TFEB controls vascular development by regulating the proliferation of endothelial cells.

Transcription factor TFEB is thought to control cellular functions-including in the vascular bed-primarily via regulation of lysosomal biogenesis and autophagic flux. Here, we report that TFEB also orchestrates a non-canonical program that controls the cell cycle/VEGFR2 pathway in the developing vasculature. In endothelial cells, TFEB depletion halts proliferation at the G1-S transition by inhibiting the CDK4/Rb pathway. TFEB-deficient cells attempt to compensate for this limitation by increasing VEGFR2 levels at the plasma membrane via microRNA-mediated mechanisms and controlled membrane trafficking. TFEB stimulates expression of the miR-15a/16-1 cluster, which limits VEGFR2 transcript stability and negatively modulates expression of MYO1C, a regulator of VEGFR2 trafficking to the cell surface. Altered levels of miR-15a/16-1 and MYO1C in TFEB-depleted cells cause increased expression of plasma membrane VEGFR2, but in a manner associated with low signaling strength. An endothelium-specific Tfeb-knockout mouse model displays defects in fetal and newborn mouse vasculature caused by reduced endothelial proliferation and by anomalous function of the VEGFR2 pathway. These previously unrecognized functions of TFEB expand its role beyond regulation of the autophagic pathway in the vascular system.

Recent advances in the regulation of plant miRNA biogenesis.

MicroRNAs (miRNAs) are essential non-coding riboregulators of gene expression in plants and animals. In plants, miRNAs guide their effector protein named ARGONAUTE (AGO) to find target RNAs for gene silencing through target RNA cleavage or translational inhibition. miRNAs are derived from primary miRNA transcripts (pri-miRNAs), most of which are transcribed by the DNA-dependent RNA polymerase II. In plants, an RNase III enzyme DICER-LIKE1-containing complex processes pri-miRNAs in the nucleus into miRNAs. To ensure proper function of miRNAs, plants use multiple mechanisms to control miRNA accumulation. On one hand, pri-miRNA levels are controlled through transcription and stability. On the other hand, the activities of the DCL1 complex are regulated by many protein factors at transcriptional, post-transcriptional and post-translational levels. Notably, recent studies reveal that pri-miRNA structure/sequence features and modifications also play important roles in miRNA biogenesis. In this review, we summarize recent progresses on the mechanisms regulating miRNA biogenesis.

Identification of small RNAs during cold acclimation in Arabidopsis thaliana.

BACKGROUND: Cold stress causes dynamic changes in gene expression that are partially caused by small non-coding RNAs since they regulate protein coding transcripts and act in epigenetic gene silencing pathways. Thus, a detailed analysis of transcriptional changes of small RNAs (sRNAs) belonging to all known sRNA classes such as microRNAs (miRNA) and small interfering RNA (siRNAs) in response to cold contributes to an understanding of cold-related transcriptome changes. RESULT: We subjected A. thaliana plants to cold acclimation conditions (4 °C) and analyzed the sRNA transcriptomes after 3 h, 6 h and 2 d. We found 93 cold responsive differentially expressed miRNAs and only 14 of these were previously shown to be cold responsive. We performed miRNA target prediction for all differentially expressed miRNAs and a GO analysis revealed the overrepresentation of miRNA-targeted transcripts that code for proteins acting in transcriptional regulation. We also identified a large number of differentially expressed cis- and trans-nat-siRNAs, as well as sRNAs that are derived from long non-coding RNAs. By combining the results of sRNA and mRNA profiling with miRNA target predictions and publicly available information on transcription factors, we reconstructed a cold-specific, miRNA and transcription factor dependent gene regulatory network. We verified the validity of links in the network by testing its ability to predict target gene expression under cold acclimation. CONCLUSION: In A. thaliana, miRNAs and sRNAs derived from cis- and trans-NAT gene pairs and sRNAs derived from lncRNAs play an important role in regulating gene expression in cold acclimation conditions. This study provides a fundamental database to deepen our knowledge and understanding of regulatory networks in cold acclimation.

Drosophila Trf4-1 involves in mRNA and primary miRNA transcription.

Drosophila Trf4-1 (DmTrf4-1) is a polyadenylation polymerase or terminal nucleotidyl transferase (PAP/TENT) that has been reported to add poly(A) tails to snRNAs in nucleus or mRNAs in cytoplasm. Here, we found that the loss of Trf4-1 resulted in the reduction of mRNAs and primary miRNAs (pri-miRNAs) in both Drosophila S2 cells and adult flies. Interestingly, the role of Trf4-1 in transcription is independent of its PAP/TENT activity. Moreover, using the chromatin immunoprecipitation assay, we uncovered that the loss of Trf4-1 led to abnormal RNA polymerase II accumulation and reduced H3K4me3 binding in promoter regions. Thus, our study indicates a positive role of Trf4-1 in the transcription of mRNAs and pri-miRNAs.

ELF4 is a critical component of a miRNA-transcription factor network and is a bridge regulator of glioblastoma receptor signaling and lipid dynamics.

BACKGROUND: The loss of neurogenic tumor suppressor microRNAs miR-124, miR-128, and miR-137 is associated with glioblastoma's undifferentiated state. Most of their impact comes via the repression of a network of oncogenic transcription factors. We conducted a high-throughput functional siRNA screen in glioblastoma cells and identify E74 like ETS transcription factor 4 (ELF4) as the leading contributor to oncogenic phenotypes. METHODS: In vitro and in vivo assays were used to assess ELF4 impact on cancer phenotypes. We characterized ELF4's mechanism of action via genomic and lipidomic analyses. A MAPK reporter assay verified ELF4's impact on MAPK signaling, and qRT-PCR and western blotting were used to corroborate ELF4 regulatory role on most relevant target genes. RESULTS: ELF4 knockdown resulted in significant proliferation delay and apoptosis in GBM cells and long-term growth delay and morphological changes in glioma stem cells (GSCs). Transcriptomic analyses revealed that ELF4 controls two interlinked pathways: 1) Receptor tyrosine kinase signaling and 2) Lipid dynamics. ELF4 modulation directly affected receptor tyrosine kinase (RTK) signaling, as mitogen-activated protein kinase (MAPK) activity was dependent upon ELF4 levels. Furthermore, shotgun lipidomics revealed that ELF4 depletion disrupted several phospholipid classes, highlighting ELF4's importance in lipid homeostasis. CONCLUSIONS: We found that ELF4 is critical for the GBM cell identity by controlling genes of two dependent pathways: RTK signaling (SRC, PTK2B, and TNK2) and lipid dynamics (LRP1, APOE, ABCA7, PLA2G6, and PITPNM2). Our data suggest that targeting these two pathways simultaneously may be therapeutically beneficial to GBM patients.

MicroRNAs: Tiny Regulators of Gene Expression with Pivotal Roles in Normal B-Cell Development and B-Cell Chronic Lymphocytic Leukemia.

MicroRNAs (miRNAs) represent a class of small non-coding RNAs bearing regulatory potency. The implication of miRNAs in physiological cellular processes has been well documented so far. A typical process orchestrated by miRNAs is the normal B-cell development. A stage-specific expression pattern of miRNAs has been reported in the developmental procedure, as well as interactions with transcription factors that dictate B-cell development. Besides their involvement in normal hematopoiesis, miRNAs are severally implicated in hematological malignancies, a typical paradigm of which is B-cell chronic lymphocytic leukemia (B-CLL). B-CLL is a highly heterogeneous disease characterized by the accumulation of abnormal B cells in blood, bone marrow, lymph nodes, and spleen. Therefore, timely, specific, and sensitive assessment of the malignancy is vital. Several studies have attempted to highlight the remarkable significance of miRNAs as regulators of gene expression, biomarkers for diagnosis, prognosis, progression, and therapy response prediction, as well as molecules with potential therapeutic utility. This review seeks to outline the linkage between miRNA function in normal and malignant hematopoiesis by demonstrating the main benchmarks of the implication of miRNAs in the regulation of normal B-cell development, and to summarize the key findings about their value as regulators, biomarkers, or therapeutic targets in B-CLL.

p73-Governed miRNA Networks: Translating Bioinformatics Approaches to Therapeutic Solutions for Cancer Metastasis.

The transcription factor p73 synthesizes a large number of isoforms and presents high structural and functional homology with p53, a well-known tumor suppressor and a famous "Holy Grail" of anticancer targeting. p73 has attracted increasing attention mainly because (a) unlike p53, p73 is rarely mutated in cancer, (b) some p73 isoforms can inhibit all hallmarks of cancer, and (c) it has the ability to mimic oncosuppressive functions of p53, even in p53-mutated cells. These attributes render p73 and its downstream pathways appealing for therapeutic targeting, especially in mutant p53-driven cancers. p73 functions are, at least partly, mediated by microRNAs (miRNAs), which constitute nodal components of p73-governed networks. p73 not only regulates transcription of crucial miRNA genes, but is also predicted to affect miRNA populations in a transcription-independent manner by developing protein-protein interactions with components of the miRNA processing machinery. This combined effect of p73, both in miRNA transcription and maturation, appears to be isoform-dependent and can result in a systemic switch of cell miRNomes toward either an anti-oncogenic or oncogenic outcome. In this review, we combine literature search with bioinformatics approaches to reconstruct the p73-governed miRNA network and discuss how these crosstalks may be exploited to develop next-generation therapeutics.

Combined Experimental and System-Level Analyses Reveal the Complex Regulatory Network of miR-124 during Human Neurogenesis.

Non-coding RNAs regulate many biological processes including neurogenesis. The brain-enriched miR-124 has been assigned as a key player of neuronal differentiation via its complex but little understood regulation of thousands of annotated targets. To systematically chart its regulatory functions, we used CRISPR/Cas9 gene editing to disrupt all six miR-124 alleles in human induced pluripotent stem cells. Upon neuronal induction, miR-124-deleted cells underwent neurogenesis and became functional neurons, albeit with altered morphology and neurotransmitter specification. Using RNA-induced-silencing-complex precipitation, we identified 98 high-confidence miR-124 targets, of which some directly led to decreased viability. By performing advanced transcription-factor-network analysis, we identified indirect miR-124 effects on apoptosis, neuronal subtype differentiation, and the regulation of previously uncharacterized zinc finger transcription factors. Our data emphasize the need for combined experimental- and system-level analyses to comprehensively disentangle and reveal miRNA functions, including their involvement in the neurogenesis of diverse neuronal cell types found in the human brain.