Regulation Of Protein Abundance Research Articles

Omnibus proteome-wide association study identifies 43 risk genes for Alzheimer disease dementia.

Transcriptome-wide association study (TWAS) tools have been applied to conduct proteome-wide association studies (PWASs) by integrating proteomics data with genome-wide association study (GWAS) summary data. The genetic effects of PWAS-identified significant genes are potentially mediated through genetically regulated protein abundance, thus informing the underlying disease mechanisms better than GWAS loci. However, existing TWAS/PWAS tools are limited by considering only one statistical model. We propose an omnibus PWAS pipeline to account for multiple statistical models and demonstrate improved performance by simulation and application studies of Alzheimer disease (AD) dementia. We employ the Aggregated Cauchy Association Test to derive omnibus PWAS (PWAS-O) p values from PWAS p values obtained by three existing tools assuming complementary statistical models-TIGAR, PrediXcan, and FUSION. Our simulation studies demonstrated improved power, with well-calibrated type I error, for PWAS-O over all three individual tools. We applied PWAS-O to studying AD dementia with reference proteomic data profiled from dorsolateral prefrontal cortex of postmortem brains from individuals of European ancestry. We identified 43 risk genes, including 5 not identified by previous studies, which are interconnected through a protein-protein interaction network that includes the well-known AD risk genes TOMM40, APOC1, and APOC2. We also validated causal genetic effects mediated through the proteome for 27 (63%) PWAS-O risk genes, providing insights into the underlying biological mechanisms of AD dementia and highlighting promising targets for therapeutic development. PWAS-O can be easily applied to studying other complex diseases.

Functional annotation of proteins in Catharanthus roseus shoot cultures under biogenic zinc nanotreatment.

Nano-interactions are well known for their positive as well as negative impacts on the morphological and physiological systems of plants. Keeping in mind, the conformational changes in plant proteins as one of the key mechanisms for stress adaptation responses, the current project was designed to explore the effect of glutathione-capped and uncapped zinc nano-entities on Catharanthus roseus shoot cultures. Zinc nanotreatment (0.05μg/mL) significantly induced ester production in C. roseus shoots as detected by Gas Chromatography-Mass spectrometry. These nanotreated shoots were further subjected to peptide-centric nano-LC-MS/MS analysis. Mass spectrometry followed by a Heat map revealed a significant effect of zinc nanoparticles on 59 distinct classes of proteins as compared to control. Proteins involved in regulating stress scavenging, transport, and secondary metabolite biosynthesis were robustly altered under capped zinc nanotreatment. UniProt database identified majority of the localization of the abundantly altered protein in cell membranes and chloroplasts. STRING and Cytoscape analysis assessed inter and intra coordination of triosephosphate isomerase with other identified proteins and highlighted its role in the regulation of protein abundance under applied stress. This study highlights the understanding of complex underlying mechanisms and regulatory networks involved in proteomic alterations and interactions within the plant system to cope with the nano-effect.

Joint analysis of proteome, transcriptome, and multi-trait analysis to identify novel Parkinson's disease risk genes.

Genome-wide association studies (GWAS) have identified multiple risk variants for Parkinson's disease (PD). Nevertheless, how the risk variants confer the risk of PD remains largely unknown. We conducted a proteome-wide association study (PWAS) and summary-data-based mendelian randomization (SMR) analysis by integrating PD GWAS with proteome and protein quantitative trait loci (pQTL) data from human brain, plasma and CSF. We also performed a large transcriptome-wide association study (TWAS) and Fine-mapping of causal gene sets (FOCUS), leveraging joint-tissue imputation (JTI) prediction models of 22 tissues to identify and prioritize putatively causal genes. We further conducted PWAS, SMR, TWAS, and FOCUS using a multi-trait analysis of GWAS (MTAG) to identify additional PD risk genes to boost statistical power. In this large-scale study, we identified 16 genes whose genetically regulated protein abundance levels were associated with Parkinson's disease risk. We undertook a large-scale analysis of PD and correlated traits, through TWAS and FOCUS studies, and discovered 26 casual genes related to PD that had not been reported in previous TWAS. 5 genes (CD38, GPNMB, RAB29, TMEM175, TTC19) showed significant associations with PD at both the proteome-wide and transcriptome-wide levels. Our study provides new insights into the etiology and underlying genetic architecture of PD.

Identifying causal genes for migraine by integrating the proteome and transcriptome.

While previous genome-wide association studies (GWAS) have identified multiple risk variants for migraine, there is a lack of evidence about how these variants contribute to the development of migraine. We employed an integrative pipeline to efficiently transform genetic associations to identify causal genes for migraine. We conducted a proteome-wide association study (PWAS) by combining data from the migraine GWAS data with proteomic data from the human brain and plasma to identify proteins that may play a role in the risk of developing migraine. We also combined data from GWAS of migraine with a novel joint-tissue imputation (JTI) prediction model of 17 migraine-related human tissues to conduct transcriptome-wide association studies (TWAS) together with the fine mapping method FOCUS to identify disease-associated genes. We identified 13 genes in the human brain and plasma proteome that modulate migraine risk by regulating protein abundance. In addition, 62 associated genes not reported in previous migraine TWAS studies were identified by our analysis of migraine using TWAS and fine mapping. Five genes including ICA1L, TREX1, STAT6, UFL1, and B3GNT8 showed significant associations with migraine at both the proteome and transcriptome, these genes are mainly expressed in ependymal cells, neurons, and glial cells, and are potential target genes for prevention of neuronal signaling and inflammatory responses in the pathogenesis of migraine. Our proteomic and transcriptome findings have identified disease-associated genes that may give new insights into the pathogenesis and potential therapeutic targets for migraine.

Substrate-specific effects of natural genetic variation on proteasome activity.

Protein degradation is an essential biological process that regulates protein abundance and removes misfolded and damaged proteins from cells. In eukaryotes, most protein degradation occurs through the stepwise actions of two functionally distinct entities, the ubiquitin system and the proteasome. Ubiquitin system enzymes attach ubiquitin to cellular proteins, targeting them for degradation. The proteasome then selectively binds and degrades ubiquitinated substrate proteins. Genetic variation in ubiquitin system genes creates heritable differences in the degradation of their substrates. However, the challenges of measuring the degradative activity of the proteasome independently of the ubiquitin system in large samples have limited our understanding of genetic influences on the proteasome. Here, using the yeast Saccharomyces cerevisiae, we built and characterized reporters that provide high-throughput, ubiquitin system-independent measurements of proteasome activity. Using single-cell measurements of proteasome activity from millions of genetically diverse yeast cells, we mapped 15 loci across the genome that influence proteasomal protein degradation. Twelve of these 15 loci exerted specific effects on the degradation of two distinct proteasome substrates, revealing a high degree of substrate-specificity in the genetics of proteasome activity. Using CRISPR-Cas9-based allelic engineering, we resolved a locus to a causal variant in the promoter of RPT6, a gene that encodes a subunit of the proteasome's 19S regulatory particle. The variant increases RPT6 expression, which we show results in increased proteasome activity. Our results reveal the complex genetic architecture of proteasome activity and suggest that genetic influences on the proteasome may be an important source of variation in the many cellular and organismal traits shaped by protein degradation.

Integrated analysis of proteome-wide and transcriptome-wide association studies identified novel genes and chemicals for vertigo.

Vertigo is a leading symptom of various peripheral and central vestibular disorders. Although genome-wide association studies (GWASs) have identified multiple risk variants for vertigo, how these risk variants contribute to the risk of vertigo remains unknown. Discovery proteome-wide association study (PWAS) was first performed by integrating the protein quantitative trait loci from the dorsolateral prefrontal cortex (DLPFC) in the Banner Sun Health Research Institute dataset (n = 152) and GWAS summary of vertigo (n = 942 613), followed by replication PWAS using the protein quantitative trait loci from the DLPFC in Religious Orders Study or the Rush Memory and Aging Project dataset (n = 376). Transcriptome-wide association studies (TWASs) were then performed by integrating the same GWAS datasets of vertigo (n = 942 613) with mRNA expression reference from human fetal brain, and DLPFC. Chemical-related gene set enrichment analysis (GSEA) and Gene ontology/Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were finally conducted to further reveal the pathogenesis of vertigo. Permutation-based empirical P values were calculated in PWAS, TWAS, and GSEA. By integrating the GWAS of vertigo and two independent brain proteomes from human DLPFC, three genes were identified to genetically regulate protein abundance levels in vertigo, and were not previously implicated by GWAS, including MTERFD2 (P Banner = 0.045, P ROSMAP = 0.031), MGST1 (P Banner = 0.014, P ROSMAP = 0.018), and RAB3B (P Banner = 0.045, P ROSMAP = 0.035). Compared with TWAS results, we identified overlapping genes RAB3B (P TWAS = 0.017) and MTERFD2 (P TWAS = 0.003) that showed significant associations with vertigo at both proteome-wide and transcriptome-wide levels. Chemical-related GSEA identified multiple chemicals that might be associated with vertigo, such as nickel (P = 0.007), glycidamide (P = 0.005), and proanthocyanidins (P = 0.015). Our study provides novel clues for understanding the biological mechanism of vertigo, and highlights several possible risks and therapeutic chemicals for vertigo.

A heterobifunctional molecule recruits cereblon to an RNA scaffold and activates its PROTAC function

The ability to regulate protein abundance provides huge opportunities for the development of treatments for various diseases. The proteolysis-targeting chimera (PROTAC) provides powerful tools to degrade these undruggable targets, becoming a promising technology. Herein, we report the development of aptamer-based PROTACs as a novel strategy to broaden the spectrum of targets. The RNA aptamer of the target protein applied in targeted degradation. A heterobifunctional molecule can recruit E3 ubiquitin ligase to a genetically encoded tandem aptamer RNA scaffold, promoting ubiquitination and degradation of target proteins. Partial degradations of different target proteins, including p50, p65, and E2F1, have been achieved by simply replacing the 3′ module on the RNA scaffold with the corresponding RNA aptamers. Besides, simultaneous degradation of multiple target proteins is realized through inserting more aptamer into RNA scaffold. Thus, the aptamer-based PROTAC technology offers a general strategy for targeted protein degradation in drug discovery and biological research. • RNA aptamers of a target protein are applied in targeted degradation • A strategy of aptamer-based PROTAC to broaden the range of target degradation • The heterobifunctional molecule can simultaneously degrade multiple target proteins Designing heterobifunctional molecules capable of inducing targeted protein degradation can be challenging. Here, Xu et al. use an RNA scaffold to recruit cereblon to induce degradation of a target protein.

An Easy-to-Use Plasmid Toolset for Efficient Generation and Benchmarking of Synthetic Small RNAs in Bacteria.

Synthetic biology approaches life from the perspective of an engineer. Standardized and de novo design of genetic parts to subsequently build reproducible and controllable modules, for example, for circuit design, is a key element. To achieve this, natural systems and elements often serve as a blueprint for researchers. Regulation of protein abundance is controlled at DNA, mRNA, and protein levels. Many tools for the activation or repression of transcription or the destabilization of proteins are available, but easy-to-handle minimal regulatory elements on the mRNA level are preferable when translation needs to be modulated. Regulatory RNAs contribute considerably to regulatory networks in all domains of life. In particular, bacteria use small regulatory RNAs (sRNAs) to regulate mRNA translation. Slowly, sRNAs are attracting the interest of using them for broad applications in synthetic biology. Here, we promote a “plug and play” plasmid toolset to quickly and efficiently create synthetic sRNAs to study sRNA biology or their application in bacteria. We propose a simple benchmarking assay by targeting the acrA gene of Escherichia coli and rendering cells sensitive toward the β-lactam antibiotic oxacillin. We further highlight that it may be necessary to test multiple seed regions and sRNA scaffolds to achieve the desired regulatory effect. The described plasmid toolset allows quick construction and testing of various synthetic sRNAs based on the user’s needs.

Brain Proteome-Wide Association Study Identifies Candidate Genes that Regulate Protein Abundance Associated with Post-Traumatic Stress Disorder.

Although previous genome-wide association studies (GWASs) on post-traumatic stress disorder (PTSD) have identified multiple risk loci, how these loci confer risk of PTSD remains unclear. Through the FUSION pipeline, we integrated two human brain proteome reference datasets (ROS/MAP and Banner) with the PTSD GWAS dataset, respectively, to conduct a proteome-wide association study (PWAS) analysis. Then two transcriptome reference weights (Rnaseq and Splicing) were applied to a transcriptome-wide association study (TWAS) analysis. Finally, the PWAS and TWAS results were investigated through brain imaging analysis. In the PWAS analysis, 8 and 13 candidate genes were identified in the ROS/MAP and Banner reference weight groups, respectively. Examples included ADK (pPWAS-ROS/MAP = 3.00 × 10−5) and C3orf18 (pPWAS-Banner = 7.07 × 10−31). Moreover, the TWAS also detected multiple candidate genes associated with PTSD in two different reference weight groups, including RIMS2 (pTWAS-Splicing = 3.84 × 10−2), CHMP1A (pTWAS-Rnaseq = 5.09 × 10−4), and SIRT5 (pTWAS-Splicing = 4.81 × 10−3). Further comparison of the PWAS and TWAS results in different populations detected the overlapping genes: MADD (pPWAS-Banner = 4.90 × 10−2, pTWAS-Splicing = 1.23 × 10−2) in the total population and GLO1(pPWAS-Banner = 4.89 × 10−3, pTWAS-Rnaseq = 1.41 × 10−3) in females. Brain imaging analysis revealed several different brain imaging phenotypes associated with MADD and GLO1 genes. Our study identified multiple candidate genes associated with PTSD in the proteome and transcriptome levels, which may provide new clues to the pathogenesis of PTSD.

Utility of the DHFR-based destabilizing domain across mouse models of retinal degeneration and aging

SummaryThe Escherichia coli dihydrofolate reductase (DHFR) destabilizing domain (DD) serves as a promising approach to conditionally regulate protein abundance in a variety of tissues. To test whether this approach could be effectively applied to a wide variety of aged and disease-related ocular mouse models, we evaluated the DHFR DD system in the eyes of aged mice (up to 24 months), a light-induced retinal degeneration (LIRD) model, and two genetic models of retinal degeneration (rd2 and Abca4−/− mice). The DHFR DD was effectively degraded in all model systems, including rd2 mice, which showed significant defects in chymotrypsin proteasomal activity. Moreover, trimethoprim (TMP) administration stabilized the DHFR DD in all mouse models. Thus, the DHFR DD-based approach allows for control of protein abundance in a variety of mouse models, laying the foundation to use this strategy for the conditional control of gene therapies to potentially treat multiple eye diseases.

Gene Expression of miRNAs Let-7aAssociated with Diabetes in Iraqi Population.

miRNAs regulate protein abundance and control diverse aspects of cellular processes and biological functions in metabolic diseases, such as obesity and diabetes. Lethal-7(Let-7) miRNAs specifically target genes associated with diabetes and have a role in the regulation of peripheral glucose metabolism. The present study aimed to describe the gene expressions of the let-7a gene with the development of diabetes in Iraq and the difference in the expression of this gene in patients with diabetes and healthy individuals. The association between age and gender with the development of diabetes was studied in this study and the results were compared with those of healthy individuals in the group of control. Based on the obtained results, there was a lack in the mean of gene expression level (ΔCt) in patients, compared to controls. Moreover, the gene expression folding (2-∆∆Ct) of the let-7a reflects significant differences in terms of gene expression between groups of patients and controls, and the level of let-7a expression was reported to be 12.97 in patients with diabetes. On the other hand, significant difference was observed in terms of age and gender between diabetic patients and controls. The findings suggest that diabetes can affect individuals in all age groups and occur regardless of gender in both males and females. Based on the obtained results in this study, the gene expression level of miRNA let-7a was lower in diabetic patients compared to healthy individuals in the group of control. This also reflects differences in the gene expression fold (2-∆∆Ct) of gene let-7a between both groups of patients and controls.

Regulation of protein abundance in genetically diverse mouse populations

SUMMARYGenetically diverse mouse populations are powerful tools for characterizing the regulation of the proteome and its relationship to whole-organism phenotypes. We used mass spectrometry to profile and quantify the abundance of 6,798 proteins in liver tissue from mice of both sexes across 58 Collaborative Cross (CC) inbred strains. We previously collected liver proteomics data from the related Diversity Outbred (DO) mice and their founder strains. We show concordance across the proteomics datasets despite being generated from separate experiments, allowing comparative analysis. We map protein abundance quantitative trait loci (pQTLs), identifying 1,087 local and 285 distal in the CC mice and 1,706 local and 414 distal in the DO mice. We find that regulatory effects on individual proteins are conserved across the mouse populations, in particular for local genetic variation and sex differences. In comparison, proteins that form complexes are often co-regulated, displaying varying genetic architectures, and overall show lower heritability and map fewer pQTLs. We have made this resource publicly available to enable quantitative analyses of the regulation of the proteome.

Role of miRNAs in Breast Cancer-induced Bone Disease

AbstractBone is the most common site of breast cancer recurrence. Despite the increasing knowledge about the metastatic process and treatment advances, the disease still remains incurable once the cancer cells actively proliferate in bone. Complex interactions between cancer cells and cells of the bone microenvironment (BME) regulate the initiation and progression of metastatic tumor growth in bone. In particular, breast cancer cells shift the otherwise tightly balanced bone remodeling towards increased bone resorption by osteoclasts. Cellular interactions in the metastatic BME are to a large extent regulated by secreted molecules. These include various cytokines as well as microRNAs (miRNAs), small non-coding RNAs that post transcriptionally regulate protein abundance in several cell types. Through this mechanism, miRNAs modulate physiological and pathological processes including bone remodeling, tumorigenesis and metastasis. Consequently, miRNAs have been identified as important regulators of cellular communication in the metastatic BME. Disruption of the crosstalk between cancer cells and the BME has emerged as a promising therapeutic target to prevent the establishment and progression of breast cancer bone metastasis. In this context, miRNA mimics or antagonists present innovative therapeutic approaches of high potential for interfering with pathological bone – cancer cell interactions. This review will discuss the role of miRNAs in the tumor-BME crosstalk in vivo and will emphasize how this could be targeted by miRNAs to improve therapeutic outcome for patients with breast cancer bone metastases.

Proteome-wide Association Study Provides Insights Into the Genetic Component of Protein Abundance in Psychiatric Disorders

BackgroundGenome-wide association studies have identified multiple risk variants for psychiatric disorders. Nevertheless, how the risk variants confer risk of psychiatric disorders remains largely unknown. MethodsWe performed proteome-wide association studies to identify genes whose cis-regulated protein abundance change in the human brain were associated with psychiatric disorders. ResultsBy integrating genome-wide associations of four common psychiatric disorders and two independent brain proteomes (n = 376 and n = 152, respectively) from the dorsolateral prefrontal cortex, we identified 61 genes (including 48 genes for schizophrenia, 12 genes for bipolar disorder, 5 genes for depression, and 2 genes for attention-deficit/hyperactivity disorder) whose genetically regulated protein abundance levels were associated with risk of psychiatric disorders. Comparison with transcriptome-wide association studies identified 18 overlapping genes that showed significant associations with psychiatric disorders at both proteome-wide and transcriptome-wide levels, suggesting that genetic risk variants likely confer risk of psychiatric disorders by regulating messenger RNA expression and protein abundance of these genes. ConclusionsOur study not only provides new insights into the genetic component of protein abundance in psychiatric disorders but also highlights several high-confidence risk proteins (including CNNM2 and CTNND1) for schizophrenia and depression. These high-confidence risk proteins represent promising therapeutic targets for future drug development.

Abstract 1008: Comparative proteomics and transcriptomics analysis of the impact of androgen stimulation and darolutamide inhibition in a prostate cancer model

Abstract Androgen receptor (AR) signaling is the major driver in prostate cancer. Darolutamide is a high-affinity AR inhibitor recently approved for non-metastatic castration-resistant prostate cancer. The impact of the AR on gene transcription has been extensively examined but the downstream effects on protein levels are still poorly understood. Here we generated for the first time comprehensive quantitative proteomic and transcriptomic data from the androgen-sensitive, prostate cancer cell line VCaP treated with the androgen R1881 and darolutamide. For the proteomic analysis, a comprehensive and quantitative label-free protein expression profiling was performed using nanoscale liquid chromatography coupled to tandem mass spectrometry. For the transcriptomic analysis, sequencing was performed via single-end, 50 base-pair reads with an average depth of 21 million reads per sample. FASTQ reads were mapped with STAR aligner to the human genome GRCh38 and quantified with RSEM. Further analysis was performed in R statistical software. We investigated the expression levels of 12900 protein-coding genes and 8721 translated proteins. Androgen treatment resulted in increased expression of known androgen response genes, as well as of genes from the mTORC1 pathway, cell cycle and other pathways previously shown to be affected by androgens. This was paralleled by upregulation of the corresponding protein levels in the majority of cases. Importantly, a detailed comparative analysis of transcriptomic and proteomic data revealed a few notable differences in regulation. In androgen-treated prostate cancer cells, there was a strong increase of FOLH1, AMACR and FKBP5 transcript levels (10 to 40-fold) but only a moderate increase of the corresponding protein levels (2 to 6-fold). Additional darolutamide treatment led to strong down-regulation of androgen-induced gene expression and of the corresponding protein levels. Here also there were a few instances where protein and transcript levels were differentially regulated. The discrepancy in transcript and protein levels seen after androgen and anti-androgen treatment suggests differential post-transcriptional regulation of protein abundance to occur in some instances. In conclusion, we found that darolutamide strongly reversed androgen-induced activation of gene expression programs related to cell proliferation and survival, both at the transcript and protein levels. We furthermore identified for the first time androgen-activated genes whose function is additionally regulated at the post-transcription level. Citation Format: Ekaterina Nevedomskaya, Simon J. Baumgart, Tatsuo Sugawara, Ralf Lesche, Dominik Mumberg, Bernard Haendler. Comparative proteomics and transcriptomics analysis of the impact of androgen stimulation and darolutamide inhibition in a prostate cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1008.

Estrogen Differentially Regulates Protein Abundance in Exosomes Released From Immortalized Kisspeptin Neurons in Vitro

Estrogen (E2) is essential for multiple physiological effects in females, ensuring maximum reproductive fitness and maintaining skeletal homeostasis. E2 has been shown to stimulate cancellous bone formation via activation of estrogen receptor alpha (ERα), an effect widely accepted to be mediated directly at bone. A recent landmark study (Herber et al., Nat Commun 2019) demonstrated bone density increases in female mice harboring ERα-deletions specifically in arcuate Kiss-1 neurons. In this study, bone from transgenic females showed higher osteoblast functioning and increases in the expression of sp7 and runx2, positing a direct neural-bone regulatory axis altered by circulating E2 acting in brain. Our laboratory has used two immortalized Kisspeptin (Kiss1)-expressing and -secreting cell lines, KTaR-1 (representative of female arcuate Kiss-1 neurons) and KTaV-3 cells (representative of female AVPV Kiss-1 neurons) as models to explore the role of Kiss-1 in multiple physiological regulatory contexts. We recently determined that factors in the media of female ARC-derived KTaR-1 cells can affect parameters of osteoblast function in vitro, including increases in sp7 and runx2 expression, and formation of bone matrix (evaluated by Alizarin Red assay). Exposure of canine osteosarcoma cells to conditioned media from KTaR-1 cells led to increases in sp7 expression in an E2-dependent manner, and 24h E2-deprivation of these neurons stimulated secretion of osteogenic factors. In this current study, we have used LCMS-MS proteomic analysis to determine the contents of exosomes isolated from Kisspeptin neurons under varying E2 exposure conditions in vitro. Preliminary results reveal ~150-170 proteins up-regulated by E2 exposure and ~200-220 proteins downregulated by E2 exposure in exosomes of both KTaR-1 and KTaV-3 Kisspeptin neurons. Estrogen-regulated Kiss-1 exosomal proteins include several candidates involved in bone remodeling (pentraxin, osteonectin, osteoclast-stimulating factor-1) and neuronal synaptic plasticity and signaling (annexins, semaphorins, connexins). Current work is exploring the effects of exposure of purified exosomes on morphology and gene expression in immortalized GnRH neurons and osteoblasts. While further study is required, initial results suggest that exosomes may represent additional cellular communication pathways utilized by Kisspeptin neurons to elicit changes in brain and bone.

Multiscale Modeling of Cross-Regulatory Transcript and Protein Influences.

With the popularity of high-throughput transcriptomic techniques like RNAseq, models of gene regulatory networks have been important tools for understanding how genes are regulated. These transcriptomic datasets are usually assumed to reflect their associated proteins. This assumption, however, ignores post-transcriptional, translational, and post-translational regulatory mechanisms that regulate protein abundance but not transcript abundance. Here we describe a method to model cross-regulatory influences between the transcripts and proteins of a set of genes using abundance data collected from a series of transgenic experiments. The developed model can capture the effects of regulation that impacts transcription as well as regulatory mechanisms occurring after transcription. This approach uses a sparse maximum likelihood algorithm to determine relationships that influence transcript and protein abundance. An example of how to explore the network topology of this type of model is also presented. This model can be used to predict how the transcript and protein abundances will change in novel transgenic modification strategies.

Generation of 3'UTR knockout cell lines by CRISPR/Cas9-mediated genome editing.

In addition to the protein code, messenger RNAs (mRNAs) also contain untranslated regions (UTRs). 3'UTRs span the region between the translational stop codon and the poly(A) tail. Sequence elements located in 3'UTRs are essential for pre-mRNA processing. 3'UTRs also contain elements that can regulate protein abundance, localization, and function. At least half of all human genes use alternative cleavage and polyadenylation (APA) to further diversify the regulatory potential of protein functions. Traditional gene editing approaches are designed to disrupt the production of functional proteins. Here, we describe a method that allows investigators to manipulate 3'UTR sequences of endogenous genes for both single- 3'UTR and multi-3'UTR genes. As 3'UTRs can regulate individual functions of proteins, techniques to manipulate 3'UTRs at endogenous gene loci will help to disentangle multi-functionality of proteins. Furthermore, the ability to directly examine the impact of gene regulatory elements in 3'UTRs will provide further insights into their functional significance.

The long noncoding RNA NARL regulates immune responses via microRNA-mediated NOD1 downregulation in teleost fish

Increasing evidence shows that the long noncoding RNA (lncRNA) is a major regulator and participates in the regulation of various physiological and pathological processes, such as cell proliferation, differentiation, metastasis, and apoptosis. Unlike mammals, however, the study of lncRNA in lower invertebrates is just beginning and the extent of lncRNA-mediate regulation remains unclear. Here, we for the first time identify an lncRNA, termed nucleotide oligomerization domain 1 (NOD1) antibacterial and antiviral-related lncRNA (NARL), as a key regulator for innate immunity in teleost fish. We found that NOD1 plays an important role in the antibacterial and antiviral process in fish and that the microRNA miR-217-5p inhibits NOD1 expression and thus weakens the NF-κB and the IRF3-driven signaling pathway. Furthermore, our results indicated that NARL functions as a competing endogenous RNA (ceRNA) for miR-217-5p to regulate protein abundance of NOD1; thus, invading microorganisms are eliminated and immune responses are promoted. Our study also demonstrates the regulation mechanism that lncRNA NARL can competitive adsorption miR-217-5p to regulate the miR-217-5p/NOD1 axis is widespread in teleost fish. Taken together, our results reveal that NARL in fish is a critical positive regulator of innate immune responses to viral and bacterial infection by suppressing a feedback to NOD1-NF-κB/IRF3-mediated signaling.

Long noncoding RNA IRL regulates NF-κB-mediated immune responses through suppression of miR-27c-3p-dependent IRAK4 downregulation in teleost fish

Growing pieces of evidence show that the long noncoding RNAs (lncRNAs) as new regulators participate in the regulation of various physiological and pathological processes. The study of lncRNA in lower invertebrates is still unclear compared with that in mammals. Here, we identified a novel lncRNA, termed IRAK4-related lncRNA (IRL), as a key regulator for innate immunity in teleost fish. We find that miR-27c-3p inhibits IRAK4 expression and thus weakens the NF-κB-mediated signaling pathway. Furthermore, the Gram-negative bacterium Vibrio anguillarum and lipopolysaccharide significantly upregulated host lncRNA IRL expression. Results indicate that IRL functions as a competing endogenous RNA for miR-27c-3p to regulate protein abundance of IRAK4; thus, invading microorganisms are eliminated and immune responses are promoted. Our study also demonstrates the regulation mechanism that lncRNA IRL can competitively adsorb miRNA to regulate the miR-27c-3p/IRAK4 axis that is widespread in teleost fish.