisense targeting of miR-128-1 or miR-148a in high-fat diet–fed C57BL/6J and Apoe-null mice resulted in altered hepatic expression of proteins involved in lipid trafficking and metabolism, and in modulated levels of circulating lipoprotein-cholesterol and triglycerides. Taken together, these findings support the notion that altered expression of miRNAs may contribute to abnormal blood lipid levels, predisposing individuals to human cardiometabolic disorders.
PIWI proteins are expressed in germ cells in a wide variety of metazoans, where they participate in the synthesis and function of a novel class of small RNAs called PIWI associated RNAs (piRNAs). One function of piRNAs is to preserve the integrity of the germline genome by silencing transposons, though they also participate in epigenetic and post-transcriptional gene regulation. In the planarian Schmidtea mediterranea, the PIWI proteins SMEDWI-1 and SMEDWI-2 are expressed in neoblasts and SMEDWI-2 is required for regeneration and homeostasis. Here, we identify a diverse population of ;32-nucleotide small RNAs that strongly resemble vertebrate and insect piRNAs and map to hundreds of thousands of loci in the S. mediterranea genome. The expression of these RNAs occurs predominantly in neoblasts and is not restricted to the germline. RNAi knockdown of either SMEDWI-2 or a newly identified PIWI protein, SMEDWI-3, impairs regeneration and homeostasis and decreases the levels of both piRNAs and neoblasts. Therefore, SMEDWI-2 and SMEDWI-3 are required for piRNA expression, regeneration, and neoblast function in S. mediterranea.
The blood disorder, β-thalassaemia, is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming peptide nucleic acids (PNAs) substituted at the γ position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human β-thalassaemia. Injection of thalassemic mice with SCF plus nanoparticles containing γPNAs and donor DNAs ameliorated the disease phenotype, with sustained elevation of blood haemoglobin levels into the normal range, reduced reticulocytosis, reversal of splenomegaly and up to 7% β-globin gene correction in HSCs, with extremely low off-target effects. The combination of nanoparticle delivery, next generation γPNAs and SCF treatment may offer a minimally invasive treatment for genetic disorders of the blood that can be achieved safely and simply by intravenous administration.
• Purification strategies developed for human Mk-E progenitors, as well as committed Mk and E progenitors.• MYB regulates the biphenotypic fate decision of human MEPs.Bipotent megakaryocyte/erythroid progenitors (MEPs) give rise to progeny limited to the megakaryocyte (Mk) and erythroid (E) lineages. We developed a novel dual-detection functional in vitro colony-forming unit (CFU) assay for single cells that differentiates down both the Mk and E lineages (CFU-Mk/E), which allowed development and validation of a novel purification strategy for the identification and quantitation of primary functional human MEPs from granulocyte colony-stimulating factor-mobilized peripheral blood and bone marrow. Applying this assay to fluorescence-activated cell sorter-sorted cell populations, we found that the Lin2 population is much more highly enriched for bipotent MEPs than any previously reported subpopulations. We also developed purification strategies for primary human lineage-committed Mk and E progenitors identified as CFU-Mk and burst forming unit-E. Comparative expression analyses in MEP, MkP, and ErP populations revealed differential expression of MYB. We tested whether alterations in MYB concentration affect the Mk-E fate decision at the single cell level in MEPs and found that short hairpin RNA-mediated MYB knockdown promoted commitment of MEPs to the Mk lineage, further defining its role in MEP lineage fate. There are numerous applications for these novel enrichment strategies, including facilitating mechanistic studies of MEP lineage commitment, improving approaches for in vitro expansion of Mk and E cells, and developing improved therapies for benign and malignant hematologic disease. (Blood. 2016;128(7):923-933)
SUMMARY Megakaryocytic-erythroid progenitors (MEPs) give rise to the cells that produce red blood cells and platelets. Although the mechanisms underlying megakaryocytic (MK) and erythroid (E) maturation have been described, those controlling their specification from MEPs are unknown. Single-cell RNA sequencing of primary human MEPs, common myeloid progenitors (CMPs), megakaryocyte progenitors, and E progenitors revealed a distinct transitional MEP signature. Inferred regulatory transcription factors (TFs) were associated with differential expression of cell cycle regulators. Genetic manipulation of selected TFs validated their role in lineage specification and demonstrated coincident modulation of the cell cycle. Genetic and pharmacologic modulation demonstrated that cell cycle activation is sufficient to promote E versus MK specification. These findings, obtained from healthy human cells, lay a foundation to study the mechanisms underlying benign and malignant disease states of the megakaryocytic and E lineages.
MicroRNAs (miRNAs) play important roles in directing the differentiation of cells down a variety of cell lineage pathways. The planarian Schmidtea mediterranea can regenerate all lost body tissue after amputation due to a population of pluripotent somatic stem cells called neoblasts, and is therefore an excellent model organism to study the roles of miRNAs in stem cell function. Here, we use a combination of deep sequencing and bioinformatics to discover 66 new miRNAs in S. mediterranea. We also identify 21 miRNAs that are specifically expressed in either sexual or asexual animals. Finally, we identified five miRNAs whose expression is sensitive to g-irradiation, suggesting they are expressed in neoblasts or early neoblast progeny. Together, these results increase the known repertoire of S. mediterranea miRNAs and identify numerous regulated miRNAs that may play important roles in regeneration, homeostasis, neoblast function, and reproduction.
SUMMARY Post-transcriptional gene regulation by miRNAs and RNA binding proteins (RBP) is important in development, physiology and disease. To examine the interplay between miRNAs and the RBP ELAVL1 (a.k.a. HuR), we mapped miRNA binding sites at the transcriptome-wide scale in WT and Elavl1 knockout murine bone marrow-derived macrophages. Proximity of ELAVL1 binding sites attenuated miRNA binding to transcripts and promoted gene expression. Transcripts that regulate angiogenesis and macrophage/endothelial cross talk were preferentially targeted by miRNAs, suggesting that ELAVL1 promotes angiogenesis, at least in part, by antagonism of miRNA function. We found that ELAVL1 antagonized binding of miR-27 to the 3′UTR of Zfp36 mRNA and alleviated miR-27-mediated suppression of the RBP ZFP36 (a.k.a. Tristetraprolin). Thus the miR-27-regulated mechanism synchronizes the expression of ELAVL1 and ZFP36. This study provides a resource for systems-level interrogation of post-transcriptional gene regulation in macrophages, a key cell type in inflammation, angiogenesis and tissue homeostasis.
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