Post-transcriptional Gene Regulation Research Articles

Method for the Enrichment of N6-Methyladenosine-Modified Cellular and HIV-1 RNA.

N6-methyladenosine (m6A) modification of RNA is an important area in studying viral replication, cellular responses, and host immunity. HIV-1 RNA contains multiple m6A modifications that regulate viral replication and gene expression. HIV-1 infection of CD4+ T-cells or HIV-1 envelope protein treatment upregulates m6A levels of cellular RNA. Changes in the m6A modification of cellular transcripts in response to HIV-1 infection provide new insights into the mechanisms of posttranscriptional gene regulation in the host cell. To better investigate the functions of m6A modification in HIV-1 infection and innate immune responses, it is helpful to standardize basic protocols. Here, we describe a method for the selective enrichment of m6A-modified RNA from HIV-1-infected primary CD4+ T-cells based on immunoprecipitation. The enriched RNA with m6A modifications can be used in a variety of downstream applications to determine the methylation status of viral or cellular RNA at resolution from transcript level down to single nucleotide.

Intron retention is a mechanism of erythropoietin regulation in brain cell models

Intron retention is a mechanism of post-transcriptional gene regulation, including genes involved in erythropoiesis. Erythropoietin (EPO) is a hormone without evidence of intracellular vesicle storage that regulates erythropoiesis. We hypothesize that EPO uses intron retention as a mechanism of post-transcriptional regulation in response to hypoxia and ischemia. Cell models of hypoxia and ischemia for kidney, liver, and brain cells were examined for intron retention by real time quantitative PCR. EPO expression increased in most cells except for blood brain barrier and liver cells. The intron retained transcript ratio decreased in brain cells, except for Astrocytes, but showed no change in kidney or liver after 24 h of ischemia. The shift in intron ratio was maintained when using poly (A) enriched cDNA, suggesting that intron retention is not due to immature transcripts. The expression of EPO was elevated at variable time points amongst cell models with the intron ratio also changing over a time course of 2 to 16 h after ischemia. We conclude that intron retention is a mechanism regulating EPO expression in response to ischemia in a tissue specific manner.

Low-Dose Ionizing Radiation-Crosslinking Immunoprecipitation (LDIR-CLIP) Identified Irradiation-Sensitive RNAs for RNA-Binding Protein HuR-Mediated Decay.

Although ionizing radiation (IR) is widely used for therapeutic and research purposes, studies on low-dose ionizing radiation (LDIR) are limited compared with those on other IR approaches, such as high-dose gamma irradiation and ultraviolet irradiation. High-dose IR affects DNA damage response and nucleotide-protein crosslinking, among other processes; however, the molecular consequences of LDIR have been poorly investigated. Here, we developed a method to profile RNA species crosslinked to an RNA-binding protein, namely, human antigen R (HuR), using LDIR and high-throughput RNA sequencing. The RNA fragments isolated via LDIR-crosslinking and immunoprecipitation sequencing were crosslinked to HuR and protected from RNase-mediated digestion. Upon crosslinking HuR to target mRNAs such as PAX6, ZFP91, NR2F6, and CAND2, the transcripts degraded rapidly in human cell lines. Additionally, PAX6 and NR2F6 downregulation mediated the beneficial effects of LDIR on cell viability. Thus, our approach provides a method for investigating post-transcriptional gene regulation using LDIR.

Targeting IGF2BP3 enhances antileukemic effects of menin-MLL inhibition in MLL-AF4 leukemia

AbstractRNA-binding proteins (RBPs) are emerging as a novel class of therapeutic targets in cancer, including in leukemia, given their important role in posttranscriptional gene regulation, and have the unexplored potential to be combined with existing therapies. The RBP insulin-like growth factor 2 messenger RNA–binding protein 3 (IGF2BP3) has been found to be a critical regulator of MLL-AF4 leukemogenesis and represents a promising therapeutic target. Here, we study the combined effects of targeting IGF2BP3 and menin-MLL interaction in MLL-AF4–driven leukemia in vitro and in vivo, using genetic inhibition with CRISPR-Cas9–mediated deletion of Igf2bp3 and pharmacologic inhibition of the menin-MLL interaction with multiple commercially available inhibitors. Depletion of Igf2bp3 sensitized MLL-AF4 leukemia to the effects of menin-MLL inhibition on cell growth and leukemic initiating cells in vitro. Mechanistically, we found that both Igf2bp3 depletion and menin-MLL inhibition led to increased differentiation in vitro and in vivo, seen in functional readouts and by gene expression analyses. IGF2BP3 knockdown had a greater effect on increasing survival and attenuating disease than pharmacologic menin-MLL inhibition with small molecule MI-503 alone and showed enhanced antileukemic effects in combination. Our work shows that IGF2BP3 is an oncogenic amplifier of MLL-AF4–mediated leukemogenesis and a potent therapeutic target, providing a paradigm for targeting leukemia at both the transcriptional and posttranscriptional level.

MiR-221 and Parkinson's disease: A biomarker with therapeutic potential.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, leading to various motor and non-motor symptoms. Several cellular and molecular mechanisms such as alpha-synuclein (α-syn) accumulation, mitochondrial dysfunction, oxidative stress and neuroinflammation are involved in the pathogenesis of this disease. MicroRNAs (miRNAs) play important roles in post-transcriptional gene regulation. They are typically about 21-25 nucleotides in length and are involved in the regulation of gene expression by binding to the messenger RNA (mRNA) molecules. miRNAs like miR-221 play important roles in various biological processes, including development, cell proliferation, differentiation and apoptosis. miR-221 promotes neuronal survival against oxidative stress and neurite outgrowth and neuronal differentiation. Additionally, the role of miR-221 in PD has been investigated in several studies. According to the results of these studies, (1) miR-221 protects PC12 cells against oxidative stress induced by 6-hydroxydopamine; (2) miR-221 prevents Bax/caspase-3 signalling activation by stopping Bim; (3) miR-221 has moderate predictive power for PD; (4) miR-221 directly targets PTEN, and PTEN over-expression eliminates the protective action of miR-221 on p-AKT expression in PC12 cells; and (5) miRNA-221 controls cell viability and apoptosis by manipulating the Akt signalling pathway in PD. This review study suggested that miR-221 has the potential to be used as a clinical biomarker for PD diagnosis and stage assignment.

Identifying Potential Conserved Cyperus rotundus microRNAs and their Human Targets by Transcriptome Mining: A Computational Genomics Assessment

For centuries, Cyperus rotundus has been a dependable natural cure for various conditions, such as diarrhea, diabetes, inflammation, malaria, and stomach and intestinal issues. However, this plant has become a weed in many tropical and subtropical areas, leading to significant economic damage in the agricultural industry. Plant consumption can lead to plant-derived microRNAs in the human body, affecting human posttranscriptional gene regulation. Recent research has shown that microRNAs can regulate across different kingdoms, indicating that miRNAs from external sources function similarly to those found in mammals. According to the present study, five conserved Cyperus rotundus microRNAs have been identified that could have targeted 129 human messenger RNAs, and three (cro-miR160a-5p, cro-miR168, and cro-miR160c-3p) have shown function homology with seven human microRNAs (hsa-miR-585-3p, hsa-miR-431-5p, hsa-miR-4730, hsa-miR-7854-3p, hsa-miR-4259, hsa-miR-2682-3p and hsa-miR-937-3p). These targets are linked to health conditions such as high blood pressure, Angina Pectoris, Dysarthria, Extrapyramidal Disorders, and Muscle hypotonia. Employing functional enrichment analysis, we revealed that the target genes were considerably associated with Hedgehog signaling and other signaling pathways. The top hub proteins ATXN1, UCHL5, CBX5, UBC, CSNK1A1, USP8, PPP1R12A, APOB, ARID1A, and CORO1C show their interaction with human diseases, including hepatocellular carcinoma. The noteworthy discovery of utilizing Cyperus rotundus microRNAs to predict target messenger RNAs in humans has the potential to revolutionize the way we approach researching and treating various human diseases. This knowledge provides us the confidence to identify new candidates for further study and, ultimately, to improve the lives of those afflicted by these diseases.

Mechanism and Therapeutic Prospect of miRNAs in Neurodegenerative Diseases.

MicroRNAs (miRNAs) are the smallest class of noncoding RNAs, which widely exist in animals and plants. They can inhibit translation or overexpression by combining with mRNA and participate in posttranscriptional regulation of genes, resulting in reduced expression of target proteins, affecting the development, growth, aging, metabolism, and other physiological and pathological processes of animals and plants. It is a powerful negative regulator of gene expression. It mediates the information exchange between different cellular pathways in cellular homeostasis and stress response and regulates the differentiation, plasticity, and neurotransmission of neurons. In neurodegenerative diseases, in addition to the complex interactions between genetic susceptibility and environmental factors, miRNAs can serve as a promising diagnostic tool for diseases. They can also increase or reduce neuronal damage by regulating the body's signaling pathways, immune system, stem cells, gut microbiota, etc. They can not only affect the occurrence of diseases and exacerbate disease progression but also promote neuronal repair and reduce apoptosis, to prevent and slow down the development of diseases. This article reviews the research progress of miRNAs on the mechanism and treatment of neurodegenerative diseases in the nervous system. This trial is registered with NCT01819545, NCT02129452, NCT04120493, NCT04840823, NCT02253732, NCT02045056, NCT03388242, NCT01992029, NCT04961450, NCT03088839, NCT04137926, NCT02283073, NCT04509271, NCT02859428, and NCT05243017.

Diadenosine Tetraphosphate (Ap4 A) Serves as a 5' RNA Cap in Mammalian Cells.

The recent expansion of the field of RNA chemical modifications has changed our understanding of post-transcriptional gene regulation. Apart from internal nucleobase modifications, 7-methylguanosine was long thought to be the only eukaryotic RNA cap. However, the discovery of non-canonical RNA caps in eukaryotes revealed a new niche of previously undetected RNA chemical modifications. We are the first to report the existence of a new non-canonical RNA cap, diadenosine tetraphosphate (Ap4 A), in human and rat cell lines. Ap4 A is the most abundant dinucleoside polyphosphate in eukaryotic cells and can be incorporated into RNA by RNA polymerases as a non-canonical initiating nucleotide (NCIN). Using liquid chromatography-mass spectrometry (LC-MS), we show that the amount of capped Ap4 A-RNA is independent of the cellular concentration of Ap4 A. A decapping enzyme screen identifies two enzymes cleaving Ap4 A-RNA,NUDT2 and DXO, both of which also cleave other substrate RNAs in vitro. We further assess the translatability and immunogenicity of Ap4 A-RNA and show that although it is not translated, Ap4 A-RNA is recognized as self by the cell and does not elicit an immune response, making it a natural component of the transcriptome. Our findings open a previously unexplored area of eukaryotic RNA regulation.

LPI-IBWA: Predicting lncRNA-protein interactions based on an improved Bi-Random walk algorithm

Many studies have shown that long-chain noncoding RNAs (lncRNAs) are involved in a variety of biological processes such as post-transcriptional gene regulation, splicing, and translation by combining with corresponding proteins. Predicting lncRNA-protein interactions is an effective approach to infer the functions of lncRNAs. The paper proposes a new computational model named LPI-IBWA. At first, LPI-IBWA uses similarity kernel fusion (SKF) to integrate various types of biological information to construct lncRNA and protein similarity networks. Then, a bounded matrix completion model and a weighted k-nearest known neighbors algorithm are utilized to update the initial sparse lncRNA-protein interaction matrix. Based on the updated lncRNA-protein interaction matrix, the lncRNA similarity network and the protein similarity network are integrated into a heterogeneous network. Finally, an improved Bi-Random walk algorithm is used to predict novel latent lncRNA-protein interactions. 5-fold cross-validation experiments on a benchmark dataset showed that the AUC and AUPR of LPI-IBWA reach 0.920 and 0.736, respectively, which are higher than those of other state-of-the-art methods. Furthermore, the experimental results of case studies on a novel dataset also illustrated that LPI-IBWA could efficiently predict potential lncRNA-protein interactions.

Do RNA modifications contribute to modulation of immune responses in allergic diseases?

RNA modifications have emerged as a fundamental mechanism of post-transcriptional gene regulation, playing vital roles in cellular physiology and the development of various diseases. While the investigation of RNA modifications has seen significant advancements, the exploration of their implication in allergic diseases has been comparatively overlooked. Allergic reactions, including hay fever, asthma, eczema and food allergies, result from hypersensitive immune responses, affecting a considerable population worldwide. Despite the high prevalence, the molecular mechanisms underlying these responses remain partially understood. The potential role of RNA modifications in modulating the hypersensitive immune responses has yet to be fully elucidated. This mini-review seeks to shed light on potential connections between RNA modifications and allergy, highlighting recent findings and potential future research directions. By expanding our understanding of the complex interplay between RNA modifications and allergic responses, we hope to unlock new avenues for allergy diagnosis, prognosis, and therapeutic intervention.

MicroRNAs: Midfielders of Cardiac Health, Disease and Treatment.

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that play a role in post-transcriptional gene regulation. It is generally accepted that their main mechanism of action is the negative regulation of gene expression, through binding to specific regions in messenger RNA (mRNA) and repressing protein translation. By interrupting protein synthesis, miRNAs can effectively turn genes off and influence many basic processes in the body, such as developmental and apoptotic behaviours of cells and cardiac organogenesis. Their importance is highlighted by inhibiting or overexpressing certain miRNAs, which will be discussed in the context of coronary artery disease, atrial fibrillation, bradycardia, and heart failure. Dysregulated levels of miRNAs in the body can exacerbate or alleviate existing disease, and their omnipresence in the body makes them reliable as quantifiable markers of disease. This review aims to provide a summary of miRNAs as biomarkers and their interactions with targets that affect cardiac health, and intersperse it with current therapeutic knowledge. It intends to succinctly inform on these topics and guide readers toward more comprehensive works if they wish to explore further through a wide-ranging citation list.

Heterogenous nuclear ribonucleoprotein D-like controls endothelial cell functions.

HnRNPs are ubiquitously expressed RNA-binding proteins, tightly controlling posttranscriptional gene regulation. Consequently, hnRNP networks are essential for cellular homeostasis and their dysregulation is associated with cancer and other diseases. However, the physiological function of hnRNPs in non-cancerous cell systems are poorly understood. We analyzed the importance of HNRNPDL in endothelial cell functions. Knockdown of HNRNPDL led to impaired proliferation, migration and sprouting of spheroids. Transcriptome analysis identified cyclin D1 (CCND1) and tropomyosin 4 (TPM4) as targets of HNRNPDL, reflecting the phenotypic changes after knockdown. Our findings underline the importance of HNRNPDL for the homeostasis of physiological processes in endothelial cells.

Dysregulation of Non-Coding RNAs: Roles of miRNAs and lncRNAs in the Pathogenesis of Multiple Myeloma.

The dysregulation of non-coding RNAs (ncRNAs), specifically microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), leads to the development and advancement of multiple myeloma (MM). miRNAs, in particular, are paramount in post-transcriptional gene regulation, promoting mRNA degradation and translational inhibition. As a result, miRNAs can serve as oncogenes or tumor suppressors depending on the target genes. In MM, miRNA disruption could result in abnormal gene expression responsible for cell growth, apoptosis, and other biological processes pertinent to cancer development. The dysregulated miRNAs inhibit the activity of tumor suppressor genes, contributing to disease progression. Nonetheless, several miRNAs are downregulated in MM and have been identified as gene regulators implicated in extracellular matrix remodeling and cell adhesion. miRNA depletion potentially facilitates the tumor advancement and resistance of therapeutic drugs. Additionally, lncRNAs are key regulators of numerous cellular processes, such as gene expression, chromatin remodeling, protein trafficking, and recently linked MM development. The lncRNAs are uniquely expressed and influence gene expression that supports MM growth, in addition to facilitating cellular proliferation and viability via multiple molecular pathways. miRNA and lncRNA alterations potentially result in anomalous gene expression and interfere with the regular functioning of MM. Thus, this review aims to highlight the dysregulation of these ncRNAs, which engender novel therapeutic modalities for the treatment of MM.

Influence of Maternal BLV Infection on miRNA and tRF Expression in Calves.

Small non-coding RNAs, such as microRNAs (miRNA) and tRNA-derived fragments (tRF), are known to be involved in post-transcriptional gene regulation. Research has provided evidence that small RNAs may influence immune development in calves. Bovine leukosis is a disease in cattle caused by Bovine Leukemia Virus (BLV) that leads to increased susceptibility to opportunistic pathogens. No research has addressed the potential influence that a maternal BLV infection may have on gene regulation through the differential expression of miRNAs or tRFs in progeny. Blood samples from 14-day old Holstein calves born to BLV-infected dams were collected. Antibodies for BLV were assessed using ELISA and levels of BLV provirus were assessed using qPCR. Total RNA was extracted from whole blood samples for small RNA sequencing. Five miRNAs (bta-miR-1, bta-miR-206, bta-miR-133a, bta-miR-133b, and bta-miR-2450d) and five tRFs (tRF-36-8JZ8RN58X2NF79E, tRF-20-0PF05B2I, tRF-27-W4R951KHZKK, tRF-22-S3M8309NF, and tRF-26-M87SFR2W9J0) were dysregulated in calves born to BLV-infected dams. The miRNAs appear to be involved in the gene regulation of immunological responses and muscle development. The tRF subtypes and parental tRNA profiles in calves born to infected dams appear to be consistent with previous publications in adult cattle with BLV infection. These findings offer insight into how maternal BLV infection status may impact the development of offspring.

Unlocking the Potential of Carbon Quantum Dots for Cell Imaging, Intracellular Localization, and Gene Expression Control in Arabidopsis thaliana (L.) Heynh.

Utilizing carbon quantum dots (CQDs) as biomaterials for delivering small substances has gained significant attention in recent research. However, the interactions and mechanisms of action of CQDs on plants have received relatively little focus. Herein, we investigated the transportation of CQDs into various organs of Arabidopsis thaliana (L.) Heynh. via the vessel system, leading to the epigenetic inheritance of Argonaute family genes. Our findings reveal that CQDs may interact with microRNAs (miRNAs), leading to the repression of post-transcriptional regulation of target genes in the cytoplasm. Transcriptome and quantitative PCR analyses demonstrated consistent gene expression levels in offspring. Moreover, microscopic observations illustrated rapid CQD localization on cell membranes and nuclei, with increased nuclear entry at higher concentrations. Notably, our study identified an alternative regulatory microRNA, microRNA172D, for the Argonaute family genes through methylation analysis, shedding light on the connection between CQDs and microRNAs.

Integrative genome-scale analyses reveal post-transcriptional signatures of early human small intestinal development in a directed differentiation organoid model

BackgroundMicroRNAs (miRNAs) are important post-transcriptional gene regulators controlling cellular lineage specification and differentiation during embryonic development, including the gastrointestinal system. However, miRNA-mediated regulatory mechanisms involved in early embryonic development of human small intestine (SI) remains underexplored. To explore candidate roles for miRNAs in prenatal SI lineage specification in humans, we used a multi-omic analysis strategy in a directed differentiation model that programs human pluripotent stem cells toward the SI lineage.ResultsWe leveraged small RNA-seq to define the changing miRNA landscape, and integrated chromatin run-on sequencing (ChRO-seq) and RNA-seq to define genes subject to significant post-transcriptional regulation across the different stages of differentiation. Small RNA-seq profiling revealed temporal dynamics of miRNA signatures across different developmental events of the model, including definitive endoderm formation, SI lineage specification and SI regional patterning. Our multi-omic, integrative analyses showed further that the elevation of miR-182 and reduction of miR-375 are key events during SI lineage specification. We demonstrated that loss of miR-182 leads to an increase in the foregut master marker SOX2. We also used single-cell analyses in murine adult intestinal crypts to support a life-long role for miR-375 in the regulation of Zfp36l2. Finally, we uncovered opposing roles of SMAD4 and WNT signaling in regulating miR-375 expression during SI lineage specification. Beyond the mechanisms highlighted in this study, we also present a web-based application for exploration of post-transcriptional regulation and miRNA-mediated control in the context of early human SI development.ConclusionThe present study uncovers a novel facet of miRNAs in regulating prenatal SI development. We leveraged multi-omic, systems biology approaches to discover candidate miRNA regulators associated with early SI developmental events in a human organoid model. In this study, we highlighted miRNA-mediated post-transcriptional regulation relevant to the event of SI lineage specification. The candidate miRNA regulators that we identified for the other stages of SI development also warrant detailed characterization in the future.

Graph neural networks for the identification of novel inhibitors of a small RNA

MicroRNAs (miRNAs) play a crucial role in post-transcriptional gene regulation and have been implicated in various diseases, including cancers and lung disease. In recent years, Graph Neural Networks (GNNs) have emerged as powerful tools for analyzing graph-structured data, making them well-suited for the analysis of molecular structures. In this work, we explore the application of GNNs in ligand-based drug screening for small molecules targeting miR-21. By representing a known dataset of small molecules targeting miR-21 as graphs, GNNs can learn complex relationships between their structures and activities, enabling the prediction of potential miRNA-targeting small molecules by capturing the structural features and similarity between known miRNA-targeting compounds. The use of GNNs in miRNA-targeting drug screening holds promise for the discovery of novel therapeutic agents and provides a computational framework for efficient screening of large chemical libraries.

Analysis of Human Endogenous mRNA Deadenylation Complexes by High-Resolution Gel Electrophoresis.

In eukaryotic cells, poly(A) tails stabilize mRNA molecules and play a pivotal role in enhancing translational efficiency. Consequently, the enzymatic shortening of these poly(A) tails by deadenylase enzymes has a critical role in post-transcriptional gene regulation. However, deadenylases are usually large, multisubunit, and multifunctional complexes, which complicates their biochemical analysis. This chapter presents a methodology for isolating human deadenylation complexes from endogenous sources and conducting an in vitro deadenylation assay to examine their enzymatic activity. The reactions involving fluorescently labeled synthetic polyadenylated RNAs are subsequently analyzed using high-resolution denaturing polyacrylamide gel electrophoresis.

A global study of miRNAome dynamics in response to Fusarium basal rot infection in onion (Allium cepa L.)

Fusarium oxysporum f. sp. cepae (Foc), the cause of Fusarium basal rot (FBR) is one of the most virulent fungal phytopathogens that damages onion and related crops worldwide. miRNAs have a crucial role in the transcriptional and post-transcriptional regulation of genes throughout the development of defense responses. However, the miRNA repertoire of onion and its associated regulation of onion-Foc interaction is yet unclear. In the current work, we compared the miRNA profiles of resistant (Ac77) and susceptible (Arka Kirthiman-AK) onion cultivars and detected 37 differentially expressed miRNAs that could be categorized as prominent regulators of defense against Foc. Intriguingly, the expression of a novel microRNA-ace-miRn3a, was markedly increased in Ac77 but mostly suppressed in AK genotype after Foc infection. Subsequently, the upregulated expression of ace-miRn3a in Ac77 displayed improved resistance to Foc, as demonstrated by reduced growth of the fungus, negligible disease symptoms and induction of host defense genes. As expected, the expression of its two target genes encoding the TIR1 type F-box proteins was reduced in Ac77 as well as in the miRn3a over-expressed AK lines. Moreover, the transient co-expression of the novel onion miRNAs and their target genes in Nicotiana benthamiana confirmed the specific binding and cleavage of the two-onion specific TIR1 type F-box genes by ace-miRn3a. Overall, our research suggest that ace-miRn3a may offer a miRNA-mediated strategy for engineering Foc resistance in onions by suppressing TIR1 F-box proteins and reducing fungal colonization.

Dysregulation of microRNAs may contribute to neurosensory impairment in Arctic cod (Boreogadus saida) following CO2 exposure

MicroRNAs (miRNAs) are epigenetic markers with a key role in post-transcriptional gene regulation. Several studies have described the dysregulation of miRNAs in temperature and hypoxic stress responses of marine organisms, but their role in the response to acidification conditions has remained relatively underexplored. We investigated the differential expression of miRNAs in whole brain tissue of Arctic cod (Boregogadus saida) exposed to elevated aqueous CO2 levels representative of future climate change predictions. We detected the expression of 17 miRNAs of interest that are either directly or indirectly associated with reduced auditory performance; 12 of the 17 miRNAs showed significant differential expression in high treatment vs. low (control) aqueous CO2 conditions. Target gene predictions indicated that these miRNAs are likely involved with inner ear maintenance, hair cell degradation, age-related hearing loss, neural inflammation, and injury. The highest differential expression was observed in mir-135b, which is linked with increased neural inflammation and injury that may be associated with neurosensory dysfunction. Collectively, these results elucidate the contributions of miRNA mechanisms underlying CO2-induced sensory deficits in fishes facing abiotic environmental change and suggest strong potential for this approach to yield novel insights into the mechanistic effects of climate change on marine organisms.