Mohamed Fareh

TUT7 controls the fate of precursor microRNAs by using three different uridylation mechanisms

Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis. Using biochemistry, single‐molecule, and deep sequencing techniques, we here investigate the mechanism by which human TUT7 (also known as ZCCHC6) recognizes and uridylates precursor miRNAs (pre‐miRNAs) in the absence of Lin28. We find that the overhang of a pre‐miRNA is the key structural element that is recognized by TUT7 and its paralogues, TUT4 (ZCCHC11) and TUT2 (GLD2/PAPD4). For group II pre‐miRNAs, which have a 1‐nt 3′ overhang, TUT7 restores the canonical end structure (2‐nt 3′ overhang) through mono‐uridylation, thereby promoting miRNA biogenesis. For pre‐miRNAs where the 3′ end is further recessed into the stem (as in 3′ trimmed pre‐miRNAs), TUT7 generates an oligo‐U tail that leads to degradation. In contrast to Lin28‐stimulated oligo‐uridylation, which is processive, a distributive mode is employed by TUT7 for both mono‐ and oligo‐uridylation in the absence of Lin28. The overhang length dictates the frequency (but not duration) of the TUT7‐RNA interaction, thus explaining how TUT7 differentiates pre‐miRNA species with different overhangs. Our study reveals dual roles and mechanisms of uridylation in repair and removal of defective pre‐miRNAs.

Subversion of Autophagy in Adherent Invasive Escherichia coli-Infected Neutrophils Induces Inflammation and Cell Death

Invading bacteria are recognized, captured and killed by a specialized form of autophagy, called xenophagy. Recently, defects in xenophagy in Crohn’s disease (CD) have been implicated in the pathogenesis of human chronic inflammatory diseases of uncertain etiology of the gastrointestinal tract. We show here that pathogenic adherent-invasive Escherichia coli (AIEC) isolated from CD patients are able to adhere and invade neutrophils, which represent the first line of defense against bacteria. Of particular interest, AIEC infection of neutrophil-like PLB-985 cells blocked autophagy at the autolysosomal step, which allowed intracellular survival of bacteria and exacerbated interleukin-8 (IL-8) production. Interestingly, this block in autophagy correlated with the induction of autophagic cell death. Likewise, stimulation of autophagy by nutrient starvation or rapamycin treatment reduced intracellular AIEC survival and IL-8 production. Finally, treatment with an inhibitor of autophagy decreased cell death of AIEC-infected neutrophil-like PLB-985 cells. In conclusion, excessive autophagy in AIEC infection triggered cell death of neutrophils.

Tumorigenic Potential of miR‐18A* in Glioma Initiating Cells Requires NOTCH‐1 Signaling

Stem cell‐like properties of glioma initiating cells (GiCs) fuel glioblastoma (GBM) development by providing the different cell types that comprise the tumor. It is therefore likely that the molecular circuitries that regulate their decision to self‐renew or commit to a more differentiated state may offer targets for future innovative therapies. In previous micro‐RNA profiling studies to search for regulators of stem cell plasticity, we identified miR‐18a* as a potential candidate and its expression correlated with the stemness state. Here, using human GiCs we found that miR‐18a* expression promotes clonal proliferation in vitro and tumorigenicity in vivo. Mechanistically, ERK‐dependent induction of miR‐18a* directly represses expression of DLL3, an autocrine inhibitor of NOTCH, thus enhancing the level of activated NOTCH‐1. Activated NOTCH‐1 in turn is required for sustained ERK activation. This feed‐forward loop, driven by miR‐18a*, is required to turn on the SHH‐GLI‐NANOG network, essential for GiC self‐renewal. Hence, by tightly regulating expression of DLL3, miR‐18a* constitutes an important signaling mediator for fine tuning the level of GiC self‐renewal. STEM Cells2013;31:1252–1265

Bringing single-molecule spectroscopy to macromolecular protein complexes

Single-molecule fluorescence spectroscopy offers real-time, nanometer-resolution information. Over the past two decades, this emerging single-molecule technique has been rapidly adopted to investigate the structural dynamics and biological functions of proteins. Despite this remarkable achievement, single-molecule fluorescence techniques must be extended to macromolecular protein complexes that are physiologically more relevant for functional studies. In this review, we present recent major breakthroughs for investigating protein complexes within cell extracts using single-molecule fluorescence. We outline the challenges, future prospects and potential applications of these new single-molecule fluorescence techniques in biological and clinical research.

Cell-based therapy using miR-302-367 expressing cells represses glioblastoma growth

Glioblastomas are incurable primary brain tumors that affect patients of all ages. The aggressiveness of this cancer has been attributed in part to the persistence of treatment-resistant glioblastoma stem-like cells. We have previously discovered the tumor-suppressor properties of the microRNA cluster miR-302-367, representing a potential treatment for glioblastoma. Here, we attempted to develop a cell-based therapy by taking advantage of the capability of glioma cells to secrete exosomes that enclose small RNA molecules. We engineered primary glioma cells to stably express the miR-302-367. Remarkably, these cells altered, in a paracrine-dependent manner, the expression of stemness markers, the proliferation and the tumorigenicity of neighboring glioblastoma cells. Further characterization of the secretome derived from miR-302-367 expressing cells showed that a large amount of miR-302-367 was enclosed in exosomes, which were internalized by the neighboring glioblastoma cells. This miR-302-367 cell-to-cell transfer resulted in the inhibition of its targets such as CXCR4/SDF1, SHH, cyclin D, cyclin A and E2F1. Orthotopic xenograft of miR-302-367-expressing cells together with glioblastoma stem-like cells efficiently altered the tumor development in mice brain.

A driver role for GABA metabolism in controlling stem and proliferative cell state through GHB production in glioma

Cell populations with differing proliferative, stem-like and tumorigenic states co-exist in most tumors and especially malignant gliomas. Whether metabolic variations can drive this heterogeneity by controlling dynamic changes in cell states is unknown. Metabolite profiling of human adult glioblastoma stem-like cells upon loss of their tumorigenicity revealed a switch in the catabolism of the GABA neurotransmitter toward enhanced production and secretion of its by-product GHB (4-hydroxybutyrate). This switch was driven by succinic semialdehyde dehydrogenase (SSADH) downregulation. Enhancing GHB levels via SSADH downregulation or GHB supplementation triggered cell conversion into a less aggressive phenotypic state. GHB affected adult glioblastoma cells with varying molecular profiles, along with cells from pediatric pontine gliomas. In all cell types, GHB acted by inhibiting α-ketoglutarate-dependent Ten–eleven Translocations (TET) activity, resulting in decreased levels of the 5-hydroxymethylcytosine epigenetic mark. In patients, low SSADH expression was correlated with high GHB/α-ketoglutarate ratios, and distinguished weakly proliferative/differentiated glioblastoma territories from proliferative/non-differentiated territories. Our findings support an active participation of metabolic variations in the genesis of tumor heterogeneity.

TRBP ensures efficient Dicer processing of precursor microRNA in RNA-crowded environments.

The RNA-binding protein TRBP is a central component of the Dicer complex. Despite a decade of biochemical and structural studies, the essential functionality of TRBP in microRNA (miRNA) biogenesis remains unknown. Here we show that TRBP is an integral cofactor for time-efficient Dicer processing in RNA-crowded environments. We competed for Dicer processing of pre-miRNA with a large amount of cellular RNA species and found that Dicer-TRBP, but not Dicer alone, remains resilient. To apprehend the mechanism of this substrate selectivity, we use single-molecule fluorescence. The real-time observation reveals that TRBP acts as a gatekeeper, precluding Dicer from engaging with pre-miRNA-like substrates. TRBP acquires the selectivity using the PAZ domain of Dicer, whereas Dicer moderates the RNA-binding affinity of TRBP for fast turnover. This coordinated action between TRBP and Dicer accomplishes an efficient way of discarding pre-miRNA-like substrates.

Single-molecule pull-down for investigating protein-nucleic acid interactions.

The genome and transcriptome are constantly modified by proteins in the cell. Recent advances in single-molecule techniques allow for high spatial and temporal observations of these interactions between proteins and nucleic acids. However, due to the difficulty of obtaining functional protein complexes, it remains challenging to study the interactions between macromolecular protein complexes and nucleic acids. Here, we combined single-molecule fluorescence with various protein complex pull-down techniques to determine the function and stoichiometry of ribonucleoprotein complexes. Through the use of three examples of protein complexes from eukaryotic cells (Drosha, Dicer, and TUT4 protein complexes), we provide step-by-step guidance for using novel single-molecule techniques. Our single-molecule methods provide sub-second and nanometer resolution and can be applied to other nucleoprotein complexes that are essential for cellular processes.

Viral suppressors of RNAi employ a rapid screening mode to discriminate viral RNA from cellular small RNA

Abstract RNA interference (RNAi) is an indispensable mechanism for antiviral defense in insects, including mosquitoes that transmit human diseases. To escape this antiviral defense system, viruses encode suppressors of RNAi that prevent elimination of viral RNAs, and thus ensure efficient virus accumulation. Although the first animal Viral Suppressor of RNAi (VSR) was identified more than a decade ago, the molecular basis of RNAi suppression by these viral proteins remains unclear. Here, we developed a single-molecule fluorescence assay to investigate how VSRs inhibit the recognition of viral RNAs by Dcr-2, a key endoribonuclease enzyme in the RNAi pathway. Using VSRs from three insect RNA viruses (Culex Y virus, Drosophila X virus and Drosophila C virus), we reveal bimodal physical interactions between RNA molecules and VSRs. During initial interactions, these VSRs rapidly discriminate short RNA substrates from long dsRNA. VSRs engage nearly irreversible binding with long dsRNAs, thereby shielding it from recognition by Dcr-2. We propose that the length-dependent switch from rapid screening to irreversible binding reflects the main mechanism by which VSRs distinguish viral dsRNA from cellular RNA species such as microRNAs.

Probing RNA–Protein Interactions with Single-Molecule Pull-Down Assays

Recent advances in single-molecule techniques allow for dynamic observations of the interactions between various protein assemblies and RNA molecules with high spatiotemporal resolution. However, it remains challenging to obtain functional eukaryotic protein complexes and cost-effective fluorescently labeled RNAs to study their interactions at the single-molecule level. Here, we describe protocols combining single-molecule fluorescence with various protein complex pull-down techniques to determine the function of RNA-interacting protein complexes of interest. We provide step-by-step guidance for using novel single-molecule techniques including RNA labeling, protein complexes purification, and single-molecule imaging. As a proof-of-concept of the utility of our single-molecule approaches, we show how human Dicer and its cofactor TRBP orchestrate the biogenesis of microRNA in real time. These single-molecule pull-down and fluorescence assays provide sub-second time resolution and can be applied to various ribonucleoprotein complexes that are essential for cellular processes.