Post-translational Regulation Research Articles

MicroRNA Analysis of In Vitro Differentiation of Spermatogonial Stem Cells Using a 3D Human Testis Organoid System

Spermatogenesis produces male gametes from spermatogonial stem cells (SSC), beginning at puberty. Modern-day laboratory techniques allow for the long-term culture of SSC and in vitro spermatogenesis. The specific biochemical processes that occur during spermatogenesis remain poorly understood. One particular element of spermatogenesis that has yet to be characterized is the role of microRNAs (miRNA), short, non-transcribed RNAs that act as post-translational regulators of gene activity. In this study, we seek to describe the presence of miRNA in a two-dimensional (2D) SSC culture and a 3D human testis organoid (HTO) system. Testicular cells were isolated from the frozen tissue of three brain-dead subjects, propagated in cultures for four to five weeks, and used to form 3D HTOs. Following organoid formation, differentiation of testicular cells was induced. RNA was isolated from the whole testis tissue (WT) showing in vivo conditions, HTO Day Zero (2D SSC culture), Day 2 HTOs, and Day 23 differentiated HTOs, then analyzed for changes in miRNA expression using the Nanostring nCounter miRNA panel. One hundred ninety-five miRNAs met the criteria for expression in WT, 186 in 2D culture, 190 in Day 2 HTOs, and 187 in differentiated HTOs. One hundred thirty-three miRNAs were common across all conditions, and 41, 17, 6, and 11 miRNAs were unique for WT, 2D culture, Day 2 HTOs, and differentiated HTOs, respectively. Twenty-two miRNAs were similar between WT and differentiated HTOS. We evaluated the miRNA expression profiles of progressively complex stages of testicular cell culture, culminating in a 3D organoid model capable of meiotic differentiation, and compared these to WT. We identified a great variance between the native tissue and the culture system; however, some miRNAs are preserved. These data may provide avenues for deeper understanding of spermatogenesis and the ability to improve this process in the laboratory. Research on miRNA continues to be an essential avenue for understanding human spermatogenesis.

Phytochelatin Synthase: An In Silico Comparative Analysis in Cyanobacteria and Eukaryotic Microalgae

Phytochelatins (PCs) are small cysteine-rich peptides involved in metal detoxification, not genetically encoded but enzymatically synthesized by phytochelatin synthases (PCSs) starting from glutathione. The constitutive PCS expression even in the absence of metal contamination, the wide phylogenetic distribution and the similarity between PCSs and the papain-type cysteine protease catalytic domain suggest a wide range of functions for PCSs. These proteins, widely studied in land plants, have not been fully analyzed in algae and cyanobacteria, although these organisms are the first to cope with heavy-metal stress in aquatic environments and can be exploited for phytoremediation. To fill this gap, we compared the features of the PCS proteins of different cyanobacterial and algal taxa by phylogenetic linkage. The analyzed sequences fall into two main, already known groups of PCS-like proteins. Contrary to previous assumptions, they are not classed as prokaryotic and eukaryotic sequences, but rather as sequences characterized by the alternative presence of asparagine and aspartic/glutamic acid residues in proximity of the catalytic cysteine. The presence of these enzymes with peculiar features suggests differences in their post-translational regulation related to cell/environmental requirements or different cell functions rather than to differences due to their belonging to different phylogenetic taxa.

Global lysine succinylation analysis unveils post-translational regulation effect on phenylpropanoid metabolism remodeling during Lonicera japonica flower development

Lonicera japonica plays a significant role in traditional Chinese medicine and as a food source, making it a focus of studies on protein succinylation and its potential role in regulating secondary metabolism during flower development. This study aimed to clarify the regulatory mechanism of protein succinylation on phenylpropanoid-related phenotypic changes by conducting a global lysine succinylation proteomic analysis across different flowering stages. A total of 586 lysine succinylated peptides in 303 proteins were identified during early and late floral stages. Functional enrichment analysis revealed that succinylated proteins primarily participated in the tricarboxylic acid (TCA) cycle, amino acid metabolism, and secondary metabolism. The abundance of succinylated aspartate transaminase (AT), 4-coumarate-CoA ligase (4CL), and phenylalanine N-hydroxylase (CYP79A2) in phenylpropanoid metabolism varied during flower development. In vitro experiments demonstrated that succinylation increased AT activity while inhibited 4CL activity. Decreased levels of total flavonoids and phenolic acids indicated significant alterations in phenylpropanoid metabolism during later floral stages. These results suggest that succinylation of TCA cycle proteins not only influences flower development but also, together with AT-4CL-CYP79A2 co-succinylation, redirects phenylpropanoid metabolism during flower development in L. japonica.

Transcriptome analysis and molecular characterization of novel small RNAs in Mycobacterium tuberculosis Lineage 1.

Mycobacterium tuberculosis (Mtb), the tuberculosis-causing agent, exhibits diverse genetic lineages, with known links to virulence. While genomic and transcriptomic variations between modern and ancient Mtb lineages have been explored, the role of small non-coding RNA (sRNA) in post-translational gene regulation remains largely uncharted. In this study, Mtb Lineage 1 (L1) Sabahan strains (n = 3) underwent sRNA sequencing, revealing 351 sRNAs, including 23 known sRNAs and 328 novel ones identified using ANNOgesic. Thirteen sRNAs were selected based on the best average cut-off value of 300, with RT-qPCR revealing significant expression differences for sRNA 1 (p = 0.0132) and sRNA 29 (p = 0.0012) between Mtb L1 and other lineages (L2 and L4, n = 3) (p > 0.05). Further characterization using RACE (rapid amplification of cDNA ends), followed by target prediction with TargetRNA3 unveils that sRNA 1 (55 base pairs) targets Rv0506, Rv0697, and Rv3590c, and sRNA 29 (86 base pairs) targets Rv33859c, Rv3345c, Rv0755c, Rv0107c, Rv1817, Rv2950c, Rv1181, Rv3610c, and Rv3296. Functional characterization with Mycobrowser reveals these targets involved in regulating intermediary metabolism and respiration, cell wall and cell processes, lipid metabolism, information pathways, and PE/PPE. In summary, two novel sRNAs, sRNA 1 and sRNA 29, exhibited differential expression between L1 and other lineages, with predicted roles in essential Mtb functions. These findings offer insights into Mtb regulatory mechanisms, holding promise for the development of improved tuberculosis treatment strategies in the future.

Ceramide Ehux-C22 Targets the miR-199a-3p/mTOR Signaling Pathway to Regulate Melanosomal Autophagy in Mouse B16 Cells

Melanosomes are specialized membrane-bound organelles where melanin is synthesized and stored. The levels of melanin can be effectively reduced by inhibiting melanin synthesis or promoting melanosome degradation via autophagy. Ceramide, a key component in the metabolism of sphingolipids, is crucial for preserving the skin barrier, keeping it hydrated, and warding off the signs of aging. Our preliminary study indicated that a long-chain C22-ceramide compound (Ehux-C22) isolated from the marine microalga Emiliania huxleyi, reduced melanin levels via melanosomal autophagy in B16 cells. Recently, microRNAs (miRNAs) were shown to act as melanogenesis-regulating molecules in melanocytes. However, whether the ceramide Ehux-C22 can induce melanosome autophagy at the post-transcriptional level, and which potential autophagy-dependent mechanisms are involved, remains unknown. Here, miR-199a-3p was screened and identified as a novel upregulated miRNA in Ehux-C22-treated B16 cells. An in vitro high melanin expression model in cultured mouse melanoma cells (B16 cells) was established by using 0.2 μM alpha-melanocyte-stimulating hormone(α-MSH) and used for subsequent analyses. miR-199a-3p overexpression significantly enhanced melanin degradation, as indicated by a reduction in the melanin level and an increase in melanosome autophagy. Further investigation demonstrated that in B16 cells, Ehux-C22 activated miR-199a-3p and inhibited mammalian target of rapamycin(mTOR) level, thus activating the mTOR-ULK1 signaling pathway by promoting the expression of unc-51-like autophagy activating kinase 1 (ULK1), B-cell lymphoma-2 (Bcl-2), Beclin-1, autophagy-related gene 5 (ATG5), and microtubule-associated protein light chain 3 (LC3-II) and degrading p62. Therefore, the roles of Ehux-C22-regulated miR-199a-3p and the mTOR pathway in melanosomal autophagy were elucidated. This research may provide novel perspectives on the post-translational regulation of melanin metabolism, which involves the coordinated control of melanosomes.

Resolutive Treatment of Actinic Keratosis through Peptides Extracted from Zebrafish Conveyed Across Laser-assisted Transcutaneous Delivery (Cryopass Therapy).

Actinic keratoses (AKs) are humans' most common keratinocyte-derived precancerous lesions. They can be observed predominantly in fair-skinned individuals on sun-exposed surfaces. Another name for AKs is solar keratosis. The primary risk factor for AKs is cumulative UV exposure from sunlight and/or tanning salons. AKs may present on a patient as a few detectable lesions. Clinically, they present as scaly erythema lesions with various pigmentations. Surgery treatment is the only approach that can definitively resolve the illness. The research aims to demonstrate the effectiveness of treating relapsing AKs through the use of factors present during organogenesis extracted from zebrafish caviar conveyed through a cryopass therapy device (cryolaser phoresis). These factors are peptides present during specific phases of organogenesis and have shown the capacity to reverse cancer and neurodegeneration processes through gene, transcriptional, translational, and post-translational regulation. We found that 90% of patients responded. The percentage of responding patients was 100% among females and 80% among men. Our results confirm previous findings about the effectiveness of stem cell differentiation stage factors (SCDFs) in cancer and open new ways to integrate treatment.

RACK1 enhances STAT3 stability and promotes T follicular helper cell development and function during blood-stage Plasmodium infection in mice.

CD4+ T cells are central mediators of protective immunity to blood-stage malaria, particularly for their capacity in orchestrating germinal center reaction and generating parasite-specific high-affinity antibodies. T follicular helper (Tfh) cells are predominant CD4+ effector T cell subset implicated in these processes, yet the factors and detailed mechanisms that assist Tfh cell development and function during Plasmodium infection are largely undefined. Here we provide evidence that receptor for activated C kinase 1 (RACK1), an adaptor protein of various intracellular signals, is not only important for CD4+ T cell expansion as previously implied but also plays a prominent role in Tfh cell differentiation and function during blood-stage Plasmodium yoelii 17XNL infection. Consequently, RACK1 in CD4+ T cells contributes significantly to germinal center formation, parasite-specific IgG production, and host resistance to the infection. Mechanistic exploration detects specific interaction of RACK1 with STAT3 in P. yoelii 17XNL-responsive CD4+ T cells, ablation of RACK1 leads to defective STAT3 phosphorylation, accompanied by substantially lower amount of STAT3 protein in CD4+ T cells, whereas retroviral overexpression of RACK1 or STAT3 in RACK1-deficient CD4+ T cells greatly restores STAT3 activity and Bcl-6 expression under the Tfh polarization condition. Further analyses suggest RACK1 positively regulates STAT3 stability by inhibiting the ubiquitin-proteasomal degradation process, thus promoting optimal STAT3 activity and Bcl-6 induction during Tfh cell differentiation. These findings uncover a novel mechanism by which RACK1 participates in posttranslational regulation of STAT3, Tfh cell differentiation, and subsequent development of anti-Plasmodium humoral immunity.

Identification of tomato F-box proteins functioning in phenylpropanoid metabolism.

Phenylpropanoids, a class of specialized metabolites, play crucial roles in plant growth and stress adaptation and include diverse phenolic compounds such as flavonoids. Phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) are essential enzymes functioning at the entry points of general phenylpropanoid biosynthesis and flavonoid biosynthesis, respectively. In Arabidopsis, PAL and CHS are turned over through ubiquitination-dependent proteasomal degradation. Specific kelch domain-containing F-Box (KFB) proteins as components of ubiquitin E3 ligase directly interact with PAL or CHS, leading to polyubiquitinated PAL and CHS, which in turn influences phenylpropanoid and flavonoid production. Although phenylpropanoids are vital for tomato nutritional value and stress responses, the post-translational regulation of PAL and CHS in tomato remains unknown. We identified 31 putative KFB-encoding genes in the tomato genome. Our homology analysis and phylogenetic study predicted four PAL-interacting SlKFBs, while SlKFB18 was identified as the sole candidate for the CHS-interacting KFB. Consistent with their homolog function, the predicted four PAL-interacting SlKFBs function in PAL degradation. Surprisingly, SlKFB18 did not interact with tomato CHS and the overexpression or knocking out of SlKFB18 did not affect phenylpropanoid contents in tomato transgenic lines, suggesting its irreverence with flavonoid metabolism. Our study successfully discovered the post-translational regulatory machinery of PALs in tomato while highlighting the limitation of relying solely on a homology-based approach to predict interacting partners of F-box proteins.

The Spatial Extracellular Proteomic Tumor Microenvironment Distinguishes Molecular Subtypes of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) mortality rates continue to increase faster than those of other cancer types due to high heterogeneity, which limits diagnosis and treatment. Pathological and molecular subtyping have identified that HCC tumors with poor outcomes are characterized by intratumoral collagenous accumulation. However, the translational and post-translational regulation of tumor collagen, which is critical to the outcome, remains largely unknown. Here, we investigate the spatial extracellular proteome to understand the differences associated with HCC tumors defined by Hoshida transcriptomic subtypes of poor outcome (Subtype 1; S1; n = 12) and better outcome (Subtype 3; S3; n = 24) that show differential stroma-regulated pathways. Collagen-targeted mass spectrometry imaging (MSI) with the same-tissue reference libraries, built from untargeted and targeted LC-MS/MS was used to spatially define the extracellular microenvironment from clinically-characterized, formalin-fixed, paraffin-embedded tissue sections. Collagen α-1(I) chain domains for discoidin-domain receptor and integrin binding showed distinctive spatial distribution within the tumor microenvironment. Hydroxylated proline (HYP)-containing peptides from the triple helical regions of fibrillar collagens distinguished S1 from S3 tumors. Exploratory machine learning on multiple peptides extracted from the tumor regions could distinguish S1 and S3 tumors (with an area under the receiver operating curve of ≥0.98; 95% confidence intervals between 0.976 and 1.00; and accuracies above 94%). An overall finding was that the extracellular microenvironment has a high potential to predict clinically relevant outcomes in HCC.

Distinct phases of cellular signaling revealed by time-resolved protein synthesis.

The post-translational regulation of protein function is involved in most cellular processes. As such, synthetic biology tools that operate at this level provide opportunities for manipulating cellular states. Here we deploy proximity-triggered protein trans-splicing technology to enable the time-resolved synthesis of target proteins from premade parts. The modularity of the strategy allows for the addition or removal of various control elements as a function of the splicing reaction, in the process permitting the cellular location and/or activity state of starting materials and products to be differentiated. The approach is applied to a diverse set of proteins, including the kinase oncofusions breakpoint cluster region-Abelson (BCR-ABL) and DNAJ-PKAc where dynamic cellular phosphorylation events are dissected, revealing distinct phases of signaling and identifying molecular players connecting the oncofusion to cancer transformation as new therapeutic targets of cancer cells. We envision that the tools and control strategies developed herein will allow the activity of both naturally occurring and designer proteins to be harnessed for basic and applied research.

Insights into the role of FoxL2 in tebuconazole-induced male- biased sex differentiation of zebrafish

Tebuconazole (TEB) is a commonly used fungicide that inhibits the aromatase Cyp19A and downregulates the transcription factor forkhead box L2 (FoxL2), leading to male-biased sex differentiation in zebrafish larvae. However, the specific mechanism by which FoxL2 functions following TEB exposure remains unclear. In this study, the phosphorylation sites and kinase-specific residues in zebrafish FoxL2 protein (zFoxL2) were predicted. Subsequently, recombinant zFoxL2 was prepared via prokaryotic expression, and a polyclonal rabbit-anti-zFoxL2 antibody was generated. Zebrafish fibroblast (ZF4) cells were exposed to 100-μM TEB alone for 8 h, after which changes in the expression of genes involved in the foxl2 regulatory pathway (akt1, pi3k, cyp19a1b, c/ebpb and sox9a) were detected. When co-exposed to 1-μM estradiol and 100-μM TEB, the expression of these key genes tended to be restored. Interestingly, TEB did not affect the expression of the foxl2 gene or protein but it significantly suppressed the phosphorylation of FoxL2 (pFoxL2) at serine 238 (decreased by 43.64 %, p = 0.009). Co-immunoprecipitation assays showed that, following exposure to 100-μM TEB, the total precipitated proteins in ZF4 cells decreased by 17.02 % (p = 0.029) and 31.39 % (p = 0.027) in the anti-zFoxL2 antibody group and anti-pFoxL2 (ser238) antibody group, respectively, indicating that TEB suppressed the capacity of the FoxL2 protein to bind to other proteins via repression of its own phosphorylation. The pull-down assay confirmed this conclusion. This study preliminarily elucidated that the foxl2 gene functions via post-translational regulation through hypophosphorylation of its encoded protein during TEB-induced male-biased sex differentiation.

Insights into the role of FoxL2 in tebuconazole-induced male- biased sex differentiation of zebrafish

Tebuconazole (TEB) is a commonly used fungicide that inhibits the aromatase Cyp19A and downregulates the transcription factor forkhead box L2 (FoxL2), leading to male-biased sex differentiation in zebrafish larvae. However, the specific mechanism by which FoxL2 functions following TEB exposure remains unclear. In this study, the phosphorylation sites and kinase-specific residues in zebrafish FoxL2 protein (zFoxL2) were predicted. Subsequently, recombinant zFoxL2 was prepared via prokaryotic expression, and a polyclonal rabbit-anti-zFoxL2 antibody was generated. Zebrafish fibroblast (ZF4) cells were exposed to 100-μM TEB alone for 8 h, after which changes in the expression of genes involved in the foxl2 regulatory pathway (akt1, pi3k, cyp19a1b, c/ebpb and sox9a) were detected. When co-exposed to 1-μM estradiol and 100-μM TEB, the expression of these key genes tended to be restored. Interestingly, TEB did not affect the expression of the foxl2 gene or protein but it significantly suppressed the phosphorylation of FoxL2 (pFoxL2) at serine 238 (decreased by 43.64 %, p = 0.009). Co-immunoprecipitation assays showed that, following exposure to 100-μM TEB, the total precipitated proteins in ZF4 cells decreased by 17.02 % (p = 0.029) and 31.39 % (p = 0.027) in the anti-zFoxL2 antibody group and anti-pFoxL2 (ser238) antibody group, respectively, indicating that TEB suppressed the capacity of the FoxL2 protein to bind to other proteins via repression of its own phosphorylation. The pull-down assay confirmed this conclusion. This study preliminarily elucidated that the foxl2 gene functions via post-translational regulation through hypophosphorylation of its encoded protein during TEB-induced male-biased sex differentiation.

Participation of CWINV and SUS Genes in Sucrose Utilization in the Disruption of Cambium Derivatives Differentiation of Silver Birch.

The mechanisms that control the accumulation of woody biomass are of great interest to the study. Invertase and sucrose synthase are enzymes that are vital for distributing carbon in various biosynthetic pathways. Karelian birch (Betula pendula var. carelica) is a form of silver birch (B. pendula Roth) and is characterized by disruption of the differentiation of cambium derivatives towards both the xylem and phloem, which leads to a change in the proportion of the conducting tissues' structural elements and the figured wood formation. We researched the expression profiles of genes encoding sucrose-cleaving enzymes (CWINV and SUS gene families) and genes encoding CVIF protein, which is responsible for the post-translational regulation of the cell wall invertase activity. In our study, 16-year-old common silver birch (Betula pendula var. pendula) and Karelian birch were used for sampling non-figured and figured trunk section tissues, respectively. Samples were selected for the research based on the radial vector: non-conductive, conductive phloem, cambial zone - differentiating xylem - mature xylem. The enzyme's activity was investigated by biochemical methods. RT-PCR method was used to determine the level of gene expression. Anatomical and morphological methods were used to determine the stage of differentiation of xylem cambial derivatives. Our research revealed a shift in the composition of xylem components in figured Karelian birch, characterized by increased parenchymatization and reduced vessel quantity. In all studied trunk tissues of Karelian birch, compared with common silver birch, an increase in the expression of the CWINV gene family and the SUS3 gene and a decrease in the expression of SUS4 were shown. Therefore, the increase in parenchymatization in figured Karelian birch is linked to a shift in sucrose metabolism towards the apoplastic pathway, indicated by a higher cell wall invertase activity and gene expression. The expression of the SUS4 gene correlates with the decrease in xylem increments and vessel proportion. The research findings will enhance our understanding of how sucrose breaking enzymes regulate secondary growth in woody plants and aid in developing practical timber cultivation methods.

Exploring the role of microRNAs as diagnostic and prognostic biomarkers in canine mammary tumors.

Canine mammary tumors (CMTs) represent a significant health concern in dogs, with a high incidence among intact female dogs. CMTs are a promising comparative model for human breast cancer, due to sharing several pathophysiological features. Additionally, CMTs have a strong genetic correlation with their human counterpart, including the expression of microRNAs (miRNAs). MiRNAs are a class of non-coding RNAs that play important roles in post-translational regulation of gene expression, being implicated in carcinogenesis, tumor progression, and metastasis. Moreover, miRNAs hold promise as diagnostic, prognostic, and metastatic biomarkers. Understanding the molecular mechanisms underlying CMTs is crucial for improving diagnosis, prognosis, and monitoring of treatments. Herein, we provide a comprehensive overview of the current knowledge on miRNAs in CMTs, highlighting their roles in carcinogenesis and their potential as biomarkers. Additionally, we highlight the current limitations and critically discuss the overarching challenges in this field, emphasizing the need for future research to translate miRNA findings into veterinary clinical practice.

Rab7 GTPase-Mediated stress signaling enhances salinity tolerance in AlRabring7 tobacco transgenics by modulating physio-biochemical parameters

The RING-type E3 ligases play a significant role in stress signaling, primarily through post-translational regulation. Ubiquitination is a crucial post-translational modification that regulates the turnover and activity of proteins. The overexpression of AlRabring7, RING-HC E3 Ub ligase in tobacco provides insights into the regulation of salinity and ABA signaling in transgenic tobacco. The seed germination potential of AlRabring7 transgenics was higher than WT, with NaCl and ABA treatments. The transgenics showed improved morpho-physio-biochemical parameters in response to salinity and ABA treatments. The photosynthetic pigments, soluble sugars, reducing sugars and proline increased in transgenics in response to NaCl and ABA treatments. The decreased ROS accumulation in transgenics on NaCl and ABA treatments can be co-related to improved activity of enzymatic and non-enzymatic antioxidants. The potential of transgenics to maintain ABA levels with ABA treatment, highlights the active participation of ABA feedback loop mechanism. Interestingly, the ability of AlRabring7 transgenics to upregulate Rab7 protein, suggests its role in facilitating vacuolar transport. Furthermore, the improved potassium accumulation and reduced sodium content indicate an efficient ion regulation mechanism in transgenic plants facilitating higher stomatal opening. The expression of downstream ion transporter (NbNHX1 and NbVHA1), ABA signaling (NbABI2 and NbABI5) and vesicle trafficking (NbMON1) responsive genes were upregulated with stress. The present study, reports that AlRabring7 participates in maintaining vacuolar transport, ion balance, ROS homeostasis, stomatal regulation through activation of Rab7 protein and regulation of downstream stress-responsive during stress. This emphasizes the potential of AlRabring7 gene for improved performance and resilience in challenging environments.

LbSakA-mediated phosphorylation of the scaffolding protein LbNoxR in the ectomycorrhizal basidiomycete Laccaria bicolor regulates NADPH oxidase activity, ROS accumulation and symbiosis development.

Ectomycorrhizal symbiosis, which involves mutually beneficial interactions between soil fungi and tree roots, is essential for promoting tree growth. To establish this symbiotic relationship, fungal symbionts must initiate and sustain mutualistic interactions with host plants while avoiding host defense responses. This study investigated the role of reactive oxygen species (ROS) generated by fungal NADPH oxidase (Nox) in the development of Laccaria bicolor/Populus tremula × alba symbiosis. Our findings revealed that L. bicolor LbNox expression was significantly higher in ectomycorrhizal roots than in free-living mycelia. RNAi was used to silence LbNox, which resulted in decreased ROS signaling, limited formation of the Hartig net, and a lower mycorrhizal formation rate. Using Y2H library screening, BiFC and Co-IP, we demonstrated an interaction between the mitogen-activated protein kinase LbSakA and LbNoxR. LbSakA-mediated phosphorylation of LbNoxR at T409, T477 and T480 positively modulates LbNox activity, ROS accumulation and upregulation of symbiosis-related genes involved in dampening host defense reactions. These results demonstrate that regulation of fungal ROS metabolism is critical for maintaining the mutualistic interaction between L. bicolor and P. tremula × alba. Our findings also highlight a novel and complex regulatory mechanism governing the development of symbiosis, involving both transcriptional and posttranslational regulation of gene networks.

Disentangling the splicing factor programs underlying complex molecular phenotypes.

The regulation of exon inclusion through alternative splicing tunes the cell's behavior by increasing the functional diversity of the transcriptome and the proteome. Splicing factors work in concert to generate gene isoform pools that contribute to cell phenotypes yet their activity is controlled by multiple regulatory and signaling layers. This hinders identification of functional, phenotype-specific splicing factors using traditional single-omic measurements, such as their mutational state or expression. To address this challenge, we propose repurposing the virtual inference of protein activity by enriched regulon analysis (VIPER) to measure splicing factor activity solely from their downstream exon transcriptomic inclusion signatures. This approach is effective in assessing the effect of co-occurring splicing factor perturbations, as well as their post-translational regulation. As proof of concept, we dissect recurrent splicing factor programs underlying tumorigenesis including aberrantly activated factors acting as oncogenes and inactivated ones acting as tumor suppressors, which are undetectable by more conventional methodologies. Activation and inactivation of these cancer splicing programs effectively stratifies overall survival, as well as cancer hallmarks such as proliferation and immune evasion. Altogether, repurposing network-based inference of protein activity for splicing factor networks distills common, functionally relevant splicing programs in otherwise heterogeneous molecular contexts.

Omeprazole Induces CYP3A4 mRNA Expression but Not CYP3A4 Protein Expression in HepaRG Cells.

Unknown interactions between drugs remain the limiting factor for clinical application of drugs, and the induction and inhibition of drug-metabolizing CYP enzymes are considered the key to examining the drug-drug interaction (DDI). In this study, using human HepaRG cells as an in vitro model system, we analyzed the potential DDI based on the expression levels of CYP3A4 and CYP1A2. Rifampicin and omeprazole, the potent inducers for CYP3A4 and CYP1A2, respectively, induce expression of the corresponding CYP enzymes at both the mRNA and protein levels. We noticed that, in addition to inducing CYP1A2, omeprazole induced CYP3A4 mRNA expression in HepaRG cells. However, unexpectedly, CYP3A4 protein expression levels were not increased after omeprazole treatment. Concurrent administration of rifampicin and omeprazole showed an inhibitory effect of omeprazole on the CYP3A4 protein expression induced by rifampicin, while its mRNA induction remained intact. Cycloheximide chase assay revealed increased CYP3A4 protein degradation in the cells exposed to omeprazole. The data presented here suggest the potential importance of broadening the current DDI examination beyond conventional transcriptional induction and enzyme-activity inhibition tests to include post-translational regulation analysis of CYP enzyme expression.

Precise measurement of molecular phenotypes with barcode-based CRISPRi systems.

Genome-wide CRISPR-Cas9 screens have untangled regulatory networks and revealed the genetic underpinnings of diverse biological processes. Their success relies on experimental designs that interrogate specific molecular phenotypes and distinguish key regulators from background effects. Here, we realize these goals with a generalizable platform for CRISPR interference with barcoded expression reporter sequencing (CiBER-seq) that dramatically improves the sensitivity and scope of genome-wide screens. We systematically address technical factors that distort phenotypic measurements by normalizing expression reporters against closely-matched control promoters, integrated together into the genome at single copy. To test our ability to capture post-transcriptional and post-translational regulation through sequencing, we screened for genes that affected nonsense-mediated mRNA decay and Doa10-mediated cytosolic protein decay. Our optimized CiBER-seq screens accurately capture the known components of well-studied RNA and protein quality control pathways with minimal background. These results demonstrate the precision and versatility of CiBER-seq for dissecting the genetic networks controlling cellular behaviors.

The new kids on the block: RNA-binding proteins regulate autophagy in disease.

Mammalian autophagy is a highly regulated and conserved cellular homeostatic process. Its existence allows the degradation of self-components to mediate cell survival in different stress conditions. Autophagy is involved in the regulation of cellular metabolic needs, protecting the cell or tissue from starvation through the degradation and recycling of cytoplasmic materials and organelles to basic molecular building blocks. It also plays a critical role in eliminating damaged or harmful proteins, organelles, and intracellular pathogens. Thus, a deterioration of the process may result in pathological conditions, such as aging-associated disorders and cancer. Understanding the crucial role of autophagy in maintaining the normal physiological function of cells, tissue, or organs has led to copious and expansive research regarding the regulation of this process. So far, most of the research has revolved around transcriptional and post-translational regulation. Here, we discuss the regulation of autophagy-related (ATG) mRNA transcripts by RNA-binding proteins (RBPs). This analysis focuses on how RBPs modulate autophagy in disease. A deeper understanding of the involvement of RBPs in autophagy can facilitate further research and treatment of a variety of human diseases.