ABSTRACT Precise control of transcription factor dosage is critical for lung mesenchymal development. The forkhead box transcription factor FOXF1 is a dosage-sensitive regulator of pulmonary vascularization and fibroblast differentiation, with haploinsufficiency causing the lethal neonatal disorder alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). A syntenically conserved long noncoding RNA (lncRNA), FENDRR , is divergently transcribed ∼1.7 kb upstream of FOXF1. Notably, ACDMPV-associated chromosomal deletions disrupting FENDRR or a distal enhancer regulating both FENDRR and FOXF1 have been identified. Consistent with a role in lung development, selective deletion of Fendrr in mice causes neonatal lethality with lung growth defects, as well as alveolar and vascular abnormalities. Here, we define the conserved expression patterns, isoform diversity, and subcellular localization of FENDRR and FOXF1 across human and murine embryonic lung cell types. Functional perturbation reveals a bidirectional regulatory circuit in which FOXF1 promotes FENDRR transcription, while FENDRR limits FOXF1 protein abundance without affecting mRNA levels. Transcriptomic analyses demonstrate overlapping target gene networks and opposing effects on fibroblast-to-myofibroblast differentiation. These findings uncover a rheostat-like regulatory layer by which the lncRNA FENDRR fine-tunes FOXF1 protein dosage to influence lung fibroblast cell fate response and offers additional context for the significance of FOXF1- FENDRR dysregulation in ACDMPV.

Long non-coding RNAs (lncRNAs) are increasingly recognized as critical regulators of gene expression underlying various cellular functions, however, the functional and mechanistic contributions of most lncRNAs to tumorigenesis remain poorly defined, and targeting of lncRNAs is challenging with conventional therapeutic approaches. Here, we uncover human PAN3-AS1 and its murine ortholog Lnc35682, previously uncharacterized lncRNAs embedded within a conserved syntenic genomic locus, as highly expressed in acute myeloid leukemia (AML). Using genetic mouse models, human cell lines and primary patient samples, we show that PAN3-AS1 is essential for leukemia maintenance but dispensable for normal hematopoiesis. Mechanistically, we find that elevated PAN3-AS1 influences chromatin accessibility, thus promoting leukemia gene expression programs. This is mediated, at least in part, by PAN3-AS1 association with the nuclear lamina through a defined functional region that is required for its leukemogenic function. We further characterize a feed-forward regulatory circuit between PAN3-AS1 and its neighboring gene FLT3 that directly links the aberrant lncRNA functions to the FLT3-mutant AML subtype. To therapeutically exploit the regulatory node, we engineer a myeloid leukemia-preferentially targeted lipid nanoparticle (LNP) formulation and demonstrate effective delivery of siRNAs against endogenous targets into leukemia cells in experimental animals. LNP-siPAN3-AS1 alone or in combination with a clinically used FLT3 inhibitor, Gilteritinib, reduces leukemia burden and significantly delay leukemogenesis in vivo. Overall, our study uncovers a therapeutic vulnerable lncRNA-centric circuitry and provides compelling preclinical evidence for the development and application of a novel RNA targeting-LNP based therapy for treatment of myeloid leukemia.

Accurate prediction of RNA secondary structures is essential for understanding the evolutionary conservation and functional roles of long noncoding RNAs (lncRNAs) across diverse species. In this study, we benchmarked two leading tools for predicting evolutionarily conserved RNA secondary structures (ECSs), SISSIz and R-scape, using two distinct experimental frameworks: one focusing on well-characterized mitochondrial RNA structures and the other on experimentally validated Rfam structures embedded within simulated genome alignments. While both tools performed similarly overall, each displayed subtle preferences in detecting ECSs. To overcome their individual limitations, we evaluated two interpretable machine learning approaches that integrate the strengths of both methods. By balancing thermodynamic stability features from RNALalifold and SISSIz with robust covariation metrics from R-scape, a random forest model classifier significantly outperformed individual tools in identifying ECSs. This classifier was implemented in ECSFinder, a new tool designed for large-scale comparative genomics applications, that can provide robust, genome-wide identification of conserved RNA structures, and offer valuable insights into the modular elements and evolutionary conservation of lncRNAs.

The diheteroarylamide-based compound 1C8 and the aminothiazole carboxamide-related compound GPS167 inhibit the CLK kinases, and affect the proliferation of a broad range of cancer cell lines. A chemogenomic screen previously performed with GPS167 revealed that the depletion of components associated with mitotic spindle assembly altered sensitivity to GPS167. Here, a similar screen performed with 1C8 also established the impact of components involved in mitotic spindle assembly. Accordingly, transcriptome analyses of cells treated with 1C8 and GPS167 indicated that the expression and RNA splicing of transcripts encoding mitotic spindle assembly components were affected. The functional relevance of the microtubule connection was confirmed by showing that subtoxic concentrations of drugs affecting mitotic spindle assembly increased sensitivity to GPS167. 1C8 and GPS167 impacted the expression and splicing of transcripts in pathways relevant to tumor progression, including MYC targets and the epithelial mesenchymal transition (EMT). Finally, 1C8 and GPS167 altered the expression and alternative splicing of transcripts involved in the antiviral immune response. Consistent with this observation, depleting the double-stranded RNA sensor DHX33 suppressed GPS167-mediated cytotoxicity on HCT116 cells. Our study uncovered molecular mechanisms through which 1C8 and GPS167 affect cancer cell proliferation as well as processes critical for metastasis.

BACKGROUND:Epidemiological evidence links the proprotein convertase subtilisin/kexin 7 (PCSK7) to triglyceride (TG) metabolism. We associated the known PCSK7 gain-of-function non-coding SNP rs236918 with higher levels of plasma apolipoprotein B (apoB) and the loss-of-function coding variant p.Pro777Leu (SNP rs201598301) with lower apoB and TG. Herein, we aimed to unravel the in vivo role of liver PCSK7. METHODS:We biochemically defined the functional role of PCSK7 in lipid metabolism using hepatic cell lines and Pcsk7-/- mice. Our findings were validated following subcutaneous administration of hepatocyte-targeted N-acetylgalactosamine (GalNAc)-antisense oligonucleotides (ASOs) against Pcsk7. RESULTS:Independent of its proteolytic activity, membrane-bound PCSK7 binds apoB100 in the endoplasmic reticulum and enhances its secretion. Mechanistically, the loss of PCSK7/Pcsk7 leads to apoB100 degradation, triggering an unfolded protein response, autophagy, and β-oxidation, eventually reducing lipid accumulation in hepatocytes. Non-alcoholic fatty liver disease (NAFLD) was induced by a 12-week high fat/fructose/cholesterol diet in wild type (WT) and Pcsk7-/- mice that were then allowed to recover on a 4-week control diet. Pcsk7-/- mice recovered more effectively than WT mice from all NAFLD-related liver phenotypes. Finally, subcutaneous administration of GalNAc-ASOs targeting hepatic Pcsk7 to WT mice validated the above results. CONCLUSIONS:Our data reveal hepatic PCSK7 as one of the major regulators of apoB, and its absence reduces apoB secretion from hepatocytes favoring its ubiquitination and degradation by the proteasome. This results in a cascade of events, eventually reducing hepatic lipid accumulation, thus supporting the notion of silencing PCSK7 mRNA in hepatocytes for targeting NAFLD.

Myoblast fusion is fundamental for the development of multinucleated myofibers. Evolutionarily conserved proteins required for myoblast fusion include RAC1 and its activator DOCK1. In the current study we analyzed the contribution of the DOCK1-interacting ELMO scaffold proteins to myoblast fusion. When Elmo1 −/− mice underwent muscle-specific Elmo2 genetic ablation, they exhibited severe myoblast fusion defects. A mutation in the Elmo2 gene that reduced signaling resulted in a decrease in myoblast fusion. Conversely, a mutation in Elmo2 coding for a protein with an open conformation increased myoblast fusion during development and in muscle regeneration. Finally, we showed that the dystrophic features of the Dysferlin -null mice, a model of limb-girdle muscular dystrophy type 2B, were reversed when expressing ELMO2 in an open conformation. These data provide direct evidence that the myoblast fusion process could be exploited for regenerative purposes and improve the outcome of muscle diseases.

RNA-protein interactions are essential to a variety of biological processes. The realization that mammalian genomes are pervasively transcribed brought a tidal wave of tens of thousands of newly identified long noncoding RNAs (lncRNAs) and raised questions about their purpose in cells. The vast majority of lncRNAs have yet to be studied, and it remains to be determined to how many of these transcripts a function can be ascribed. However, results gleaned from studying a handful of these macromolecules have started to reveal common themes of biological function and mechanism of action involving intricate RNA-protein interactions. Some lncRNAs were shown to regulate the chromatin and transcription of distant and neighboring genes in the nucleus, while others regulate the translation or localization of proteins in the cytoplasm. Some lncRNAs were found to be crucial during development, while mutations and aberrant expression of others have been associated with several types of cancer and a plethora of diseases. Over the last few years, the establishment of new technologies has been key in providing the tools to decode the rules governing lncRNA-protein interactions and functions. This chapter will highlight the general characteristics of lncRNAs, their function, and their mode of action, with a special focus on protein interactions. It will also describe the methods at the disposition of scientists to help them cross this next frontier in our understanding of lncRNA biology.

Resistance to chemotherapy plays a significant role in cancer mortality. To identify genetic units affecting sensitivity to cytarabine, the mainstay of treatment for acute myeloid leukemia (AML), we developed a comprehensive and integrated genome-wide platform based on a dual protein-coding and non-coding integrated CRISPRa screening (DICaS). Putative resistance genes were initially identified using pharmacogenetic data from 760 human pan-cancer cell lines. Subsequently, genome scale functional characterization of both coding and long non-coding RNA (lncRNA) genes by CRISPR activation was performed. For lncRNA functional assessment, we developed a CRISPR activation of lncRNA (CaLR) strategy, targeting 14,701 lncRNA genes. Computational and functional analysis identified novel cell-cycle, survival/apoptosis, and cancer signaling genes. Furthermore, transcriptional activation of the GAS6-AS2 lncRNA, identified in our analysis, leads to hyperactivation of the GAS6/TAM pathway, a resistance mechanism in multiple cancers including AML. Thus, DICaS represents a novel and powerful approach to identify integrated coding and non-coding pathways of therapeutic relevance.

The Linc-p21 locus, encoding a long non-coding RNA, plays an important role in p53 signaling, cell-cycle regulation, and tumor suppression. However, despite extensive study, confusion exists regarding its mechanism of action: is activity driven by the transcript acting in trans, in cis, or by an underlying functional enhancer? Here, using a knockout mouse model and a massively parallel enhancer assay, we delineate the functional elements at this locus. We observe that, even in tissues with no detectable Linc-p21 transcript, deletion of the locus significantly affects local gene expression, including of the cell-cycle regulator Cdkn1a. To characterize this RNA-independent regulatory effect, we systematically interrogated the underlying DNA sequence for enhancer activity at nucleotide resolution and confirmed the existence of multiple enhancer elements. Together, these data suggest that, in vivo, the cis-regulatory effects mediated by Linc-p21, in the presence or absence of transcription, are due to DNA enhancer elements.

Long noncoding RNAs ( lncRNAs) have been implicated in numerous cellular processes including brain development. However, the in vivo expression dynamics and molecular pathways regulated by these loci are not well understood. Here, we leveraged a cohort of 13 lncRNA-null mutant mouse models to investigate the spatiotemporal expression of lncRNAs in the developing and adult brain and the transcriptome alterations resulting from the loss of these lncRNA loci. We show that several lncRNAs are differentially expressed both in time and space, with some presenting highly restricted expression in only selected brain regions. We further demonstrate altered regulation of genes for a large variety of cellular pathways and processes upon deletion of the lncRNA loci. Finally, we found that 4 of the 13 lncRNAs significantly affect the expression of several neighboring protein-coding genes in a cis-like manner. By providing insight into the endogenous expression patterns and the transcriptional perturbations caused by deletion of the lncRNA locus in the developing and postnatal mammalian brain, these data provide a resource to facilitate future examination of the specific functional relevance of these genes in neural development, brain function, and disease.

Eukaryotic cells contain small, highly abundant, nuclear-localized non-coding RNAs [snRNAs (small nuclear RNAs)] which play important roles in splicing of introns from primary genomic transcripts. Through a combination of RNA–RNA and RNA–protein interactions, two of the snRNPs, U1 and U2, recognize the splice sites and the branch site of introns. A complex remodelling of RNA–RNA and protein-based interactions follows, resulting in the assembly of catalytically competent spliceosomes, in which the snRNAs and their bound proteins play central roles. This process involves formation of extensive base-pairing interactions between U2 and U6, U6 and the 5′ splice site, and U5 and the exonic sequences immediately adjacent to the 5′ and 3′ splice sites. Thus RNA–RNA interactions involving U2, U5 and U6 help position the reacting groups of the ! rst and second steps of splicing. In addition, U6 is also thought to participate in formation of the spliceosomal active site. Furthermore, emerging evidence suggests additional roles for snRNAs in regulation of various aspects of RNA biogenesis, from transcription to polyadenylation and RNA stability. These snRNP-mediated regulatory roles probably serve to ensure the co-ordination of the different processes involved in biogenesis of RNAs and point to the central importance of snRNAs in eukaryotic gene expression.

Multipotent long-term repopulating hematopoietic stem cells (LT-HSCs) can self-renew or differentiate into the less primitive short-term repopulating stem cells (ST-HSCs), which themselves produce progenitors that ensure the daily supply of all essential blood components. The Polycomb group (PcG) protein BMI1 is essential for the activity of both HSCs and progenitor cells. Although BMI1 operates by suppressing the Ink4a/Arf locus in progenitors and ST-HSCs, the mechanisms through which this gene regulates the activity of LT-HSCs remain poorly understood. Toward this goal, we isolated BMI1-containing protein complexes and identified UBAP2L as a novel BMI1-interacting protein. We also showed that UBAP2L is preferentially expressed in mouse and human HSC-enriched populations when compared with more mature cell types, and that this gene is essential for the activity of LT-HSCs. In contrast to what is observed for Bmi1 knockdown, we found that UBAP2L depletion does not affect the Ink4a/Arf locus. Given that we demonstrated that BMI1 overexpression is able to rescue the deleterious effects of Ubap2l downregulation on LT-HSC activity and that UBAP2L is part of a PcG subcomplex comprising BMI1, we propose a model in which at least 2 different BMI1-containing PcG complexes regulate HSC activity, which are distinguishable by the presence of UBAP2L.

RNA, including long noncoding RNA (lncRNA), is known to be an abundant and important structural component of the nuclear matrix. However, the molecular identities, functional roles and localization dynamics of lncRNAs that influence nuclear architecture remain poorly understood. Here, we describe one lncRNA, Firre, that interacts with the nuclear-matrix factor hnRNPU through a 156-bp repeating sequence and localizes across an ~5-Mb domain on the X chromosome. We further observed Firre localization across five distinct trans-chromosomal loci, which reside in spatial proximity to the Firre genomic locus on the X chromosome. Both genetic deletion of the Firre locus and knockdown of hnRNPU resulted in loss of colocalization of these trans-chromosomal interacting loci. Thus, our data suggest a model in which lncRNAs such as Firre can interface with and modulate nuclear architecture across chromosomes.

Histone methylation is a dynamic and reversible process proposed to directly impact on stem cell fate. The Jumonji (JmjC) domain-containing family of demethylases comprises 27 members that target mono-, di-, and trimethylated lysine residues of histone (or nonhistone) proteins. To evaluate their role in regulation of hematopoietic stem cell (HSC) behavior, we performed an in vivo RNAi-based functional screen and demonstrated that Jarid1b and Jhdm1f play opposing roles in regulation of HSC activity. Decrease in Jarid1b levels correlated with an in vitro expansion of HSCs with preserved long-term in vivo lymphomyeloid differentiation potential. Through RNA sequencing analysis, Jarid1b knockdown was associated with increased expression levels of several HSC regulators (Hoxa7, Hoxa9, Hoxa10, Hes1, Gata2) and reduced levels of differentiation-associated genes. shRNA against Jhdmlf, in contrast, impaired hematopoietic reconstitution of bone marrow cells. Together, our studies identified Jarid1b as a negative regulator of HSC activity and Jhdmlf as a positive regulator of HSC activity.

The polycomb group protein Bmi-1 is a well known determinant of hematopoietic stem cell function. Bmi-1-/- mice display severe hematopoietic defects, including progressive loss of hematopoietic cells from the bone marrow. Bmi-1 is dispensable for hematopoietic stem cell specification, but essential for their maintenance, an effect attributable to its ability to promote HSC self-renewal. The mechanism by which Bmi-1 regulates this process is not completely understood. Bmi-1 has been shown to repress the INK4A/ARF locus encoding the cell cycle inhibitors p16ink4a and p19arf , to interact with the E4F1 protein and to regulate the DNA damage response pathway, however experimental manipulation of these proteins/pathways only partially rescues the hematopoietic defects of the Bmi-1-/-mice. It thus appears that the mechanism by which Bmi-1 regulates HSC self-renewal remains to be determined. Towards this goal, we purified Bmi-1 containing protein complexes from cellular extracts and identified Bmi-1 interaction partners by mass spectrometry. We observed that the protein Ubap2l, which has never been shown to associate with Bmi-1 and for which no link with polycomb group protein function has been described, was consistently found in Bmi-1-containing protein complexes. Immunoprecipitation experiments revealed that Ubap2l indirectly associates with Bmi-1 via an interaction with the polycomb group protein Rnf2. We then evaluated the possibility that Ubap2l might be involved in the regulation of HSC activity. We observed that Ubap2l transcripts are more abundant in primitive HSC populations compared to total BM. CFC assays performed with BM cells infected with Ubap2l shRNAs revealed that Ubap2l knockdown causes a modest and progressive loss of progenitor activity when cells are kept in culture, with multipotent and bipotent progenitors being substantially more affected than unipotent progenitors. We transplanted these cells in mice and observed a gradual decrease in the percentage of donor derived cells expressing Ubap2l shRNAs in the peripheral blood of the recipient mice, with the most striking effect observed 16 weeks post-transplantation in the BM. Bmi-1 has been shown to regulate the proliferative capacity of both progenitor and stem cells, and its deletion in BM cells is known to dramatically reduce the reconstitution activity of these cells at early time points following transplantation. In contrast, Ubap2l appears to preferentially regulate LTR-HSC activity. We tested the effects of Ubap2l silencing on leukemic cells in vivo and observed that a reduction of Ubap2l levels in these cells had an important impact on their ability to reconstitute recipient mice, suggesting that Ubap2l also plays a role in leukemic stem cell activity. We determined if the mechanism by which Ubap2l regulates HSC activity is related to Bmi-1 function by simultaneously introducing Bmi-1 cDNA and Ubap2l shRNAs in BM cells and found that Bmi-1 is able to rescue the long-term reconstitution defect caused by Ubap2l downregulation in these cells. We observed that Ubap2l silencing does not significantly affect the expression of the known Bmi-1 targets p16ink4a and p19arf, implying that Ubap2l regulates HSC activity via a Bmi-1-dependent mechanism that does not involve repression of the INK4A/ARF locus. One explanation for the two Bmi-1 dependent mechanisms at play in the regulation of HSC activity could be that Bmi-1 is part of two separate protein complexes, each regulating different aspects of hematopoietic cell function. To test this hypothesis, we fractionated cellular extracts and were indeed able to resolve two distinct Bmi-1 containing protein complexes, distinguishable by the presence of Ubap2l. Based on the results we obtained, we propose a model in which two different Bmi-1 containing protein complexes regulate hematopoietic stem cell function. An Ubap2l-independent complex, which is most likely involved in the repression of the INK4A/ARF locus, and could be responsible for the effects of Bmi-1 on multipotent progenitors and STR-HSCs, and an Ubap2l-dependent complex, which operates via a yet to be defined mechanism unrelated to p16Ink4a and p19Arf, and would account for the effects of Bmi-1 on LTR-HSC activity. These results position Ubap2l as a key regulator of LTR-HSC activity and unveil a novel protein complex mediating the effects of Bmi-1 on LTR-HSCs. Disclosures: No relevant conflicts of interest to declare.

Many studies are uncovering functional roles for long noncoding RNAs (lncRNAs), yet few have been tested for in vivo relevance through genetic ablation in animal models. To investigate the functional relevance of lncRNAs in various physiological conditions, we have developed a collection of 18 lncRNA knockout strains in which the locus is maintained transcriptionally active. Initial characterization revealed peri- and postnatal lethal phenotypes in three mutant strains (Fendrr, Peril, and Mdgt), the latter two exhibiting incomplete penetrance and growth defects in survivors. We also report growth defects for two additional mutant strains (linc-Brn1b and linc-Pint). Further analysis revealed defects in lung, gastrointestinal tract, and heart in Fendrr(-/-) neonates, whereas linc-Brn1b(-/-) mutants displayed distinct abnormalities in the generation of upper layer II-IV neurons in the neocortex. This study demonstrates that lncRNAs play critical roles in vivo and provides a framework and impetus for future larger-scale functional investigation into the roles of lncRNA molecules. DOI: http://dx.doi.org/10.7554/eLife.01749.001.

The prevalence of obesity has led to a surge of interest in understanding the detailed mechanisms underlying adipocyte development. Many protein-coding genes, mRNAs, and microRNAs have been implicated in adipocyte development, but the global expression patterns and functional contributions of long noncoding RNA (lncRNA) during adipogenesis have not been explored. Here we profiled the transcriptome of primary brown and white adipocytes, preadipocytes, and cultured adipocytes and identified 175 lncRNAs that are specifically regulated during adipogenesis. Many lncRNAs are adipose-enriched, strongly induced during adipogenesis, and bound at their promoters by key transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (CEBPα). RNAi-mediated loss of function screens identified functional lncRNAs with varying impact on adipogenesis. Collectively, we have identified numerous lncRNAs that are functionally required for proper adipogenesis.

Differential analysis of gene and transcript expression using high-throughput RNA sequencing (RNA-seq) is complicated by several sources of measurement variability and poses numerous statistical challenges. We present Cuffdiff 2, an algorithm that estimates expression at transcript-level resolution and controls for variability evident across replicate libraries. Cuffdiff 2 robustly identifies differentially expressed transcripts and genes and reveals differential splicing and promoter-preference changes. We demonstrate the accuracy of our approach through differential analysis of lung fibroblasts in response to loss of the developmental transcription factor HOXA1, which we show is required for lung fibroblast and HeLa cell cycle progression. Loss of HOXA1 results in significant expression level changes in thousands of individual transcripts, along with isoform switching events in key regulators of the cell cycle. Cuffdiff 2 performs robust differential analysis in RNA-seq experiments at transcript resolution, revealing a layer of regulation not readily observable with other high-throughput technologies.

We recently generated 2 phenotypically similar Hoxa9+Meis1 overexpressing acute myeloid leukemias that differ by their in vivo biologic behavior. The first leukemia, named FLA2, shows a high frequency of leukemia stem cells (LSCs; 1 in 1.4 cells), whereas the second, FLB1, is more typical with a frequency of LSCs in the range of 1 per several hundred cells. To gain insights into possible mechanisms that determine LSC self-renewal, we profiled and compared the abundance of nuclear and cytoplasmic proteins and phosphoproteins from these leukemias using quantitative proteomics. These analyses revealed differences in proteins associated with stem cell fate, including a hyperactive p38 MAP kinase in FLB1 and a differentially localized Polycomb group protein Ezh2, which is mostly nuclear in FLA2 and predominantly cytoplasmic in FLB1. Together, these newly documented proteomes and phosphoproteomes represent a unique resource with more than 440 differentially expressed proteins and 11 543 unique phosphopeptides, of which 80% are novel and 7% preferentially phosphorylated in the stem cell-enriched leukemia.