Conditional RNA interference in mammalian cells via RNA transactivation.

RNA interference (RNAi) is a powerful tool for sequence-specific gene knockdown in therapeutic and research applications. However, spatiotemporal control of RNAi is required to decrease nonspecific targeting, potential toxicity, and allow targeting of essential genes. Herein we describe a class of de-novo-designed RNA switches that enable sequence-specific regulation of RNAi in mammalian cells. Using cis-repressing RNA elements, we engineer RNA devices that only initiate microRNA biogenesis when binding with cognate trigger RNAs. We demonstrate that this conditional RNAi system, termed Orthogonal RNA Interference induced by Trigger RNA (ORIENTR), provides up to 14-fold increases in artificial miRNA biogenesis upon activation in orthogonal libraries. We show that integration of ORIENTR triggers with dCas13d enhances dynamic range to up to 31-fold. We further demonstrate that ORIENTR can be applied to detect endogenous RNA signals and to conditionally knockdown endogenous genes, thus enabling regulatory possibilities including cell-type-specific RNAi and rewiring of transcriptional networks via RNA profile.

Gra-CRC-miRTar: The pre-trained nucleotide-to-graph neural networks to identify potential miRNA targets in colorectal cancer.

Colorectal cancer (CRC) is the third most diagnosed cancer and the second deadliest cancer worldwide representing a major public health problem. In recent years, increasing evidence has shown that microRNA (miRNA) can control the expression of targeted human messenger RNA (mRNA) by reducing their abundance or translation, acting as oncogenes or tumor suppressors in various cancers, including CRC. Due to the significant up-regulation of oncogenic miRNAs in CRC, elucidating the underlying mechanism and identifying dysregulated miRNA targets may provide a basis for improving current therapeutic interventions. In this paper, we proposed Gra-CRC-miRTar, a pre-trained nucleotide-to-graph neural network framework, for identifying potential miRNA targets in CRC. Different from previous studies, we constructed two pre-trained models to encode RNA sequences and transformed them into de Bruijn graphs. We employed different graph neural networks to learn the latent representations. The embeddings generated from de Bruijn graphs were then fed into a Multilayer Perceptron (MLP) to perform the prediction tasks. Our extensive experiments show that Gra-CRC-miRTar achieves better performance than other deep learning algorithms and existing predictors. In addition, our analyses also successfully revealed 172 out of 201 functional interactions through experimentally validated miRNA-mRNA pairs in CRC. Collectively, our effort provides an accurate and efficient framework to identify potential miRNA targets in CRC, which can also be used to reveal miRNA target interactions in other malignancies, facilitating the development of novel therapeutics.

An Interpretable Population Graph Network to Identify Rapid Progression of Alzheimer's Disease Using UK Biobank.

Alzheimer's disease (AD) manifests with varying progression rates across individuals, necessitating the understanding of their intricate patterns of cognition decline that could contribute to effective strategies for risk monitoring. In this study, we propose an innovative interpretable population graph network framework for identifying rapid progressors of AD by utilizing patient information from electronic health-related records in the UK Biobank. To achieve this, we first created a patient similarity graph, in which each AD patient is represented as a node; and an edge is established by patient clinical characteristics distance. We used graph neural networks (GNNs) to predict rapid progressors of AD and created a GNN Explainer with SHAP analysis for interpretability. The proposed model demonstrates superior predictive performance over the existing benchmark approaches. We also revealed several clinical features significantly associated with the prediction, which can be used to aid in effective interventions for the progression of AD patients.

Target-directed microRNA degradation: Mechanisms, significance, and functional implications.

MicroRNAs (miRNAs) are small non-coding RNAs that play a fundamental role in enabling miRNA-mediated target repression, a post-transcriptional gene regulatory mechanism preserved across metazoans. Loss of certain animal miRNA genes can lead to developmental abnormalities, disease, and various degrees of embryonic lethality. These short RNAs normally guide Argonaute (AGO) proteins to target RNAs, which are in turn translationally repressed and destabilized, silencing the target to fine-tune gene expression and maintain cellular homeostasis. Delineating miRNA-mediated target decay has been thoroughly examined in thousands of studies, yet despite these exhaustive studies, comparatively less is known about how and why miRNAs are directed for decay. Several key observations over the years have noted instances of rapid miRNA turnover, suggesting endogenous means for animals to induce miRNA degradation. Recently, it was revealed that certain targets, so-called target-directed miRNA degradation (TDMD) triggers, can "trigger" miRNA decay through inducing proteolysis of AGO and thereby the bound miRNA. This process is mediated in animals via the ZSWIM8 ubiquitin ligase complex, which is recruited to AGO during engagement with triggers. Since its discovery, several studies have identified that ZSWIM8 and TDMD are indispensable for proper animal development. Given the rapid expansion of this field of study, here, we summarize the key findings that have led to and followed the discovery of ZSWIM8-dependent TDMD. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA in Disease and Development > RNA in Development.

Ethical leadership and TMT decision-making of corporate social responsibility - a perspective of self-determination theory.

This study examines the impact of ethical leadership on top management team (TMT) decision-making regarding corporate social responsibility (CSR), considering the mediating role of TMT passion and the moderating role of performance stress. The study distinguishes between TMT harmonious and obsessive work passion and categorizes CSR as proactive and reactive. The findings reveal the following: (1) Ethical leadership positively influences proactive CSR, with TMT harmonious work passion acting as a positive mediator and TMT obsessive work passion playing a negative mediating role; (2) ethical leadership positively affects reactive CSR, with both TMT harmonious and obsessive work passion serving as positive mediators; (3) performance stress diminishes the impact of ethical leadership on TMT harmonious work passion; however, it amplifies the effect on TMT obsessive work passion. Consequently, the mediating effect of TMT harmonious work passion weakens, while the mediating effect of TMT obsessive work passion strengthens. This study emphasizes the significant role of TMT in CSR strategic decision-making and proposes a novel mediating mechanism through which ethical leadership drives CSR decision-making by considering TMT work passion. These findings reconcile the theoretical-practical conflict and have important theoretical and practical implications for enterprises in fulfilling their social responsibility.

Meet the authors: Yuzhi Wang, Conner Traugot, and Mingyi Xie.

We talk to authors Yuzhi Wang, Conner Traugot, and Mingyi Xie about their paper "N6-methyladenosine in 7SK small nuclear RNA underlies RNA polymerase II transcription regulation" (this issue of Molecular Cell), their path to research science, and the interesting findings that keep bringing them back to the bench.

N6-methyladenosine in 7SK small nuclear RNA underlies RNA polymerase II transcription regulation.

N6-methyladenosine (m6A) modifications play crucial roles in RNA metabolism. How m6A regulates RNA polymerase II (RNA Pol II) transcription remains unclear. We find that 7SK small nuclear RNA (snRNA), a regulator of RNA Pol II promoter-proximal pausing, is highly m6A-modified in non-small cell lung cancer (NSCLC) cells. In A549 cells, we identified eight m6A sites on 7SK and discovered methyltransferase-like 3 (METTL3) and alkB homolog 5 (ALKBH5) as the responsible writer and eraser. When the m6A-7SK is specifically erased by a dCasRx-ALKBH5 fusion protein, A549 cell growth is attenuated due to reduction of RNA Pol II transcription. Mechanistically, removal of m6A leads to 7SK structural rearrangements that facilitate sequestration of the positive transcription elongation factor b (P-TEFb) complex, which results in reduction of serine 2 phosphorylation (Ser2P) in the RNA Pol II C-terminal domain and accumulation of RNA Pol II in the promoter-proximal region. Taken together, we uncover that m6A modifications of a non-coding RNA regulate RNA Pol II transcription and NSCLC tumorigenesis.

YAP at the Crossroads of Biomechanics and Drug Resistance in Human Cancer.

Biomechanical forces are of fundamental importance in biology, diseases, and medicine. Mechanobiology is an emerging interdisciplinary field that studies how biological mechanisms are regulated by biomechanical forces and how physical principles can be leveraged to innovate new therapeutic strategies. This article reviews state-of-the-art mechanobiology knowledge about the yes-associated protein (YAP), a key mechanosensitive protein, and its roles in the development of drug resistance in human cancer. Specifically, the article discusses three topics: how YAP is mechanically regulated in living cells; the molecular mechanobiology mechanisms by which YAP, along with other functional pathways, influences drug resistance of cancer cells (particularly lung cancer cells); and finally, how the mechanical regulation of YAP can influence drug resistance and vice versa. By integrating these topics, we present a unified framework that has the potential to bring theoretical insights into the design of novel mechanomedicines and advance next-generation cancer therapies to suppress tumor progression and metastasis.

Functional Interrogation of Ca2+ Signals in Human Cancer Cells In Vitro and Ex Vivo by Fluorescent Microscopy and Molecular Tools.

Genetically encoded calcium indicators (GECIs) and high-resolution confocal microscopy enable dynamic visualization of calcium signals in cells and tissues. Two-dimensional and 3D biocompatible materials mimic the mechanical microenvironments of tumor and healthy tissues in a programmable manner. Cancer xenograft models and ex vivo functional imaging of tumor slices reveal physiologically relevant functions of calcium dynamics in tumors at different progression stages. Integration of these powerful techniques allows us to quantify, diagnose, model, and understand cancer pathobiology. Here, we describe detailed materials and methods used to establish this integrated interrogation platform, from generating transduced cancer cell lines that stably express CaViar (GCaMP5G + QuasAr2) to in vitro and ex vivo calcium imaging of the cells in 2D/3D hydrogels and tumor tissues. These tools open the possibility for detailed explorations of mechano-electro-chemical network dynamics in living systems.

Fluorescent In Situ Detection of RNA-Protein Interactions in Intact Cells by RNA-PLA.

RNA-protein proximity ligation assay (RNA-PLA) enables the detection of specific RNA-protein interactions in fixed cells. In RNA-PLA, bridging and ligation of a circular DNA template occurs if the target RNA and protein are within 40 nanometers of each other. The resulting circular template is amplified by rolling circle amplification and abundantly recognized by fluorescent antisense DNA oligonucleotides. This strategy therefore enables localization of RNA-protein interactions in situ with high specificity and sensitivity. Here, we describe the use of RNA-PLA to detect interactions between a nuclear viral RNA and a host RNA-binding protein in Epstein-Barr virus (EBV)-infected B cells.

Investigating miR-9 as a mediator in laryngeal cancer health disparities.

Background: For several decades, Black patients have carried a higher burden of laryngeal cancer among all races. Even when accounting for sociodemographics, a disparity remains. Differentially expressed microRNAs have been linked to racially disparate clinical outcomes in breast and prostate cancers, yet an association in laryngeal cancer has not been addressed. In this study, we present our computational analysis of differentially expressed miRNAs in Black compared with White laryngeal cancer and further validate microRNA-9-5p (miR-9-5p) as a potential mediator of cancer phenotype and chemoresistance. Methods: Bioinformatic analysis of 111 (92 Whites, 19 Black) laryngeal squamous cell carcinoma (LSCC) specimens from the TCGA revealed miRNAs were significantly differentially expressed in Black compared with White LSCC. We focused on miR-9-5 p which had a significant 4-fold lower expression in Black compared with White LSCC (p<0.05). After transient transfection with either miR-9 mimic or inhibitor in cell lines derived from Black (UM-SCC-12) or White LSCC patients (UM-SCC-10A), cellular migration and cell proliferation was assessed. Alterations in cisplatin sensitivity was evaluated in transient transfected cells via IC50 analysis. qPCR was performed on transfected cells to evaluate miR-9 targets and chemoresistance predictors, ABCC1 and MAP1B. Results: Northern blot analysis revealed mature miR-9-5p was inherently lower in cell line UM-SCC-12 compared with UM-SCC-10A. UM -SCC-12 had baseline increase in cellular migration (p < 0.01), proliferation (p < 0.0001) and chemosensitivity (p < 0.01) compared to UM-SCC-10A. Increasing miR-9 in UM-SCC-12 cells resulted in decreased cellular migration (p < 0.05), decreased proliferation (p < 0.0001) and increased sensitivity to cisplatin (p < 0.001). Reducing miR-9 in UM-SCC-10A cells resulted in increased cellular migration (p < 0.05), increased proliferation (p < 0.05) and decreased sensitivity to cisplatin (p < 0.01). A significant inverse relationship in ABCC1 and MAP1B gene expression was observed when miR-9 levels were transiently elevated or reduced in either UM-SCC-12 or UM-SCC-10A cell lines, respectively, suggesting modulation by miR-9. Conclusion: Collectively, these studies introduce differential miRNA expression in LSCC cancer health disparities and propose a role for low miR-9-5p as a mediator in LSCC tumorigenesis and chemoresistance.

Screening of Drosophila microRNA-degradation sequences reveals Argonaute1 mRNA's role in regulating miR-999.

MicroRNAs (miRNA) load onto AGO proteins to target mRNAs for translational repression or degradation. However, miRNA degradation can be triggered when extensively base-paired with target RNAs, which induces confirmational change of AGO and recruitment of ZSWIM8 ubiquitin ligase to mark AGO for proteasomal degradation. This target RNA-directed miRNA degradation (TDMD) mechanism appears to be evolutionarily conserved, but recent studies have focused on mammalian systems. Here, we performed AGO1-CLASH in Drosophila S2 cells, with Dora (ortholog of vertebrate ZSWIM8) knockout mediated by CRISPR-Cas9 to identify five TDMD triggers (sequences that can induce miRNA degradation). Interestingly, one trigger in the 3' UTR of AGO1 mRNA induces miR-999 degradation. CRISPR-Cas9 knockout of the AGO1 trigger in S2 cells and in Drosophila specifically elevates miR-999, with concurrent repression of the miR-999 targets. AGO1 trigger knockout flies respond poorly to hydrogen peroxide-induced stress, demonstrating the physiological importance of this TDMD event.

Simultaneous Photoluminescence and Photothermal Investigation of Individual CH3NH3PbBr3 Microcrystals.

The photoluminescence (PL) of CH3NH3PbBr3 (MAPbBr3), from thin films to nanoparticles, has been widely studied, providing information about charge carrier dynamics. However, the other energy dissipative channel, nonradiative relaxation, has not been thoroughly investigated due to a lack of proper technology. In this work, we simultaneously investigated the PL and photothermal (PT) properties of single MAPbBr3 microcrystals (MCs) by a home-built PL and PT microscope. In addition to the direct observation of the heterogeneity of the PL and PT images and kinetics of different MCs, we demonstrated the variation in the absorption of single MAPbBr3 MCs, which was believed to be constant. We also proved that more absorbed energy dissipated from the nonradiative channel at higher heating power. These results show that PL and PT microscopy is an effective and convenient method to investigate the charge carrier behaviors of optoelectronic materials at the single particle level for a deep understanding of their photophysical processes.

Enhancer-Mediated Formation of Nuclear Transcription Initiation Domains.

Enhancers in higher eukaryotes and upstream activating sequences (UASs) in yeast have been shown to recruit components of the RNA polymerase II (Pol II) transcription machinery. At least a fraction of Pol II recruited to enhancers in higher eukaryotes initiates transcription and generates enhancer RNA (eRNA). In contrast, UASs in yeast do not recruit transcription factor TFIIH, which is required for transcription initiation. For both yeast and mammalian systems, it was shown that Pol II is transferred from enhancers/UASs to promoters. We propose that there are two modes of Pol II recruitment to enhancers in higher eukaryotes. Pol II complexes that generate eRNAs are recruited via TFIID, similar to mechanisms operating at promoters. This may involve the binding of TFIID to acetylated nucleosomes flanking the enhancer. The resulting eRNA, together with enhancer-bound transcription factors and co-regulators, contributes to the second mode of Pol II recruitment through the formation of a transcription initiation domain. Transient contacts with target genes, governed by proteins and RNA, lead to the transfer of Pol II from enhancers to TFIID-bound promoters.

Towards an integrative understanding of cancer mechanobiology: calcium, YAP, and microRNA under biophysical forces.

An increasing number of studies have demonstrated the significant roles of the interplay between microenvironmental mechanics in tissues and biochemical-genetic activities in resident tumor cells at different stages of tumor progression. Mediated by molecular mechano-sensors or -transducers, biomechanical cues in tissue microenvironments are transmitted into the tumor cells and regulate biochemical responses and gene expression through mechanotransduction processes. However, the molecular interplay between the mechanotransduction processes and intracellular biochemical signaling pathways remains elusive. This paper reviews the recent advances in understanding the crosstalk between biomechanical cues and three critical biochemical effectors during tumor progression: calcium ions (Ca2+), yes-associated protein (YAP), and microRNAs (miRNAs). We address the molecular mechanisms underpinning the interplay between the mechanotransduction pathways and each of the three effectors. Furthermore, we discuss the functional interactions among the three effectors in the context of soft matter and mechanobiology. We conclude by proposing future directions on studying the tumor mechanobiology that can employ Ca2+, YAP, and miRNAs as novel strategies for cancer mechanotheraputics. This framework has the potential to bring insights into the development of novel next-generation cancer therapies to suppress and treat tumors.

Biological features between miRNAs and their targets are unveiled from deep learning models.

MicroRNAs (miRNAs) are ~ 22 nucleotide ubiquitous gene regulators. They modulate a broad range of essential cellular processes linked to human health and diseases. Consequently, identifying miRNA targets and understanding how they function are critical for treating miRNA associated diseases. In our earlier work, a hybrid deep learning-based approach (miTAR) was developed for predicting miRNA targets. It performs substantially better than the existing methods. The approach integrates two major types of deep learning algorithms: convolutional neural networks (CNNs) and recurrent neural networks (RNNs). However, the features in miRNA:target interactions learned by miTAR have not been investigated. In the current study, we demonstrated that miTAR captures known features, including the involvement of seed region and the free energy, as well as multiple novel features, in the miRNA:target interactions. Interestingly, the CNN and RNN layers of the model perform differently at capturing the free energy feature: the units in RNN layer is more unique at capturing the feature but collectively the CNN layer is more efficient at capturing the feature. Although deep learning models are commonly thought "black-boxes", our discoveries support that the biological features in miRNA:target can be unveiled from deep learning models, which will be beneficial to the understanding of the mechanisms in miRNA:target interactions.

Sequencing of Argonaute-bound microRNA/mRNA hybrids reveals regulation of the unfolded protein response by microRNA-320a.

MicroRNAs (miRNA) are short non-coding RNAs widely implicated in gene regulation. Most metazoan miRNAs utilize the RNase III enzymes Drosha and Dicer for biogenesis. One notable exception is the RNA polymerase II transcription start sites (TSS) miRNAs whose biogenesis does not require Drosha. The functional importance of the TSS-miRNA biogenesis is uncertain. To better understand the function of TSS-miRNAs, we applied a modified Crosslinking, Ligation, and Sequencing of Hybrids on Argonaute (AGO-qCLASH) to identify the targets for TSS-miRNAs in HCT116 colorectal cancer cells with or without DROSHA knockout. We observed that miR-320a hybrids dominate in TSS-miRNA hybrids identified by AGO-qCLASH. Targets for miR-320a are enriched for the eIF2 signaling pathway, a downstream component of the unfolded protein response. Consistently, in miR-320a mimic- and antagomir- transfected cells, differentially expressed gene products are associated with eIF2 signaling. Within the AGO-qCLASH data, we identified the endoplasmic reticulum (ER) chaperone calnexin as a direct miR-320a down-regulated target, thus connecting miR-320a to the unfolded protein response. During ER stress, but not amino acid deprivation, miR-320a up-regulates ATF4, a critical transcription factor for resolving ER stress. In summary, our study investigates the targetome of the TSS-miRNAs in colorectal cancer cells and establishes miR-320a as a regulator of unfolded protein response.

Widespread microRNA degradation elements in target mRNAs can assist the encoded proteins.

Binding of microRNAs (miRNAs) to mRNAs normally results in post-transcriptional repression of gene expression. However, extensive base-pairing between miRNAs and target RNAs can trigger miRNA degradation, a phenomenon called target RNA-directed miRNA degradation (TDMD). Here, we systematically analyzed Argonaute-CLASH (cross-linking, ligation, and sequencing of miRNA-target RNA hybrids) data and identified numerous candidate TDMD triggers, focusing on their ability to induce nontemplated nucleotide addition at the miRNA 3' end. When exogenously expressed in various cell lines, eight triggers induce degradation of corresponding miRNAs. Both the TDMD base-pairing and surrounding sequences are essential for TDMD. CRISPR knockout of endogenous trigger or ZSWIM8, a ubiquitin ligase essential for TDMD, reduced miRNA degradation. Furthermore, degradation of miR-221 and miR-222 by a trigger in BCL2L11, which encodes a proapoptotic protein, enhances apoptosis. Therefore, we uncovered widespread TDMD triggers in target RNAs and demonstrated an example that could functionally cooperate with the encoded protein.

A noncanonical microRNA derived from the snaR-A noncoding RNA targets a metastasis inhibitor.

MicroRNAs (miRNAs) are small noncoding RNAs that function as critical posttranscriptional regulators in various biological processes. While most miRNAs are generated from processing of long primary transcripts via sequential Drosha and Dicer cleavage, some miRNAs that bypass Drosha cleavage can be transcribed as part of another small noncoding RNA. Here, we develop the target-oriented miRNA discovery (TOMiD) bioinformatic analysis method to identify Drosha-independent miRNAs from Argonaute crosslinking and sequencing of hybrids (Ago-CLASH) data sets. Using this technique, we discovered a novel miRNA derived from a primate specific noncoding RNA, the small NF90 associated RNA A (snaR-A). The miRNA derived from snaR-A (miR-snaR) arises independently of Drosha processing but requires Exportin-5 and Dicer for biogenesis. We identify that miR-snaR is concurrently up-regulated with the full snaR-A transcript in cancer cells. Functionally, miR-snaR associates with Ago proteins and targets NME1, a key metastasis inhibitor, contributing to snaR-A's role in promoting cancer cell migration. Our findings suggest a functional link between a novel miRNA and its precursor noncoding RNA.