A search for general regulators of cancer metastasis has yielded a set of microRNAs for which expression is specifically lost as human breast cancer cells develop metastatic potential. Here we show that restoring the expression of these microRNAs in malignant cells suppresses lung and bone metastasis by human cancer cells in vivo. Of these microRNAs, miR-126 restoration reduces overall tumour growth and proliferation, whereas miR-335 inhibits metastatic cell invasion. miR-335 regulates a set of genes whose collective expression in a large cohort of human tumours is associated with risk of distal metastasis. miR-335 suppresses metastasis and migration through targeting of the progenitor cell transcription factor SOX4 and extracellular matrix component tenascin C. Expression of miR-126 and miR-335 is lost in the majority of primary breast tumours from patients who relapse, and the loss of expression of either microRNA is associated with poor distal metastasis-free survival. miR-335 and miR-126 are thus identified as metastasis suppressor microRNAs in human breast cancer.Although metastasis is the overwhelming cause of mortality in patients with solid tumours, our understanding of its molecular and cellular determinants is limited 1-3 . Transcriptional profiling has revealed sets of genes, or `signatures', for which expression in primary tumours correlates with metastatic relapse or poor survival 4 . Some of these genes endow cancer cells with a more invasive phenotype, enhanced angiogenic and intravasation activity, the ability to exit from the circulation, or an ability to modify the metastasis microenvironment 5,6 . Such gene sets are thus providing numerous candidate mediators of metastasis to be validated through functional and clinical studies. Much less insight, however, has been gained into the

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 DROSHA1–4. 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 product5. 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.

SUMMARY N6-methyladenosine (m6A) is the most abundant internal modification of messenger RNA. While the presence of m6A on transcripts can impact alternative splicing, a nuclear reader of this mark that mediates the processing of nuclear transcripts has not been identified. We find that the RNA-binding HNRNPA2B1 protein binds m6A-bearing RNAs in vivo and in vitro and its biochemical footprint matches the m6A consensus motif. HNRNPA2B1 directly binds a set of nuclear transcripts and modulates their alternative splicing in a similar manner as the m6A ‘writer’ METTL3. Moreover, HNRNPA2B1 binds to m6A marks in a subset of primary-miRNA transcripts, interacts with the microRNA Microprocessor complex protein DGCR8, and promotes primary miRNA processing—phenocopying the effect of METTL3 depletion on the processing of these precursor transcripts. We propose HNRNPA2B1 to be a nuclear reader of the m6A mark and to mediate, in part, this mark’s effects on primary microRNA processing and alternative splicing.

TGFβ and BMP receptor kinases activate Smad transcription factors by C-terminal phosphorylation. We have identified a subsequent agonist-induced phosphorylation that plays a central dual role in Smad transcriptional activation and turnover. As receptor-activated Smads form transcriptional complexes, they are phosphorylated at an interdomain linker region by CDK8 and CDK9, which are components of transcriptional mediator and elongation complexes. These phosphorylations promote Smad transcriptional action, which in the case of Smad1, is mediated by the recruitment of YAP to the phosphorylated linker sites. An effector of the highly conserved Hippo organ size control pathway, YAP supports Smad1-dependent transcription and is required for BMP suppression of neural differentiation of mouse embryonic stem cells. The phosphorylated linker is ultimately recognized by specific ubiquitin ligases, leading to proteasome-mediated turnover of activated Smad proteins. Thus, nuclear CDK8/9 drive a cycle of Smad utilization and disposal that is an integral part of canonical BMP and TGFβ pathways.

FGF and other Ras/MAPK pathway activators counterbalance BMP action during neurogenesis, bone formation, and other aspects of vertebrate development and homeostasis. BMP receptors signal through C-terminal phosphorylation and nuclear translocation of the transcription factor Smad1, whereas MAPKs catalyze inhibitory phosphorylation in the Smad1 linker region. Here we show that linker phosphorylation restricts Smad1 activity by enabling Smad1 recognition by the HECT-domain ubiquitin ligase Smurf1. Besides causing Smad1 polyubiquitination, Smurf1 binding inhibits the interaction of Smad1 with the nuclear translocation factor Nup214. Consequently, MAPK-dependent Smurf1 binding leads Smad1 alternatively to degradation or cytoplasmic retention. Smad1 linker phosphorylation and Smurf1 act as interdependent inputs to control BMP signaling during mouse osteoblast differentiation and Xenopus neural development. Linker phosphorylation is triggered also by BMP, providing feedback control. The interplay between linker phosphorylation, Smurf-dependent ubiquitination, and nucleoporin exclusion enables regulation of BMP action by diverse signals and biological contexts.

Summary TGFβ induces phosphorylation of the transcription factors Smad2 and Smad3 at the C-terminus as well as at an interdomain linker region. TGFβ-induced linker phosphorylation marks the activated Smad proteins for proteasome-mediated destruction. Here we identify Nedd4L as the ubiquitin ligase responsible for this step. Through its WW domain Nedd4L specifically recognizes a TGFβ-induced phosphoThr-ProTyr motif in the linker region, resulting in Smad2/3 poly-ubiquitination and degradation. Nedd4L is not interchangeable with Smurf1, a ubiquitin ligase that targets BMP-activated, linker-phosphorylated Smad1. Nedd4L limits the half-life of TGFβ activated Smads, restricts the amplitude and duration of TGFβ gene responses, and in mouse embryonic stem cells limits the induction of mesoendodermal fates by Smad2/3-activating factors. Hierarchical regulation is provided by SGK1, which phosphorylates Nedd4L to prevent binding of Smad2/3. Previously identified as a regulator of renal sodium channels, Nedd4L is shown here to play a broader role as a general modulator of Smad turnover during TGFβ signal transduction.

Breast cancers may evade the growth-inhibitory action of TGFb by accumulating defects of unknown nature that selectively eliminate cytostatic gene responses. We found the transcription factor C/EBPb to be essential for TGFb induction of the cell cycle inhibitor p15INK4b by a FoxO-Smad complex and repression of c-MYC by an E2F4/5-Smad complex in human epithelial cells. These cytostatic responses are selectively missing in metastatic breast cancer cells from half of the patients that we tested. The basis for this loss was traced to an excess of the C/EBPb inhibitory isoform LIP. We suggest that C/EBPb plays a key role in the coordination of TGFb cytostatic gene responses, and its malfunction may trigger evasion of these responses in breast cancer.

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