Rpb4 is an RNA polymerase II (Pol II) subunit that binds Pol II transcripts co-transcriptionally, accompanies them to the cytoplasm and modulates mRNA export, translation and decay by interacting with cytoplasmic RNA modulators. The importance of the cytoplasmic roles of Rpb4 was challenged by a study reporting that the phenotype of rpb2Δ rpb4Δ cells can be rescued by an Rpb2-Rpb4 fusion protein, assuming that its Rpb4 moiety cannot dissociate from Pol II and functions in the cytoplasm. Here we demonstrate that although the fusion protein supports normal transcription, it adversely affects mRNA decay, cell proliferation and adaptability–e.g., response to stress. These defects are similar, albeit milder, than the defects that characterize rpb4Δ cells. At least two mechanisms alleviate the deleterious effect of the fusion protein. First, a portion of this fusion protein is cleaved into free Rpb2 and Rpb4. The free Rpb4 is functional, as it binds mRNAs and polysomes, like WT Rpb4. Second, the fusion protein is also capable of binding poly(A)+ mRNAs in the cytoplasm, in an Rpb7-mediated manner, probably complementing the functions of the diminished Rpb4. Collectively, normal coupling between mRNA synthesis and decay requires wild-type configuration of Rpb4, and fusing Rpb4 to Rpb2 compromises this coupling.
Environmental contamination from heavy metals poses a global concern for the marine environment, as heavy metals are passed up the food chain and persist in the environment long after the pollution source is contained. Cnidarians play an important role in shaping marine ecosystems, but environmental pollution profoundly affects their vitality. Among the cnidarians, the sea anemone Nematostella vectensis is an advantageous model for addressing questions in molecular ecology and toxicology as it tolerates extreme environments and its genome has been published. Here, we employed a transcriptome-wide RNA-Seq approach to analyse N. vectensis molecular defence mechanisms against four heavy metals: Hg, Cu, Cd and Zn. Altogether, more than 4800 transcripts showed significant changes in gene expression. Hg had the greatest impact on up-regulating transcripts, followed by Cu, Zn and Cd. We identified, for the first time in Cnidaria, co-up-regulation of immediate-early transcription factors such as Egr1, AP1 and NF-κB. Time-course analysis of these genes revealed their early expression as rapidly as one hour after exposure to heavy metals, suggesting that they may complement or substitute for the roles of the metal-mediating Mtf1 transcription factor. We further characterized the regulation of a large array of stress-response gene families, including Hsp, ABC, CYP members and phytochelatin synthase, that may regulate synthesis of the metal-binding phytochelatins instead of the metallothioneins that are absent from Cnidaria genome. This study provides mechanistic insight into heavy metal toxicity in N. vectensis and sheds light on ancestral stress adaptations.
AbstractmRNA level is controlled by factors that mediate both mRNA synthesis and decay, including the 5’ to 3’ exonuclease Xrn1. Here we show that nucleocytoplasmic shuttling of several yeast mRNA decay factors plays a key role in determining both mRNA synthesis and decay. Shuttling is regulated by RNA-controlled binding of the karyopherin Kap120 to two nuclear localization sequences (NLSs) in Xrn1, location of one of which is conserved from yeast to human. The decaying RNA binds and masks NLS1, establishing a link between mRNA decay and Xrn1 shuttling. Preventing Xrn1 import, either by deleting KAP120 or mutating the two Xrn1 NLSs, compromises transcription and, unexpectedly, also cytoplasmic decay, uncovering a cytoplasmic decay pathway that initiates in the nucleus. Most mRNAs are degraded by both pathways - the ratio between them represents a full spectrum. Importantly, Xrn1 shuttling is required for proper responses to environmental changes, e.g., fluctuating temperatures, involving proper changes in mRNA abundance and in cell proliferation rate.
mRNA level is controlled by factors that mediate both mRNA synthesis and decay, including the exonuclease Xrn1 - a major mRNA synthesis and decay factor. Here we show that nucleocytoplasmic shuttling of Xrn1 and of some of its associated mRNA decay factors plays a key role in determining both mRNA synthesis and decay. Shuttling is regulated by RNA controlled binding of the karyopherin Kap120 to two nuclear localization sequences (NLSs) in Xrn1. The decaying RNA binds and masks NLS1, establishing a link between mRNA decay and Xrn1 shuttling. Mutations in the two NLSs, which prevent Xrn1 import, compromise transcription and, unexpectedly, also the cytoplasmic decay of ~50% of the cellular mRNAs - comparably to Xrn1 deletion. These findings uncover a cytoplasmic mRNA decay pathway that begins in the nucleus. Interestingly, Xrn1 shuttling is required for proper adaptation to environmental changes, in particular to ever changing environmental fluctuations.
Background: Tertiary lymphoid structures (TLS) are organized aggregates of immune cells that develop in non-lymphoid tissues and are associated with better prognosis and immunotherapy response across cancer types. Multiple IHC stainings are required for an accurate detection of TLS, making it challenging to implement as a clinical biomarker. Here, we developed a deep learning (DL) model that extracts nuclear morphology features to detect TLS from H&E slides and demonstrated its prognostic role in colorectal cancer (CRC) patients. Methods: A publicly available dataset consisting of 140 tissue cores from 35 CRC pts stained with H&E and 56 protein markers using the CODEX multiplex immunofluorescence (mIF) system was analyzed. Immune cell aggregates on the H&E were annotated by expert pathologists as either TLS or lymphocyte aggregates (LA), based on marker expression from the mIF stain on the same core. TLS were defined as dense aggregates of CD3+/CD20+/CD21+ cells, while all other immune cell aggregates were defined as LA. Next, HoVerNet was used to perform nuclear segmentation on cells within the TLS and LA on the H&E. Nuclear features including eccentricity, solidity, convexity, and nuclear intensity per cell were extracted and the mean and variance of each feature was summarized per tissue core. Based on these features, a univariate analysis comparing TLS and LA was performed, and a TLS classifier was trained using multivariate logistic regression. The classifier performance was assessed using 5 repeats of 5-fold cross validation and average accuracy and area under the ROC curve (AUC) were calculated. Overall survival (OS) was compared between patients with predicted TLS and LA using a Cox proportional hazard regression analysis. Results: From the 140 tissue cores, we identified cores with either TLS (n=18), LA (n=34) or none (n=92). No core presented both TLS and LA. In a Mann Whitney univariate analysis, cells in TLS areas demonstrated a higher mean nuclear eccentricity (p<0.0001) and solidity (p=0.01) along with lower variance in these features (p<0.0001 and p=0.001, respectively) compared to cells in LA. The multivariate classifier trained on nuclear features exhibited a 90.4% average accuracy (p<0.0001) and 94% AUC (p<0.0001) in differentiating between TLS and LA. Median OS was significantly higher in patients with at least one predicted TLS (n=13) vs. patients with at least one predicted LA (n=13) detected on H&E (NR vs. 19 months, HR=0.21, 95% CI 0.06-0.78; p=0.01). Conclusions: Nuclear based morphological features can be used to accurately detect the presence of TLS and LA from H&E slides, without the need for mIF or IHC stainings. Given the predictive value of TLS presence, this work demonstrates the potential for H&E slides to be used for patient selection for immunotherapy treatments. Citation Format: Becky Arbiv, Tal Dankovich, Sun Dagan, Yuval Shachaf, Tomer Dicker, Ron Elran, Avi Laniado, Amit Bart, Ori Zelichov, Ettai Markovits. Identification of tertiary lymphoid structures from H&E slides using deep learning analysis of nuclear morphology is associated with favorable survival in colorectal cancer patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4316.
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