7SK methylation by METTL3 promotes transcriptional activity.

A fundamental feature of cell signaling is the conversion of extracellular signals into adaptive transcriptional responses. The role of RNA modifications in this process is poorly understood. The small nuclear RNA 7SK prevents transcriptional elongation by sequestering the cyclin dependent kinase 9/cyclin T1 (CDK9/CCNT1) positive transcription elongation factor (P-TEFb) complex. We found that epidermal growth factor signaling induces phosphorylation of the enzyme methyltransferase 3 (METTL3), leading to METTL3-mediated methylation of 7SK. 7SK methylation enhanced its binding to heterogeneous nuclear ribonucleoproteins, causing the release of the HEXIM1 P-TEFb complex subunit1 (HEXIM1)/P-TEFb complex and inducing transcriptional elongation. Our findings establish the mechanism underlying 7SK activation and uncover a previously unknown function for the m6A modification in converting growth factor signaling events into a regulatory transcriptional response via an RNA methylation-dependent switch.

Performance of Medium-Rise Buildings with Reinforced Concrete Shear Walls Designed for High Seismic Hazard.

This work has evaluated the collapse fragility of a typical Chilean building for residential use, structured based on shear-resistant RC walls and inverted beams arranged along its entire perimeter, using the incremental dynamic analysis (IDA) for the evaluation of its structural behavior, using for this the 2018 version of the SeismoStruct software. This method evaluates the global collapse capacity of the building from the graphical representation of its maximum inelastic response, obtained through a non-linear time-history analysis, against the scaled intensity of a set of seismic records obtained in the subduction zone, thus creating the IDA curves of the building. The processing of the seismic records is included within the applied methodology to make them compatible with the elastic spectrum of the Chilean design, achieving an adequate seismic input in the two main structural directions. In addition, an alternative IDA method based on the elongated period is applied to calculate the seismic intensity. The results of the IDA curve obtained with this procedure and the standard IDA analysis are analyzed and compared. The results show that the method relates very well to the structure's demand and capacity and confirms the non-monotonous behavior exposed by other authors. Regarding the alternative IDA procedure, the results indicate that the method is inadequate, failing to improve the results obtained by the standard method.

[Quality of the Forensic Case Formulation in Mental Health]. / La calidad de la formulación del caso forense en salud mental.

OBJECTIVES: The "Guide to understand and assess the quality of the case formulation in forensic mental health - TECF" is tested in order to contribute to the development of the forensic reports. METHOD: Twelve mental health professionals of different Latin American traditions assessed with the TECF the quality of ten international forensic reports, both psychological and psychiatric. The guide includes the manual that describes the dimensions, criteria and guidelines to assess the quality of the forensic case formulation. RESULTS: Internal consistency indicators were in a rank of 0.34- 0.81 for the dimensions and 0.85 for the total TECF. The absolute agreement intraclass correlation coefficient among evaluators with similar tradition reached values of 0.72; 0.52; 0.76 and 0.77 for the dimensions and 0.84 for the total. The agreement among evaluators with different traditions was less. CONCLUSIONS: The TECF is a promising instrument to guide the assessment of quality and to stimulate improvement of the forensic reports. Caution is recommended in light of the traditional rating scale method due to the effect that can be produced by the different practices.

A prometastatic splicing program regulated by SNRPA1 interactions with structured RNA elements.

Aberrant alternative splicing is a hallmark of cancer, yet the underlying regulatory programs that control this process remain largely unknown. Here, we report a systematic effort to decipher the RNA structural code that shapes pathological splicing during breast cancer metastasis. We discovered a previously unknown structural splicing enhancer that is enriched near cassette exons with increased inclusion in highly metastatic cells. We show that the spliceosomal protein small nuclear ribonucleoprotein polypeptide A' (SNRPA1) interacts with these enhancers to promote cassette exon inclusion. This interaction enhances metastatic lung colonization and cancer cell invasion, in part through SNRPA1-mediated regulation of PLEC alternative splicing, which can be counteracted by splicing modulating morpholinos. Our findings establish a noncanonical regulatory role for SNRPA1 as a prometastatic splicing enhancer in breast cancer.

Nuclear TARBP2 Drives Oncogenic Dysregulation of RNA Splicing and Decay.

Post-transcriptional regulation of RNA stability is a key step in gene expression control. We describe a regulatory program, mediated by the RNA binding protein TARBP2, that controls RNA stability in the nucleus. TARBP2 binding to pre-mRNAs results in increased intron retention, subsequently leading to targeted degradation of TARBP2-bound transcripts. This is mediated by TARBP2 recruitment of the m6A RNA methylation machinery to its target transcripts, where deposition of m6A marks influences the recruitment of splicing regulators, inhibiting efficient splicing. Interactions between TARBP2 and the nucleoprotein TPR then promote degradation of these TARBP2-bound transcripts by the nuclear exosome. Additionally, analysis of clinical gene expression datasets revealed a functional role for TARBP2 in lung cancer. Using xenograft mouse models, we find that TARBP2 affects tumor growth in the lung and that this is dependent on TARBP2-mediated destabilization of ABCA3 and FOXN3. Finally, we establish ZNF143 as an upstream regulator of TARBP2 expression.

TMEM2 Is a SOX4-Regulated Gene That Mediates Metastatic Migration and Invasion in Breast Cancer.

The developmental transcription factor SOX4 contributes to the metastatic spread of multiple solid cancer types, but its direct target genes that mediate cancer progression are not well defined. Using a systematic molecular and genomic approach, we identified the TMEM2 transmembrane protein gene as a direct transcriptional target of SOX4. TMEM2 was transcriptionally activated by SOX4 in breast cancer cells where, like SOX4, TMEM2 was found to mediate proinvasive and promigratory effects. Similarly, TMEM2 was sufficient to promote metastatic colonization of breast cancer cells and its expression in primary breast tumors associated with a higher likelihood of metastatic relapse. Given earlier evidence that genetic inactivation of SOX4 or TMEM2 yield similar defects in cardiac development, our findings lead us to propose that TMEM2 may not only mediate the pathologic effects of SOX4 on cancer progression but also potentially its contributions to embryonic development. Cancer Res; 76(17); 4994-5005. ©2016 AACR.

HNRNPA2B1 Is a Mediator of m(6)A-Dependent Nuclear RNA Processing Events.

N(6)-methyladenosine (m(6)A) is the most abundant internal modification of messenger RNA. While the presence of m(6)A on transcripts can impact nuclear RNA fates, a reader of this mark that mediates processing of nuclear transcripts has not been identified. We find that the RNA-binding protein HNRNPA2B1 binds m(6)A-bearing RNAs in vivo and in vitro and its biochemical footprint matches the m(6)A consensus motif. HNRNPA2B1 directly binds a set of nuclear transcripts and elicits similar alternative splicing effects as the m(6)A writer METTL3. Moreover, HNRNPA2B1 binds to m(6)A marks in a subset of primary miRNA transcripts, interacts with the microRNA Microprocessor complex protein DGCR8, and promotes primary miRNA processing. Also, HNRNPA2B1 loss and METTL3 depletion cause similar processing defects for these pri-miRNA precursors. We propose HNRNPA2B1 to be a nuclear reader of the m(6)A mark and to mediate, in part, this mark's effects on primary microRNA processing and alternative splicing. PAPERCLIP.

N6-methyladenosine marks primary microRNAs for processing.

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. 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. 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(6)-methyladenosine (m(6)A) 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(6)A mark acts as a key post-transcriptional modification that promotes the initiation of miRNA biogenesis.

Ubiquitin ligase Nedd4L targets activated Smad2/3 to limit TGF-beta signaling.

TGF-beta induces phosphorylation of the transcription factors Smad2 and Smad3 at the C terminus as well as at an interdomain linker region. TGF-beta-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 TGF-beta-induced phosphoThr-ProTyr motif in the linker region, resulting in Smad2/3 polyubiquitination and degradation. Nedd4L is not interchangeable with Smurf1, a ubiquitin ligase that targets BMP-activated, linker-phosphorylated Smad1. Nedd4L limits the half-life of TGF-beta-activated Smads and restricts the amplitude and duration of TGF-beta gene responses, and in mouse embryonic stem cells, it 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 TGF-beta signal transduction.

Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-beta pathways.

TGF-beta 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 TGF-beta pathways.

Endogenous human microRNAs that suppress breast cancer metastasis.

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.

Balancing BMP signaling through integrated inputs into the Smad1 linker.

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.

Dephosphorylation of the linker regions of Smad1 and Smad2/3 by small C-terminal domain phosphatases has distinct outcomes for bone morphogenetic protein and transforming growth factor-beta pathways.

Smad proteins transduce bone morphogenetic protein (BMP) and transforming growth factor-beta (TGFbeta) signals upon phosphorylation of their C-terminal SXS motif by receptor kinases. The activity of Smad1 in the BMP pathway and Smad2/3 in the TGFbeta pathway is restricted by pathway cross-talk and feedback through protein kinases, including MAPK, CDK2/4, p38MAPK, JNK, and others. These kinases phosphorylate Smads 1-3 at the region that links the N-terminal DNA-binding domain and the C-terminal transcriptional domain. Phosphatases that dephosphorylate the linker region are therefore likely to play an integral part in the regulation of Smad activity. We reported previously that small C-terminal domain phosphatases 1, 2, and 3 (SCP1-3) dephosphorylate Smad1 C-terminal tail, thereby attenuating BMP signaling. Here we provide evidence that SCP1-3 also dephosphorylate the linker regions of Smad1 and Smad2/3 in vitro, in mammalian cells and in Xenopus embryos. Overexpression of SCP 1, 2, or 3 decreased linker phosphorylation of Smads 1, 2 and 3. Moreover, RNA interference-mediated knockdown of SCP1/2 increased the BMP-dependent phosphorylation of the Smad1 linker region as well as the C terminus. In contrast, SCP1/2 knockdown increased the TGFbeta-dependent linker phosphorylation of Smad2/3 but not the C-terminal phosphorylation. Consequently, SCP1/2 knockdown inhibited TGFbeta transcriptional responses, but it enhanced BMP transcriptional responses. Thus, by dephosphorylating Smad2/3 at the linker (inhibitory) but not the C-terminal (activating) site, the SCPs enhance TGFbeta signaling, and by dephosphorylating Smad1 at both sites, the SCPs reset Smad1 to the basal unphosphorylated state.

C/EBPbeta at the core of the TGFbeta cytostatic response and its evasion in metastatic breast cancer cells.

Breast cancers may evade the growth-inhibitory action of TGFbeta by accumulating defects of unknown nature that selectively eliminate cytostatic gene responses. We found the transcription factor C/EBPbeta to be essential for TGFbeta 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/EBPbeta inhibitory isoform LIP. We suggest that C/EBPbeta plays a key role in the coordination of TGFbeta cytostatic gene responses, and its malfunction may trigger evasion of these responses in breast cancer.

A FoxO-Smad synexpression group in human keratinocytes.

Transforming growth factor beta (TGF-beta) signals through activation of Smad transcription factors. Activated Smad proteins associate with different DNA-binding cofactors for the recognition and regulation of specific target genes. Members of the forkhead box O family (FoxO1, FoxO3, and FoxO4) play such a role in the induction of the cyclin-dependent kinase inhibitors p15Ink4b and p21Cip1. To delineate the organization of the TGF-beta response in human keratinocytes, we defined the set of genes whose activation by TGF-beta requires both FoxO and Smad functions. FoxO factors are shown to be essential for 11 of the 115 immediate gene activation responses to TGF-beta in these cells. FoxO1, FoxO3, and FoxO4 act redundantly as mediators of these effects. Smad4, which functions as a partner of receptor-phosphorylated Smad2/3, is required for all of these responses. These results define a FoxO-Smad synexpression group or group of genes that are jointly induced by a common mechanism in response to TGF-beta. In addition to p15INK4b and p21CIP1, these genes include mediators of stress responses (GADD45A, GADD45B, and IER1) and adaptive cell signaling responses (CTGF, JAG1, LEMD3, SGK, CDC42EP3, and OVOL1). Bioinformatic analysis of the promoter region of these genes reveals diverse configurations of Smad and FoxO binding elements, implying differences in the regulatory properties of this group of genes. Indeed, a subset of FoxO/Smad-dependent TGF-beta gene responses additionally require the transcription factor CCAAT/enhancer-binding protein beta. The composition of the FoxO-Smad synexpression group suggests that stress reactions and adaptive functions accompany the cytostatic response of keratinocytes to TGF-beta.

Unique players in the BMP pathway: small C-terminal domain phosphatases dephosphorylate Smad1 to attenuate BMP signaling.

Smad transcription factors are key signal transducers for the TGF-beta/bone morphogenetic protein (BMP) family of cytokines and morphogens. C-terminal serine phosphorylation by TGF-beta and BMP membrane receptors drives Smads into the nucleus as transcriptional regulators. Dephosphorylation and recycling of activated Smads is an integral part of this process, which is critical for agonist sensing by the cell. However, the nuclear phosphatases involved have remained unknown. Here we provide functional, biochemical, and embryological evidence identifying the SCP (small C-terminal domain phosphatase) family of nuclear phosphatases as mediators of Smad1 dephosphorylation in the BMP signaling pathway in vertebrates. Xenopus SCP2/Os4 inhibits BMP activity in the presumptive ectoderm and leads to neuralization. In Xenopus embryos, SCP2/Os4 and human SCP1, 2, and 3 cause selective dephosphorylation of Smad1 compared with Smad2, inhibiting BMP- and Smad1-dependent transcription and leading to the induction of the secondary dorsal axis. In human cells, RNAi-mediated depletion of SCP1 and SCP2 increases the extent and duration of Smad1 phosphorylation in response to BMP, the transcriptional action of Smad1, and the strength of endogenous BMP gene responses. The present identification of the SCP family as Smad C-terminal phosphatases sheds light on the events that attenuate Smad signaling and reveals unexpected links to the essential phosphatases that control RNA polymerase II in eukaryotes.

Ku70/Ku80 and DNA-dependent protein kinase catalytic subunit modulate RAG-mediated cleavage: implications for the enforcement of the 12/23 rule.

The 12/23 rule is a critical step for regulation of V(D)J recombination. To date, only the RAG proteins and high mobility group protein 1 or 2 have been implicated in 12/23 regulation. Through protein fractionation and biochemical experiments, we find that Ku70/Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) modulate RAG-mediated cleavage. Modulation of cleavage by Ku70/80 and DNA-PKcs results in preferential inhibition of 12/12 and 23/23 DNA cleavage, thus increasing 12/23 rule specificity. This observation indicates that DNA repair factors, Ku70/80 and DNA-PKcs, might be present upstream of the DNA cleavage events and not recruited downstream as is currently thought, assigning new nonrepair functions to the DNA-dependent protein kinase.