ZFP36L1 and ZFP36L2 reduce cyclin D1 expression by decreasing expression of E2F1 and long 3'UTR isoform of CCND1 transcripts.

The CCND1 mRNA possesses at least two distinct lengths of the 3'-untranslated region (3'UTR), with the long isoform containing multiple AU-rich elements (AREs). The tandem zinc finger (TZF) domains of human ZFP36 family members have the capacity to bind to AREs and promote mRNA degradation. Our previous study demonstrated that mutations in the TZF domain of ZFP36L1 or ZFP36L2 increased the CCND1 expression. In this study, we investigated whether ZFP36L1 and ZFP36L2 could downregulate the expression of the long 3'UTR isoform of CCND1 mRNA in human colorectal cancer (CRC) cells. Firstly, the Gene Expression Profiling Interactive Analysis 2 database indicated downregulation of ZFP36 and ZFP36L1, while E2F1 and CCND1 were upregulated in human CRC tissues compared to normal colorectal tissues. Overexpression of ZFP36L1 and/or ZFP36L2 in T-REx-293, DLD-1, and HCT116 cells led to a decrease in the total CCND1, long isoform ratio of CCND1 mRNA, and E2F1 expression. Conversely, knockdown of ZFP36L1 and ZFP36L2 in HCT116 cells resulted in an increase in total CCND1, long isoform ratio of CCND1 mRNA, and E2F1 expression. Knockdown of E2F1 decreased CCND1 expression, indicating a potential role for E2F1 in regulating CCND1 expression at the transcriptional level. These findings suggest that ZFP36L1 and ZFP36L2 play a negative role in CCND1 expression. The underlying mechanisms might involve the reduction of E2F1 transactivation at the transcriptional level and the promotion of AREs-mediated decay of the long 3'UTR isoform of CCND1 through posttranscriptional processes.

A promoter-RBS library for fine-tuning gene expression in Methanosarcina acetivorans.

Methanogens are the main biological producers of methane on Earth. Methanosarcina acetivorans is one of the best characterized methanogens that has powerful genetic tools for genome editing. To study the physiology of this methanogen in further detail as well as to effectively balance the flux of their engineered metabolic pathways in expansive project undertakings, there is the need for controlled gene expression, which then requires the availability of well-characterized promoters and ribosome-binding sites (RBS). In this study, we constructed a library of 33 promoter-RBS combinations that includes 13 wild-type and 14 hybrid combinations, as well as six combination variants in which the 5'-untranslated region (5'UTR) was rationally engineered. The expression strength for each combination was calculated by inducing the expression of the ß-glucuronidase reporter gene in M. acetivorans cells in the presence of the two most used growth substrates, either methanol (MeOH) or trimethyl amine (TMA). In this study, the constructed library covers a relatively wide range (140-fold) between the weakest and strongest promoter-RBS combination as well as shows a steady increase and allows different levels of gene expression. Effects on the gene expression strength were also assessed by making measurements at three distinct growth phases for all 33 promoter-RBS combinations. Our promoter-RBS library is effective in enabling the fine-tuning of gene expression in M. acetivorans for physiological studies and the design of metabolic engineering projects that, e.g., aim for the biotechnological valorization of one-carbon compounds. IMPORTANCE: Methanogenic archaea are potent producers of the greenhouse gas methane and thus contribute substantially to global warming. Under controlled conditions, these microbes can catalyze the production of biogas, which is a renewable fuel, and might help counter global warming and its effects. Engineering the primary metabolism of Methanosarcina acetivorans to render it better and more useful requires controllable gene expression, yet only a few well-characterized promoters and RBSs are presently available. Our study rectifies this situation by providing a library of 33 different promoter-RBS combinations with a 140-fold dynamic range in expression strength. Future metabolic engineering projects can take advantage of this library by using these promoter-RBS combinations as an efficient and tunable gene expression system for M. acetivorans. Furthermore, the methodologies we developed in this study could also be utilized to construct promoter libraries for other types of methanogens.

Synthetic mRNAs Containing Minimalistic Untranslated Regions Are Highly Functional In Vitro and In Vivo.

Synthetic mRNA produced by in vitro transcription (ivt mRNA) is the active pharmaceutical ingredient of approved anti-COVID-19 vaccines and of many drugs under development. Such synthetic mRNA typically contains several hundred bases of non-coding "untranslated" regions (UTRs) that are involved in the stabilization and translation of the mRNA. However, UTRs are often complex structures, which may complicate the entire production process. To eliminate this obstacle, we managed to reduce the total amount of nucleotides in the UTRs to only four bases. In this way, we generate minimal ivt mRNA ("minRNA"), which is less complex than the usual optimized ivt mRNAs that are contained, for example, in approved vaccines. We have compared the efficacy of minRNA to common augmented mRNAs (with UTRs of globin genes or those included in licensed vaccines) in vivo and in vitro and could demonstrate equivalent functionalities. Our minimal mRNA design will facilitate the further development and implementation of ivt mRNA-based vaccines and therapies.

The Engineered Drug 3'UTRMYC1-18 Degrades the c-MYC-STAT5A/B-PD-L1 Complex In Vivo to Inhibit Metastatic Triple-Negative Breast Cancer.

c-MYC is overexpressed in 70% of human cancers, including triple-negative breast cancer (TNBC), yet there is no clinically approved drug that directly targets it. Here, we engineered the mRNA-stabilizing poly U sequences within the 3'UTR of c-MYC to specifically destabilize and promote the degradation of c-MYC transcripts. Interestingly, the engineered derivative outcompetes the endogenous overexpressed c-MYC mRNA, leading to reduced c-MYC mRNA and protein levels. The iron oxide nanocages (IO-nanocages) complexed with MYC-destabilizing constructs inhibited primary and metastatic tumors in mice bearing TNBC and significantly prolonged survival by degrading the c-MYC-STAT5A/B-PD-L1 complexes that drive c-MYC-positive TNBC. Taken together, we have described a novel therapy for c-MYC-driven TNBC and uncovered c-MYC-STAT5A/B-PD-L1 interaction as the target.

Molecular detection and genotyping of bovine viral diarrhea virus in Selangor, Malaysia.

Objective: Bovine viral diarrhea (BVD) disease is a viral infection in cows caused by a single-stranded plus-sense RNA virus of the Pestivirus genus under the Flaviviridae family. The clinical manifestation of BVD mainly includes diarrhea and immunosuppression, thereby exacerbating various respiratory diseases. This study was conducted to detect and molecularly characterize the bovine viral diarrhea disease virus (BVDV) in cattle on selected farms in Selangor, Malaysia. Materials and Methods: A reverse transcription polymerase chain reaction (RT-PCR) was performed for antigen detection in 253 plasma samples collected from cows using a cross-sectional study design. We selected the 5 untranslated regions (5'-UTR) region and the E2 region to compare the genetic differences between the isolates. Results: One sample was found to be positive (1/253) following RT-PCR targeting the conserved 5'-UTR region of BVDV. Thus, BVDV antigen prevalence was 0.40% (95% confidence interval: 0.0%-2.2%). By targeting the hypervariable E2 region of the isolated virus, UPM/MAL/BVDV/D17, the virus was classified under the subgenotype BVDV-1a. Conclusion: BVDV is present and circulating on selected cattle farms in Selangor, Malaysia. Given the presence of BVDV in several subgenotypes, the screening of all incoming cattle at Malaysia's border is pertinent to prevent the entry of other BVDV subgenotypes into the country.

The dual role of a highly structured RNA (the S fragment) in the replication of foot-and-mouth disease virus.

Secondary and tertiary RNA structures play key roles in genome replication of single-stranded positive sense RNA viruses. Complex, functional structures are particularly abundant in the untranslated regions of picornaviruses, where they are involved in initiation of translation, priming of new strand synthesis and genome circularization. The 5' UTR of foot-and-mouth disease virus (FMDV) is predicted to include a c. 360 nucleotide-long stem-loop, termed the short (S) fragment. This structure is highly conserved and essential for viral replication, but the precise function(s) are unclear. Here, we used selective 2' hydroxyl acetylation analyzed by primer extension (SHAPE) to experimentally determine aspects of the structure, alongside comparative genomic analyses to confirm structure conservation from a wide range of field isolates. To examine its role in virus replication in cell culture, we introduced a series of deletions to the distal and proximal regions of the stem-loop. These truncations affected genome replication in a size-dependent and, in some cases, host cell-dependent manner. Furthermore, during the passage of viruses incorporating the largest tolerated deletion from the proximal region of the S fragment stem-loop, an additional mutation was selected in the viral RNA-dependent RNA polymerase, 3Dpol. These data suggest that the S fragment and 3Dpol interact in the formation of the FMDV replication complex.

Major-groove sequence-specific RNA recognition by LoaP, a paralog of transcription elongation factor NusG.

LoaP is a member of the universal NusG protein family. Previously, we reported that unlike other characterized homologs, LoaP binds RNA sequence-specifically, recognizing a stem-loop in the 5'-untranslated region of operons it regulates. To elucidate how this NusG homolog acquired this ability, we now determined the co-crystal structure of Thermoanaerobacter pseudethanolicus LoaP bound to its cognate 26-nucleotide dfn RNA element. Our structure reveals that the LoaP C-terminal KOW domain recognizes the helical portion of the RNA by docking into a broadened major groove, while a protruding ß-hairpin of the N-terminal NusG-like domain binds the UNCG tetraloop capping the stem-loop. Major-groove RNA recognition is unusual and is made possible by conserved features of the dfn hairpin. Superposition with structures of other NusG proteins implies that LoaP can bind concurrently to the dfn RNA and the transcription elongation complex, suggesting a new level of co-transcriptional regulation by proteins of this conserved family.

5-mC methylation study of sORFs in 3'UTR of transcription factor JUNGBRUNNEN 1-like during leaf rust pathogenesis in wheat.

BACKGROUND: JUB1, a NAC domain containing hydrogen peroxide-induced transcription factor, plays a critical role in plant immunity. Little is known about how JUB1 responds to leaf rust disease in wheat. Recent discoveries in genomics have also unveiled a multitude of sORFs often assumed to be non-functional, to argue for the necessity of including them as potential regulatory players of translation. However, whether methylation on sORFs spanning the 3'UTR of regulatory genes like JUB1 modulate gene expression, remains unclear. METHODS AND RESULTS: In this study, we identified the methylation states of two sORFs in 3'UTR of a homologous gene of JUB1 in wheat, TaJUB1-L, at cytosine residues in CpG, CHH and CHG sites at different time points of disease progression in two near-isogenic lines of wheat (HD2329), with and without Lr24 gene during leaf rust pathogenesis. Here, we report a significant demethylation of the CpG dinucleotides occurring in the sORFs of the 3'UTR in the resistant isolines after 24 h post-infection. Also, the up-regulated gene expression observed through RT-qPCR was directly proportional to the demethylation of the CpG sites in the sORFs. CONCLUSIONS: Our findings indicate that TaJUB1-L might be a positive regulator in providing tolerance during leaf rust pathogenesis and cytosine methylation at 3'UTR might act as a switch for its expression control. These results enrich the potential benefit of conventional methylation assay techniques for unraveling the unexplored enigma in epigenetics during plant-pathogen interaction in a cost-effective and confidentially conclusive manner.

Association of 3'UTR variations of EGFR and KRAS oncogenes with clinical parameters in lung cancer tumours.

BACGROUND INFORMATION: Lung cancer is one of the leading types of cancer deaths worldwide, with approximately 2 million people diagnosed with lung cancer each year. In this study, we aimed to determine the exonic and 3'UTR sequences of EGFR, PIK3CA and KRAS genes in 39 sporadic lung cancer tumors and to reveal the changes in the miRNA binding profile of tumors with somatic variation in the 3'UTR region and to examine the relationship of these changes with clinical parameters. RESULTS: A statistically significant correlation was found between the presence of miRNA that could not bind to the 3'UTR region due to variation in at least one of the EGFR or KRAS genes and the presence of metastasis in the tumor. At the same time, Kaplan-Meier analysis between those with and without alterations in the miRNA profile due to somatic variation in the 3'UTR region showed that survival was lower in those with miRNA alterations and this was statistically significant. CONCLUSIONS: In our study, it was shown that variations in the 3'UTR regions of EGFR and KRAS oncogenes may cause increased expression of these oncogenes by preventing the binding of miRNAs, and it was suggested that this may be related to metastasis, survival and drug resistance mechanism. SIGNIFICANCE: In this study, we show that hsa-miR-124-3p, hsa-miR-506-3p, hsa-miR-1290 and hsa-miR-6514-3p are particularly prominent in lung carcinoma in relation to these biological pathways and the roles that variations in the 3'UTR regions of oncogenes may play in the carcinogenesis process.

A 44-Nucleotide Region in the Chikungunya Virus 3' UTR Dictates Viral Fitness in Disparate Host Cells.

We previously reported that deletion of a 44-nucleotide element in the 3' untranslated region (UTR) of the Chikungunya virus (CHIKV) genome enhances the virulence of CHIKV infection in mice. Here, we find that while this 44-nucleotide deletion enhances CHIKV fitness in murine embryonic fibroblasts in a manner independent of the type I interferon response, the same mutation decreases viral fitness in C6/36 mosquito cells. Further, the fitness advantage conferred by the UTR deletion in mammalian cells is maintained in vivo in a mouse model of CHIKV dissemination. Finally, SHAPE-MaP analysis of the CHIKV 3' UTR revealed this 44-nucleotide element forms a distinctive two-stem-loop structure that is ablated in the mutant 3' UTR without altering additional 3' UTR RNA secondary structures.

The Roles of Cytoplasmic Polyadenylation Element Binding Protein 1 in Tumorigenesis.

BACKGROUND: CPEB1 is an alternative polyadenylation binding protein that promotes or suppresses the expression of related mRNAs and proteins by binding to a highly conserved Cytoplasmic Polyadenylation Element (CPE) in the mRNAs 3'UTR. It is found to express abnormally in multiple tumors and affect tumorigenesis through many pathways. This review summarizes the functions and mechanisms of CPEB1 in a variety of cancers and suggests new directions for future related treatments. METHODS: A total of 95 articles were eligible for inclusion based on the year, quality of the research, and the strength of association with CPEB1. In this review, current research about how CPEB1 affects the initiation and progression of glioblastoma, breast cancer, hepatocellular carcinoma, gastric cancer, colorectal cancer, non-small cell lung cancer, prostate cancer, and melanoma are dissected, and the biomedical functions and mechanisms are summarized. RESULTS: CPEB1 mostly presents as a tumor suppressor for breast cancer, endometrial carcinoma, hepatocellular carcinoma, non-small cell lung cancer, prostate cancer, and melanoma. However, glioblastoma, gastric cancer, and colorectal cancer it exhibit two opposing properties of tumorigenesis, either promoting or inhibiting it. CONCLUSION: CPEB1 is likely to serve as a target and dynamic detection index or prognostic indicator for its function of apoptosis, activity, proliferation, migration, invasion, stemness, drug resistance, and even ferroptosis in various cancers.

Evaluation of RNA Secondary Stem-Loop Structures in the UTRs of Mouse Hepatitis Virus as New Therapeutic Targets.

MHV-A59 is a beta-coronavirus that causes demyelinating encephalitis and hepatitis in mice. Recently, the mouse infection model of MHV-A59 has been used as an alternative animal infection model for SARS-CoV and SARS-CoV-2, aiding the development of new antiviral drugs. In this study, the MHV-A59 model was employed to investigate the potential of SARS-CoV-2 UTRs as new targets for antiviral drugs. Optimal targets within the MHV-A59 UTRs were identified using a shRNA and siRNA design tool, focusing on RNA secondary stem-loop (SL) structures in the UTRs. We then examined whether the designed RNAi constructs could inhibit MHV-A59 replication. In the 5'UTR, the stem-loop 1 (SL1) was identified as the most effective target, while in the 3'UTR, the minimal element for the initiation of negative-strand RNA synthesis (MIN) proved to be the most effective. Importantly, siRNAs targeting SL1 and MIN structures significantly reduced total RNA synthesis, negative-strand genomic RNA synthesis, subgenomic (sg) RNA synthesis, viral titer, and the plaque size of MHV-A59 compared to the control. Although not statistically significant, the combination of siSL1 and siMIN had a stronger effect on inhibiting MHV-A59 replication than either siRNA monotherapy. Interestingly, while the SL1 structure is present in both MHV and SARS-CoV-2, the MIN structure is unique to MHV. Thus, the SL1 of SARS-CoV-2 may represent a novel and promising target for RNAi-based antiviral drugs.

Characterization of a Molecular Clone of Deformed Wing Virus B.

Honey bees (Apis mellifera) play a crucial role in agriculture through their pollination activities. However, they have faced significant health challenges over the past decades that can limit colony performance and even lead to collapse. A primary culprit is the parasitic mite Varroa destructor, known for transmitting harmful bee viruses. Among these viruses is deformed wing virus (DWV), which impacts bee pupae during their development, resulting in either pupal demise or in the emergence of crippled adult bees. In this study, we focused on DWV master variant B. DWV-B prevalence has risen sharply in recent decades and appears to be outcompeting variant A of DWV. We generated a molecular clone of a typical DWV-B strain to compare it with our established DWV-A clone, examining RNA replication, protein expression, and virulence. Initially, we analyzed the genome using RACE-PCR and RT-PCR techniques. Subsequently, we conducted full-genome RT-PCR and inserted the complete viral cDNA into a bacterial plasmid backbone. Phylogenetic comparisons with available full-length sequences were performed, followed by functional analyses using a live bee pupae model. Upon the transfection of in vitro-transcribed RNA, bee pupae exhibited symptoms of DWV infection, with detectable viral protein expression and stable RNA replication observed in subsequent virus passages. The DWV-B clone displayed a lower virulence compared to the DWV-A clone after the transfection of synthetic RNA, as evidenced by a reduced pupal mortality rate of only 20% compared to 80% in the case of DWV-A and a lack of malformations in 50% of the emerging bees. Comparable results were observed in experiments with low infection doses of the passaged virus clones. In these tests, 90% of bees infected with DWV-B showed no clinical symptoms, while 100% of pupae infected with DWV-A died. However, at high infection doses, both DWV-A and DWV-B caused mortality rates exceeding 90%. Taken together, we have generated an authentic virus clone of DWV-B and characterized it in animal experiments.

A multiplex one-step fluorescence quantitative differential diagnosis method for severe hand, foot and mouth disease caused by coxsackievirus A16.

Hand foot and mouth disease (HFMD) is a common childhood infectious disease which is caused by human enterovirus. The objective of this study was to develop a rapid, sensitive, and accurate method for detecting severe HFMD caused by coxsackievirus A16 (CV-A16). A closed-tube sensitive multiplex one-step reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was applied to detect CV-A16 in the early stage of severe HFMD. This assay targeted the CV-A16 structure protein VP1 to distinguish CV-A16 from other coxsackieviruses The 5'UTR region of enteric viruses was used for detecting the enterovirus and ribonuclease P (RNaseP) was adopted as the internal reference gene. The multiplex MGB probe assay system was used to detect PCR amplicons with different fluorescence reporters in the same system. The limit of detection (LOD) of the RT-qPCR assay for the CV-A16 VP1 gene was 125.893 copies/µl, for the 5' UTR was 50.1187 copies/µl and for the RNaseP gene was 158.49 copies/µl. Furthermore, specificity analysis showed that the multiplex RT-PCR had no cross-reactivity with the influenza virus, herpangina virus and SARS-COV-2. In correlation analysis, the sensitivity of the multiplex RT-qPCR assay for CV-A16 detection was 100 % (288/288) and the specificity of the multiplex RT-qPCR assay was 99.94 % (3395/3397). The overall agreement between the multiplex RT-qPCR and the results of clinical diagnosis was 99.95 % (3683/3685) and kappa value was 0.996 (p<0.001). The entire procedure, from specimen processing to result reporting, could be completed within 1.5 hours. The one-step multiplex RT-qPCR assay for detecting CV-A16 developed in this study is a good laboratory diagnostic tool for rapid and reliable distinguished detection of CV-A16, especially for severe HFMD patients at an early stage in the disease with low virus load of CV-A16.

Ckip-1 3'UTR alleviates prolonged sleep deprivation induced cardiac dysfunction by activating CaMKK2/AMPK/cTNI pathway.

Sleep deprivation (SD) has emerged as a critical concern impacting human health, leading to significant damage to the cardiovascular system. However, the underlying mechanisms are still unclear, and the development of targeted drugs is lagging. Here, we used mice to explore the effects of prolonged SD on cardiac structure and function. Echocardiography analysis revealed that cardiac function was significantly decreased in mice after five weeks of SD. Real-time quantitative PCR (RT-q-PCR) and Masson staining analysis showed that cardiac remodeling marker gene Anp (atrial natriuretic peptide) and fibrosis were increased, Elisa assay of serum showed that the levels of creatine kinase (CK), creatine kinase-MB (CK-MB), ANP, brain natriuretic peptide (BNP) and cardiac troponin T (cTn-T) were increased after SD, suggesting that cardiac remodeling and injury occurred. Transcript sequencing analysis indicated that genes involved in the regulation of calcium signaling pathway, dilated cardiomyopathy, and cardiac muscle contraction were changed after SD. Accordingly, Western blotting analysis demonstrated that the cardiac-contraction associated CaMKK2/AMPK/cTNI pathway was inhibited. Since our preliminary research has confirmed the vital role of Casein Kinase-2 -Interacting Protein-1 (CKIP-1, also known as PLEKHO1) in cardiac remodeling regulation. Here, we found the levels of the 3' untranslated region of Ckip-1 (Ckip-1 3'UTR) decreased, while the coding sequence of Ckip-1 (Ckip-1 CDS) remained unchanged after SD. Significantly, adenovirus-mediated overexpression of Ckip-1 3'UTR alleviated SD-induced cardiac dysfunction and remodeling by activating CaMKK2/AMPK/cTNI pathway, which proposed the therapeutic potential of Ckip-1 3'UTR in treating SD-induced heart disease.

CGG repeats in the human FMR1 gene regulate mRNA localization and cellular stress in developing neurons.

The human genome has many short tandem repeats, yet the normal functions of these repeats are unclear. The 5' untranslated region (UTR) of the fragile X messenger ribonucleoprotein 1 (FMR1) gene contains polymorphic CGG repeats, the length of which has differing effects on FMR1 expression and human health, including the neurodevelopmental disorder fragile X syndrome. We deleted the CGG repeats in the FMR1 gene (0CGG) in human stem cells and examined the effects on differentiated neurons. 0CGG neurons have altered subcellular localization of FMR1 mRNA and protein, and differential expression of cellular stress proteins compared with neurons with normal repeats (31CGG). In addition, 0CGG neurons have altered responses to glucocorticoid receptor (GR) activation, including FMR1 mRNA localization, GR chaperone HSP90α expression, GR localization, and cellular stress protein levels. Therefore, the CGG repeats in the FMR1 gene are important for the homeostatic responses of neurons to stress signals.

Alternative 3'UTR expression induced by T cell activation is regulated in a temporal and signal dependent manner.

The length of 3' untranslated regions (3'UTR) is highly regulated during many transitions in cell state, including T cell activation, through the process of alternative polyadenylation (APA). However, the regulatory mechanisms and functional consequences of APA remain largely unexplored. Here we present a detailed analysis of the temporal and condition-specific regulation of APA following activation of primary human CD4+ T cells. We find that global APA changes are regulated temporally and CD28 costimulatory signals enhance a subset of these changes. Most APA changes upon T cell activation involve 3'UTR shortening, although a set of genes enriched for function in the mTOR pathway exhibit 3'UTR lengthening. While upregulation of the core polyadenylation machinery likely induces 3'UTR shortening following prolonged T cell stimulation; a significant program of APA changes occur prior to cellular proliferation or upregulation of the APA machinery. Motif analysis suggests that at least a subset of these early changes in APA are driven by upregulation of RBM3, an RNA-binding protein which competes with the APA machinery for binding. Together this work expands our understanding of the impact and mechanisms of APA in response to T cell activation and suggests new mechanisms by which APA may be regulated.

Posttranscriptional regulation of the T-box gene midline via the 3'UTR in Drosophila is complex and cell- and tissue-dependent.

The T-box (Tbx) proteins have a 180-230 amino acid DNA-binding domain, first reported in the Brachyury (T) protein. They are highly conserved among metazoans. They regulate a multitude of cellular functions in development and disease. Here, we report posttranscriptional and translational regulation of midline (mid), a Tbx member in Drosophila. We found that the 3'UTR of mid has mRNA degradation elements and AT-rich sequences. In Schneider S2 cells, mid-mRNA could be detected only when the transgene was without the 3'UTR. Similarly, the 3'UTR linked to the Renilla luciferase reporter significantly reduced the activity of the Luciferase, whereas deleting only the degradation elements from the 3'UTR resulted in reduced activity, but not as much. Overexpression of mid in MP2, an embryonic neuroblast, showed no significant difference in the levels of mid-mRNA between the 2 transgenes, with and without the 3'UTR, indicating the absence of posttranscriptional regulation of mid in MP2. Moreover, while elevated mid-RNA was detected in MP2 in nearly all hemisegments, only a fifth of those hemisegments had elevated levels of the protein. Overexpression of the 2 transgenes resulted in MP2-lineage defects at about the same frequency. These results indicate a translational/posttranslational regulation of mid in MP2. The regulation of ectopically expressed mid in the wing imaginal disc was complex. In the wing disc, where mid is not expressed, the ectopic expression of the transgene lacking the 3'UTR had a higher level of mid-RNA and the protein had a stronger phenotypic effect. These results indicate that the 3'UTR can subject mid-mRNA to degradation in a cell- and tissue-specific manner. We further report a balancer-mediated transgenerational modifier effect on the expression and gain of function effects of the 2 transgenes.

The 3'UTR Polymorphisms in the NLRP3 Gene Associated with the Risk of COPD and Their Putative Effects on the microRNA Mechanism.

Aims: Evaluating the association between a single nucleotide polymorphism in the 3' untranslated region (3'UTR) of the miRNA binding site of the NLRP3 gene and the occurrence and development of chronic obstructive pulmonary disease (COPD) and providing information to aid in the early detection and treatment of COPD. Materials and Methods: The regulatory single nuclear polymorphisms (SNPs) located in NLRP3 3'UTR were searched by using the dbSNP database and miRNA binding site prediction database. Meanwhile, samples from COPD patients and healthy controls in the same period were used for verification. The clinical baseline information of all subjects was collected, and the transcription level and protein expression level of NLRP3 and the expression level of inflammatory factors downstream of NLRP3 were detected. The effects of SNPs' single nucleotide changes on the transcription and expression of inflammatory factors were analyzed. Results: The study included 418 participants (249 in the COPD group and 169 in the control group). NLRP3 SNPs with miRNA binding sites include rs10754558 (G > C), rs1664774076 (ATAT > del), and rs1664775106 (C > G). Furthermore, two genotypes, GCG and GCA, were discovered to have a linkage mutation at 3'UTR 459-461. COPD susceptibility is tightly associated with the expression of the rs1664774076 del/del genotype, and the risk of COPD increased by 2.770 times (p = 0.003). Type 459-461 GCA was substantially related to the likelihood of developing COPD at various stages (p < 0.05). Except for rs10754558, all homozygous mutants increased NLRP3 mRNA and protein levels. NLRP3 had the greatest area under the receiver operating characteristic (ROC) curve for predicting the development and diagnosis of COPD when compared with its downstream inflammatory variables (AUC = 0.9291). Conclusions: The NLRP3 rs1664774076 del/del genotype is a COPD susceptibility gene, and the GCA genotype at 459-461 can be used as an early predictor of COPD exacerbation. The NLRP3 3'UTR polymorphism may alter the loss of miRNA binding sites, leading to an increase in NLRP3 expression. In the development of COPD, NLRP3 has a better diagnostic value than traditional inflammatory factors. The Clinical Trials Registration number Z: protocol KY01-2020-11-06.