UTR Site Research Articles

Genetic Variants at PRKCG Splice and UTR Sites Promote Cancer Susceptibility by Disrupting Epigenetic and miRNA Regulatory Network

Genetic Variants at PRKCG Splice and UTR Sites Promote Cancer Susceptibility by Disrupting Epigenetic and miRNA Regulatory Network

A novel variant in the 3′ UTR of the TCF4 gene likely causes Pitt-Hopkins syndrome: a case report

BackgroundPitt–Hopkins syndrome (PTHS) is a rare neurodevelopmental disorder that results from variants of TCF4 gene. PTHS follows an autosomal dominant inheritance pattern and the underlying pathological mechanisms of this disease are still unclear.MethodsWhole-genome sequencing (WGS) was conducted to screen for potential pathogenic variant in a boy highly suspected of having a genetic disorder. PCR and Sanger sequencing were used to verify the effects of the variant. Serum TCF4 levels were measured by ELISA.ResultsWe present a 4-year and 3-month-old Chinese boy clinically and molecularly diagnosed with PTHS. The proband experienced global development delay, and the preliminary clinical diagnosis was cerebral palsy. WGS identified a de novo heterozygous variant: c.*1A > G in the 3’UTR of the TCF4 gene as a potential cause of his condition. The variant was verified to cause aberrant mRNA splicing by PCR and the aberrant splicing was confirmed by Sanger sequencing.ConclusionThe study identified and demonstrated the pathogenicity of a novel 3’UTR site TCF4 variant for the first time. This research enhances understanding of pathogenetic mechanisms of PTHS and aids genetic counseling and diagnosis.

Differential expression of alpha-synuclein in the hippocampus of SHR and SLA16 isogenic rat strains

Two inbred strains, Lewis (LEW) and Spontaneously Hypertensive Rats (SHR), are well-known for their contrasting behavior related to anxiety/emotionality. Studies with these two strains led to the discovery of the Quantitative Trait Loci (QTL) on chromosome 4 (Anxrr16). To better understand the influences of this genomic region, the congenic rat strain SLA16 (SHR.LEW-Anxrr16) was developed. SLA16 rats present higher hyperactivity/impulsivity, deficits in learning and memory, and lower basal blood pressure than the SHR strain, even though genetic differences between them are only in chromosome 4. Thus, the present study proposed the alpha-synuclein and the dopaminergic system as candidates to explain the differential behavior of SHR and SLA16 strains. To accomplish this, beyond the behavioral analysis, we performed (I) the Snca gene expression and (II) quantification of the alpha-synuclein protein in the hippocampus (HPC), prefrontal cortex (PFC), and striatum (STR) of SHR and SLA16 strains; (III) sequencing of the 3′UTR of the Snca gene; and (IV) evaluation of miRNA binding in the 3′UTR site. A Single Nucleotide Polymorphism (SNP) was identified in the 3′UTR of the Snca gene, which exhibited upregulation in the HPC of SHR compared to SLA16 females. Alpha-synuclein protein was higher in the HPC of SHR males compared to SLA16 males. The results of this work suggested that differences in alpha-synuclein HPC content could be influenced by miRNA regulation and associated with behavioral differences between SHR and SLA16 animals.

ESOMIR: a curated database ofbiomarker genes andmiRNAs associated with esophageal cancer.

'Esophageal cancer' (EC) is a highly aggressive and deadly complex disease. It comprises two types, esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), with Barrett's esophagus (BE) being the only known precursor. Recent research has revealed that microRNAs (miRNAs) play a crucial role in the development, prognosis and treatment of EC and are involved in various human diseases. Biological databases have become essential for cancer research as they provide information on genes, proteins, pathways and their interactions. These databases collect, store and manage large amounts of molecular data, which can be used to identify patterns, predict outcomes and generate hypotheses. However, no comprehensive database exists for EC and miRNA relationships. To address this gap, we developed a dynamic database named 'ESOMIR (miRNA in esophageal cancer) (https://esomir.dqweilab-sjtu.com)', which includes information about targeted genes and miRNAs associated with EC. The database uses analysis and prediction methods, including experimentally endorsed miRNA(s) information. ESOMIR is a user-friendly interface that allows easy access to EC-associated data by searching for miRNAs, target genes, sequences, chromosomal positions and associated signaling pathways. The search modules are designed to provide specific data access to users based on their requirements. Additionally, the database provides information about network interactions, signaling pathways and region information of chromosomes associated with the 3'untranslated region (3'UTR) or 5'UTR and exon sites. Users can also access energy levels of specific miRNAs with targeted genes. A fuzzy term search is included in each module to enhance the ease of use for researchers. ESOMIR can be a valuable tool for researchers and clinicians to gain insight into EC, including identifying biomarkers and treatments for this aggressive tumor. Database URL https://esomir.dqweilab-sjtu.com.

A-to-I RNA editing shows dramatic up-regulation in osteosarcoma and broadly regulates tumor-related genes by altering microRNA target regions.

A-to-I RNA editing is a prevalent type of RNA modification in animals. The dysregulation of RNA editing has led to multiple human cancers. However, the role of RNA editing has never been studied in osteosarcoma, a complex bone cancer with unknown molecular basis. We retrieved the RNA-sequencing data from 24 primary osteosarcoma patients and 3 healthy controls. We systematically profiled the RNA editomes in these samples and quantitatively identified reliable differential editing sites (DES) between osteosarcoma and normal samples. RNA editing efficiency is dramatically increased in osteosarcoma, presumably due to the significant up-regulation of editing enzymes ADAR1 and ADAR2. Up-regulated DES in osteosarcoma are enriched in 3'UTRs. Strikingly, such 3'UTR sites are further enriched in microRNA binding regions of gene EMP2 and other oncogenes, abolishing the microRNA suppression on target genes. Accordingly, the expression of these tumor-promoting genes is elevated in osteosarcoma. There might be an RNA editing-dependent pathway leading to osteosarcoma. We expanded our knowledge on the potential roles of RNA editing in oncogenesis. Based on these molecular features, our work is valuable for future prognosis and diagnosis of osteosarcoma.

Accelerated Apoptosis and Down-Regulated FMRP in Human Neuroblastoma Cells with CRISPR/Cas9 Genome Editing.

Fragile X syndrome (FXS) is a genetic disease with intellectual disabilities. FXS is often caused by the CGG-repeat expansion mutation in the FMR1 gene with suppressed FMR1 transcription and decreased protein levels in the brain of the patients. The RNA-guided CRISPR/Cas9 system is a promising targeted genomic editing tool in gene therapy of FXS. In order to evaluate its feasibility, the present study used CRISPR/Cas9 system to target the FMR1 5'-UTR sites in cultured human neuroblastoma cells. PCR and DNA clone were used to construct plasmids. CRISPR function was tested by Western blot and flow cytometry. Data were analyzed by a two-tailed unpaired Student's t-test using GraphPad software. This research was conducted from 2020 to 2022 in the Second Affiliated Hospital of Soochow University, Suzhou, China. Cell cycle analysis showed significant differences in G1, S and G2/M phases between the two groups (P<0.05). In the knockout cells, apoptosis was accelerated (P<0.05) with a significantly down-regulated (P<0.05) expression of FMRP as compared with the control group. This study provides further understanding about the FMRP function and molecular mechanism of FMR1 gene in nerve cells, and suggests the feasibility of gene therapy in FXS by CRISPR/Cas9 gene editing system.

Alternative polyadenylation alters protein dosage by switching between intronic and 3'UTR sites.

Alternative polyadenylation (APA) creates distinct transcripts from the same gene by cleaving the pre-mRNA at poly(A) sites that can lie within the 3' untranslated region (3'UTR), introns, or exons. Most studies focus on APA within the 3'UTR; however, here, we show that CPSF6 insufficiency alters protein levels and causes a developmental syndrome by deregulating APA throughout the transcript. In neonatal humans and zebrafish larvae, CPSF6 insufficiency shifts poly(A) site usage between the 3'UTR and internal sites in a pathway-specific manner. Genes associated with neuronal function undergo mostly intronic APA, reducing their expression, while genes associated with heart and skeletal function mostly undergo 3'UTR APA and are up-regulated. This suggests that, under healthy conditions, cells toggle between internal and 3'UTR APA to modulate protein expression.

The Somatic Mutation Landscape of UDP-Glycosyltransferase (UGT) Genes in Human Cancers.

The human UDP-glycosyltransferase (UGTs) superfamily has a critical role in the metabolism of anticancer drugs and numerous pro/anti-cancer molecules (e.g., steroids, lipids, fatty acids, bile acids and carcinogens). Recent studies have shown wide and abundant expression of UGT genes in human cancers. However, the extent to which UGT genes acquire somatic mutations within tumors remains to be systematically investigated. In the present study, our comprehensive analysis of the somatic mutation profiles of 10,069 tumors from 33 different TCGA cancer types identified 3427 somatic mutations in UGT genes. Overall, nearly 18% (1802/10,069) of the assessed tumors had mutations in UGT genes with huge variations in mutation frequency across different cancer types, ranging from over 25% in five cancers (COAD, LUAD, LUSC, SKCM and UCSC) to less than 5% in eight cancers (LAML, MESO, PCPG, PAAD, PRAD, TGCT, THYM and UVM). All 22 UGT genes showed somatic mutations in tumors, with UGT2B4, UGT3A1 and UGT3A2 showing the largest number of mutations (289, 307 and 255 mutations, respectively). Nearly 65% (2260/3427) of the mutations were missense, frame-shift and nonsense mutations that have been predicted to code for variant UGT proteins. Furthermore, about 10% (362/3427) of the mutations occurred in non-coding regions (5' UTR, 3' UTR and splice sites) that may be able to alter the efficiency of translation initiation, miRNA regulation or the splicing of UGT transcripts. In conclusion, our data show widespread somatic mutations of UGT genes in human cancers that may affect the capacity of cancer cells to metabolize anticancer drugs and endobiotics that control pro/anti-cancer signaling pathways. This highlights their potential utility as biomarkers for predicting therapeutic efficacy and clinical outcomes.

Regulation of Iron-Ion Transporter SLC11A2 by Three Identical miRNAs.

Here, we searched for microRNAs (miRNAs) in silico that could interact with SLC11A2 mRNA, a solute carrier (SLC) iron-ion transporter, and investigated their effects on SLC11A2 gene expression using the cultured human colon carcinoma cell line, Caco-2. In silico analysis using the miRWalk2.0 database revealed that several types of miRNAs interact with the human SLC11A2 gene; we focused on three miRNAs, miR-149-5p, miR-362-5p, and miR-539-5p as candidates in this study. We first revealed that the three miRNAs interact with the SLC11A2 3'-untranslated region (3'-UTR) using a luciferase assay in a Caco-2 cell line. We then examined whether the expression of each miRNA affected the expression of SLC11A2 mRNAs and their transcribed transporter proteins. We found transiently expressed miRNAs significantly reduced the reporter activity of the SLC11A2 3'-UTR site in Caco-2 cells by significantly decreasing the SLC11A2 gene and protein expression in the miRNA-transfected Caco-2 cells. Subsequently, we investigated the effects of these miRNAs on SLC11A2's iron-ion transporting activity by measuring iron-ion concentration in Caco-2 cells. Administration of ammonium iron (II) sulfate hexahydrate to Caco-2 cells significantly increased the intracellular iron-ion concentration. However, in iron-ion-pretreated cells, overexpression of each of the three miRNAs resulted in decreased intracellular iron-ion concentration. This indicated that overexpressed miRNAs inhibited iron-ion influx into Caco-2 cells by attenuating SLC11A2 transporting activity. Using in silico analysis, we predicted that three studied miRNAs could bind to the iron-ion influx transporter SLC11A2 and revealed that they regulate SLC11A2 gene expression and iron-ion transporting function in an in vitro system.

Congested Heart Failure in Small Breeds Dogs

Mitral valve disease, commonly known as MVD, is the most common heart disease in dogs, accounting for nearly 75% of all dogs affected. The characteristic of this predominant disease includes collagen degradation, elastic fiber fragmentation and proteoglycan accumulation, leading to deterioration of the mitral valve and ultimately, congestive heart failure (CHF). Treatment to reverse or even delay the damaging effects of MVD has not yet been discovered due to a lack of awareness of the underlying processes. Furthermore, this disease in dogs has many similarities with the human MVD and may potentially serve as a translational model for studying MVD further. Therefore, it is imperative to identify the causes and underlying mechanisms associated with canine MVD in order to treat these patients more efficiently at the earliest detectable stage. MicroRNAs (miRNAs) are small, 21‐25 nucleotides in length, non‐coding RNAs that can down‐regulate the expression of protein coding genes by binding to the 3’UTR sites in their messenger RNAs (mRNA), causing either degradation or gene silencing. Prior studies have shown that one miRNA may regulate multiple mRNAs, subsequently effecting the downstream levels of gene expression. Furthermore, if miRNAs prove to be a useful biomarker of heart disease, not only may they serve as detectors of early disease onset, but they may also be useful as therapeutic agents. Given this recent data, we propose to test the hypothesis that circulating miRNAs may be useful biomarkers for detection of CHF, secondary to MVD. Liquid biopsies (blood samples) were collected in EDTA tubes from 7 healthy dogs with no prior history of heart disease and 7 dogs being treated for CHF secondary to MVD. Total serum RNA was isolated using miRNeasy Serum/Plasma Kit (Qiagen). Isolated RNA was then reverse transcribed using miScript II RT Kit (Qiagen) and lastly, miRNA was quantified using miScript SYBR Green PCR Kit, along with miScript miRNA PCR Arrays (Qiagen).

Regulation of Methylase METTL3 on Fat Deposition.

N6-methyladenosine (m6A) is the most prevalent and abundant type of internal post-transcriptional RNA modification in eukaryotic cells. METTL3 is a methylation modifying enzyme, which can directly or indirectly affect biological processes, such as RNA degradation, translation and splicing. In addition, it was found that 67% of 3’-UTR regions containing m6A sites had at least one miRNA binding site, and the number of m6A at 3’-UTR sites was closely related to the binding sites of miRNA. With the improvement of human living standards, obesity has become a very serious and urgent problem. The essence of obesity is the accumulation of excess fat. Exploring the origin and development mechanisms of adipocyte from the perspective of fat deposition has always been a hotspot in the field of adipocyte research. The aim of the present review is to focus on METTL3 regulating fat deposition through mRNA/adipocyte differentiation axis and pri-miRNA/pre-miRNA/target genes/adipocyte differentiation and to provide a theoretical basis according to the currently available literature for further exploring this association. This review may provide new insights for obesity, fat deposition disease and molecular breeding.

Identification of deleterious single nucleotide polymorphism (SNP)s in the human TBX5 gene & prediction of their structural & functional consequences: An in silico approach

T-box transcription factor 5 gene (TBX5) encodes the transcription factor TBX5, which plays a crucial role in the development of heart and upper limbs. Damaging single nucleotide variants in this gene alter the protein structure, disturb the functions of TBX5, and ultimately cause Holt-Oram Syndrome (HOS). By analyzing the available single nucleotide polymorphism information in the dbSNP database, this study was designed to identify the most deleterious TBX5 SNPs through insilico approaches and predict their structural and functional consequences.Fifty-eight missense substitutions were found damaging by sequence homology-based tools: SIFT and PROVEAN, and structure homology-based tool PolyPhen-2. Various disease association meta-predictors further scrutinized these SNPs. Additionally, conservation profile of the amino acid residues, their surface accessibility, stability, and structural integrity of the native protein upon mutations were assessed. From these analyses, finally 5 SNPs were detected as the most damaging ones: [rs1565941579 (P85S), rs1269970792 (W121R), rs772248871 (V153D), rs769113870 (E208D), and rs1318021626 (I222N)]. Analyses of stop-lost, nonsense, UTR, and splice site SNPs were also conducted.Through integrative bioinformatics analyses, this study has identified the SNPs that are deleterious to the TBX5 protein structure and have the potential to cause HOS. Further wet-lab experiments can validate these findings.

LncRNA FBXL19-AS1 promotes proliferation and metastasis of cervical cancer through upregulating COL1A1 as a sponge of miR-193a-5p

BackgroundCervical cancer (CC) is one of the most common and malignant tumors in women. In this study, we aim to explore the role and mechanism of F-box and leucine rich repeat protein 19 antisense RNA 1 (FBXL19-AS1), a novel long-chain non coding RNA (lncRNA) with marked roles in a variety of tumors, in regulating the proliferation and metastasis of CC.MethodsThe expression of FBXL19-AS1, miR-193a-5p and COL1A1 were detected by RT-PCR and western blot. Gain- and loss-of functional assays of FBXL19-AS1 and miR-193a-5p were performed in CC cell lines in vitro or in vivo. The proliferation, migration, invasion, apoptosis and epithelial-mesenchymal transition (EMT) of CC cells were determined.ResultsFBXL19-AS1 and COL1A1 were significantly up-regulated in CC tissues, while miR-193a-5p was significantly down-regulated. Overexpression of FBXL19-AS1 significantly promoted the proliferation, migration, invasion, EMT and growth of CC cells and inhibited apoptosis, while knockdown of FBXL19-AS1 had the opposite effects. On the other hand, miR-193a-5p inhibited the proliferation and metastasis of CC cells. Mechanistically, FBXL19-AS1 functioned as a competitive endogenous RNA (ceRNA) and inhibited the expression of miR-193a-5p, which targeted at the 3’-UTR site of COL1A1 and negatively regulated COL1A1 expression.ConclusionsFBXL19-AS1 promotes the proliferation and metastasis of CC cells by sponging miR-193a-5p and up-regulating COL1A1.

ELAVL1 primarily couples mRNA stability with the 3' UTRs of interferon-stimulated genes.

SUMMARYUpon pathogen detection, the innate immune system triggers signaling events leading to upregulation of pro-inflammatory and anti-microbial mRNA transcripts. RNA-binding proteins (RBPs) interact with these critical mRNAs and regulate their fates at the post-transcriptional level. One such RBP is ELAVL1. Although significant progress has been made in understanding how embryonic lethal vision-like protein 1 (ELAVL1) regulates mRNAs, its target repertoire and binding distribution within an immunological context remain poorly understood. We overlap four high-throughput approaches to define its context-dependent targets and determine its regulatory impact during immune activation. ELAVL1 transitions from binding overwhelmingly intronic sites to 3′ UTR sites upon immune stimulation of cells, binding previously and newly expressed mRNAs. We find that ELAVL1 mediates the RNA stability of genes that regulate pathways essential to pathogen sensing and cytokine production. Our findings reveal the importance of examining RBP regulatory impact under dynamic transcriptomic events to understand their post-transcriptional regulatory roles within specific biological circuitries.

Abstract 12: The Etiology Of Inverse Salt Sensitivity Of Blood Pressure: Mirna-485-5p Binds To The Dopamine Type 2 Receptor (d 2 R) Snp Rs6276 And Decreases D 2 R Expression

Inverse salt sensitivity is the paradoxical increase in blood pressure of individuals to a low sodium diet. rs6276, a single nucleotide polymorphism found in the 3’ untranslated region of the D2R gene (DRD2), which is associated with decreased expression and is also associated with the inverse salt sensitive phenotype (ISS). Urine derived renal proximal tubule cells grown from ISS participants with homozygous rs6276 SNPs vs salt resistant (SR) wild type for rs6276 SNPs have decreased expression of D2R as measured by receptor binding studies using BodipyFL-Spiperone (100 nM for 2 hrs, -36.9% ± 2.6% reduction in ISS-10 vs SR-6, n=5, p&lt;0.01). miRNA-485-5p potentially binds to the rs6276 SNP in the 3’ UTR site and therefore could be a member of a newly identified classification of SNPs called miRSNPs. We transfected a miRNA-485-5p miRNA mimic and a miRNA blocker (20 nM for 72 hrs) to determine the effect on D2R expression on both wild type and homozygous variant SNP participant cell lines. Transfection of mir-485-5p only alters the expression of D2R in the ISS cell lines with SNPs (52619 ± 2001 RFU ISS control miRNA vs 69496 ± 2108 RFU ISS-10 miRNA blocker and vs 30434 ± 1824 RFU ISS-10 miRNA mimic, n=9, p&lt;0.01, one-way ANOVA). The miRNA-485-5p blocker was able to return the D2R expression levels back to levels found in SR cells. Further studies are necessary to determine if the miRNA-485-5p mimic enhances and miRNA-485-5p blocker reverses any of the ISS cell phenotypes identified.

DAZL mediates a broad translational program regulating expansion and differentiation of spermatogonial progenitors.

Fertility across metazoa requires the germline-specific DAZ family of RNA-binding proteins. Here we examine whether DAZL directly regulates progenitor spermatogonia using a conditional genetic mouse model and in vivo biochemical approaches combined with chemical synchronization of spermatogenesis. We find that the absence of Dazl impairs both expansion and differentiation of the spermatogonial progenitor population. In undifferentiated spermatogonia, DAZL binds the 3' UTRs of ~2,500 protein-coding genes. Some targets are known regulators of spermatogonial proliferation and differentiation while others are broadly expressed, dosage-sensitive factors that control transcription and RNA metabolism. DAZL binds 3' UTR sites conserved across vertebrates at a UGUU(U/A) motif. By assessing ribosome occupancy in undifferentiated spermatogonia, we find that DAZL increases translation of its targets. In total, DAZL orchestrates a broad translational program that amplifies protein levels of key spermatogonial and gene regulatory factors to promote the expansion and differentiation of progenitor spermatogonia.

MiRNAs and Neural Alternative Polyadenylation Specify the Virgin Behavioral State.

How are diverse regulatory strategies integrated to impose appropriately patterned gene expression that underlie invivo phenotypes? Here, we reveal how coordinated miRNA regulation and neural-specific alternative polyadenylation (APA) of a single locus controls complex behaviors. Our entry was the unexpected observation that deletion of Bithorax complex (BX-C) miRNAs converts virgin female flies into a subjective post-mated behavioral state, normally induced by seminal proteins following copulation. Strikingly, this behavioral switch is directly attributable to misregulation of homothorax (hth). We localize specific CNS abdominal neurons where de-repressed Hth compromises virgin behavior in BX-C miRNA mutants. Moreover, we use genome engineering to demonstrate that precise mutation of hth 3' UTR sites for BX-C miRNAs or deletion of its neural 3' UTR extension containing most of these sites both induce post-mated behaviors in virgins. Thus, facilitation of miRNA-mediated repression by neural APA is required for virgin females to execute behaviors appropriate to their internal state.

Dexmedetomidine had neuroprotective effects on hippocampal neuronal cells via targeting lncRNA SHNG16 mediated microRNA-10b-5p/BDNF axis

Dexmedetomidine (DEX), a highly selective alpha2 adrenergic receptor agonist, is a commonly used anesthetic drug in surgical procedures. Previous studies have indicated that DEX exerts neuroprotective effects while the detailed mechanism has not been fully elucidated. Here, we aim to study the role of lncRNA SHNG16 in DEX-induced brain protection and its underlying molecular mechanism. The rats underwent middle cerebral artery occlusion (MCAO) surgery and oxygen–glucose deprivation (OGD)-treated HT22 hippocampal neurons were treated with DEX, respectively. CCK8 was used to evaluate cell viability. sh-SHNG16 as well as miR-10b-5p mimics were transfected into hippocampal neurons to further explore the bio-function of SNHG16 and miR-10b-5p in vitro. Furthermore, the interactions between SHNG16 and miR-10b-5p, miR-10b-5p and BDNF gene were confirmed by dual-luciferase report assay. Our data revealed that DEX attenuated neurological damage of the MCAO rats and also increased the cell viability of the neurons significantly. Besides, expression of SHNG16 and BDNF were both downregulated while miR-10b-5p was upregulated in MCAO brain tissues or OGD treated neurons. DEX inhibited miR-10b-5p expression but increased SHNG16 and BDNF levels with a dosage effect. After transfection with sh-SHNG16 or miR-10b-5p mimics, the expression of BDNF protein was downregulated, accompanied with decreased neuron viability. Dual-luciferase assay showed that SHNG16 targeted on miR-10b-5p, which also could bind directly to the 3′-UTR sites of BDNF and negatively regulate its expression. In conclusion, DEX exerts neuroprotective in ischemic stroke via improving neuron damage, the underlying mechanism may be upregulating SHNG16 and BDNF via sponging miR-10b-5p.

MicroRNAs as a Potential Biomarker in Feline Kidney Disease

Kidney failure is a very common disease in felines, more so than many may have thought. Due to there being very little research on kidney failure in felines there is not much understanding to what causes it and how it can be prevented. Since this disease in felines is quite similar to kidney failure in humans, this research may benefit further research on kidney failure. Therefore, it is important to identify the causes of kidney failure in felines in order to treat all patients at the earliest detectable stage. MicroRNAs are small, non‐coding RNAs that can down‐regulate the expression of protein coding genes by binding to the 3′UTR sites in their messenger RNAs. Prior research shows that kidney failure can be detected by microRNAs, making microRNAs a useful biomarker to kidney failure. The microRNAs may also help detect the early stages of kidney failure in felines making it easier to diagnose this disease. Our proposal is to test that circulating microRNAs may be useful biomarkers for detection of kidney failure. Liquid biopsies, blood samples, were collected in EDTA tubes from 8 healthy felines with no prior history of kidney failure and 8 felines who were diagnosed with kidney failure. Total serum RNS was isolated using miRNeasy Serum/Plasma kit. Isolated RNA was then reverse transcribed using miScript II RT Kit and lastly, miRNA was quantified using miScript SYBR green PCR Kit, along with miScript miRNA PCR Arrays.

M6A modification of a 3\u2032 UTR site reduces RME1 mRNA levels to promote meiosis

Despite the vast number of modification sites mapped within mRNAs, known examples of consequential mRNA modifications remain rare. Here, we provide multiple lines of evidence to show that Ime4p, an N6-methyladenosine (m6A) methyltransferase required for meiosis in yeast, acts by methylating a site in the 3′ UTR of the mRNA encoding Rme1p, a transcriptional repressor of meiosis. Consistent with this mechanism, genetic analyses reveal that IME4 functions upstream of RME1. Transcriptome-wide, RME1 is the primary message that displays both increased methylation and reduced expression in an Ime4p-dependent manner. In yeast strains for which IME4 is dispensable for meiosis, a natural polymorphism in the RME1 promoter reduces RME1 transcription, obviating the requirement for methylation. Mutation of a single m6A site in the RME1 3′ UTR increases Rme1p repressor production and reduces meiotic efficiency. These results reveal the molecular and physiological consequences of a modification in the 3′ UTR of an mRNA.