Altered abundance of phosphatidyl inositides (PIs) is a feature of cancer. Various PIs mark the identity of diverse membranes in normal and malignant cells. Phosphatidylinositol 4,5-bisphosphate ...(PI(4,5)P2) resides predominantly in the plasma membrane, where it regulates cellular processes by recruiting, activating, or inhibiting proteins at the plasma membrane. We find that PTPRN2 and PLCbeta1 enzymatically reduce plasma membrane PI(4,5)P2 levels in metastatic breast cancer cells through two independent mechanisms. These genes are upregulated in highly metastatic breast cancer cells, and their increased expression associates with human metastatic relapse. Reduction in plasma membrane PI(4,5)P2 abundance by these enzymes releases the PI(4,5)P2-binding protein cofilin from its inactive membrane-associated state into the cytoplasm where it mediates actin turnover dynamics, thereby enhancing cellular migration and metastatic capacity. Our findings reveal an enzymatic network that regulates metastatic cell migration through lipid-dependent sequestration of an actin-remodeling factor. Synopsis The phosphatidylinositol phosphatase activity harboring protein tyrosine phosphatase PTPRN2 and the phospholipase PLCbeta1 are overexpressed in breast cancer. Both enzymes reduce plasma membrane PI(4,5)P2 levels, thereby enhancing cofilin-mediated actin remodeling and breast cancer metastasis. Increased expression of PTPRN2 and PLCbeta1 clinically correlates with breast cancer metastasis. PTPRN2 and PLCbeta1 promote breast cancer cell migration. PTPRN2 and PLCbeta1 modulate plasma membrane PI(4,5)P2 levels to promote actin remodeling.
The first step in the biogenesis of microRNAs is the processing of primary microRNAs (pri-miRNAs) by the microprocessor complex, composed of the RNA-binding protein DGCR8 and the type III RNase ...DROSHA (1-4). This initial event requires recognition of the junction between the stem and the flanking single-stranded RNA of the pri-miRNA hairpin by DGCR8 followed by recruitment of DROSHA, which cleaves the RNA duplex to yield the pre-miRNA product (5). While the mechanisms underlying pri-miRNA processing have been determined, the mechanism by which DGCR8 recognizes and binds pri-miRNAs, as opposed to other secondary structures present in transcripts, is not understood. Here we find in mammalian cells that methyltransferase-like 3 (METTL3) methylates pri-miRNAs, marking them for recognition and processing by DGCR8. Consistent with this, METTL3 depletion reduced the binding of DGCR8 to pri-miRNAs and resulted in the global reduction of mature miRNAs and concomitant accumulation of unprocessed pri-miRNAs. In vitro processing reactions confirmed the sufficiency of the N.sup.6-methyladenosine (m.sup.6A) mark in promoting pri-miRNA processing. Finally, gain-of-function experiments revealed that METTL3 is sufficient to enhance miRNA maturation in a global and non-cell-type-specific manner. Our findings reveal that the m.sup.6A mark acts as a key post-transcriptional modification that promotes the initiation of miRNA biogenesis.
The first step in the biogenesis of microRNAs is the processing of primary microRNAs (pri-miRNAs) by the microprocessor complex, composed of the RNA-binding protein DGCR8 and the type III RNase ...DROSHA1-4. This initial event requires recognition of the junction between the stem and the flanking single-stranded RNA of the pri-miRNA hairpin by DGCR8 followed by recruitment of DROSHA, which cleaves the RNA duplex to yield the pre-miRNA product5. While the mechanisms underlying pri-miRNA processing have been determined, the mechanism by which DGCR8 recognizes and binds pri-miRNAs, as opposed to other secondary structures present in transcripts, is not understood. Here we find in mammalian cells that methyltransferase-like 3(METTL3) methylates pri-miRNAs, marking them for recognition and processing by DGCR8. Consistent with this, METTL3 depletion reduced the binding of DGCR8 to pri-miRNAs and resulted in the global reduction of mature miRNAs and concomitant accumulation of unprocessed pri-miRNAs. In vitro processing reactions confirmed the sufficiency of the N^sup 6^-methyladenosine (m^sup 6^A) mark in promoting pri-miRNA processing. Finally, gain-of-function experiments revealed that METTL3 is sufficient to enhance miRNA maturation ina global and non-cell-type-specific manner. Our findings reveal that them^sup 6^A mark acts as a key post-transcriptional modification that promotes the initiation of miRNA biogenesis.
The first step in the biogenesis of microRNAs is the processing of primary microRNAs (pri-miRNAs) by the microprocessor complex, composed of the RNA binding protein DGCR8 and the ribonuclease type ...III DROSHA
1
–
4
. This initial event requires the recognition of the junction between the stem and the flanking single-stranded RNA of the pri-miRNA hairpin by DGCR8 followed by recruitment of DROSHA, which cleaves the RNA duplex to yield the pre-miRNA product
5
. While the mechanisms underlying pri-miRNA processing have been elucidated, the mechanism by which DGCR8 recognizes and binds pri-miRNAs as opposed to other secondary structures present in transcripts is not understood. We find that methyltransferase like 3 (METTL3) methylates pri-miRNAs, marking them for recognition and processing by DGCR8. Consistent with this, METTL3 depletion reduced the binding of DGCR8 to pri-miRNAs and resulted in the global reduction of mature miRNAs and concomitant accumulation of unprocessed pri-miRNAs.
In vitro
processing reactions confirmed the sufficiency of the m6A mark in promoting pri-miRNA processing. Finally, gain-of-function experiments revealed that METTL3 is sufficient to enhance miRNA maturation in a global and non-cell-type specific manner. Our findings reveal that the m6A mark acts as a key post-transcriptional modification that promotes the initiation of miRNA biogenesis.
Cell lineage analysis with retroviral libraries suggests that clonal progeny disperse widely in rodent cortex. To determine whether widespread dispersion is a general mammalian plan and to ...investigate phylogenetic differences in cortical development, we analyzed cell lineage in the ferret, a carnivore and near relative of the cat. The ferret possesses a highly developed, folded cerebral cortex, characteristic of higher mammalian species. Progenitor cells of the ferret cerebral cortex were tagged with an amphotropic retroviral library encoding alkaline phosphatase, and sibling relationships were determined using the polymerase chain reaction. Neuronal clones were single neurons (52%) or large clones (48%; average, 7 neurons) containing neurons and glia in widespread cortical locations. Neuronal clones in the ferret labeled at middle to late neurogenesis (embryonic day 33-35) contained large numbers of neurons and showed little tendency to cluster. The large proportion of single neuron clones, contrasted with the large size of multicell clones, suggests that some progenitors divide asymmetrically, producing a postmitotic neuron and regenerating a multipotential cell.
The effects of two new acetylcholine receptor antagonists, α‐conotoxin MII and α‐conotoxin ImI, on nicotinic synaptic transmission in the 10th paravertebral sympathetic ganglion of the leopard frog ...(Rana pipiens) were examined. The preganglionic nerve was electrically stimulated (at low frequency, 1 min−1, to avoid use‐dependent changes) while compound action potentials of B and C neurones were monitored from the postganglionic nerve.
α‐Conotoxins MII and ImI, at low micromolar concentrations, reversibly blocked both B and C waves. α‐Conotoxin MII blocked the C wave more effectively than the B wave, whereas the potency of α‐conotoxin ImI was opposite that of MII. The observation that nicotinic antagonists can differentially block synaptic transmission of B versus C neurones with opposite selectivities strongly suggests that these neurones possess distinct nicotinic receptors.
In addition to fast and slow B waves described by others, C waves with two temporally distinguishable components were present in our recordings. Each α‐conotoxin affected fast and slow B waves similarly. Likewise, toxins did not discriminate between the two components of C waves. This suggests that all neurones within each major class (B or C) may have the same nicotinic receptors.
Synthetic forms of α‐conotoxins MII and ImI were used in the present study. Their ease of synthesis and their specificities should make these toxins useful probes to investigate the various subtypes of neuronal nicotinic acetylcholine receptors.
British Journal of Pharmacology (1997) 120, 995–1000; doi:10.1038/sj.bjp.0700993
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