Selection and validation of reference genes for normalisation of gene expression in ischaemic and toxicological studies in kidney disease.

Normalisation to standard reference gene(s) is essential for quantitative real-time polymerase chain reaction (RT-qPCR) to obtain reproducible and comparable results of a gene of interest (GOI) between subjects and under varying experimental conditions. There is limited evidence to support selection of the commonly used reference genes in rat ischaemic and toxicological kidney models. Employing these models, we determined the most stable reference genes by comparing 4 standard methods (NormFinder, qBase+, BestKeeper and comparative ΔCq) and developed a new 3-way linear mixed-effects model for evaluation of reference gene stability. This new technique utilises the intra-class correlation coefficient as the stability measure for multiple continuous and categorical covariates when determining the optimum normalisation factor. The model also determines confidence intervals for each candidate normalisation gene to facilitate selection and allow sample size calculation for designing experiments to identify reference genes. Of the 10 candidate reference genes tested, the geometric mean of polyadenylate-binding nuclear protein 1 (PABPN1) and beta-actin (ACTB) was the most stable reference combination. In contrast, commonly used ribosomal 18S and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were the most unstable. We compared the use of PABPN1×ACTB and 2 commonly used genes 18S and GAPDH on the expression of 4 genes of interest know to vary after renal injury and expressed by different kidney cell types (KIM-1, HIF1α, TGFß1 and PECAM1). The less stable reference genes gave varying patterns of GOI expression in contrast to the use of the least unstable reference PABPN1×ACTB combination; this improved detection of differences in gene expression between experimental groups. Reduced within-group variation of the now more accurately normalised GOI may allow for reduced experimental group size particularly for comparison between various models. This objective selection of stable reference genes increased the reliability of comparisons within and between experimental groups.

Transthyretin Upregulates Long Non-Coding RNA MEG3 by Affecting PABPC1 in Diabetic Retinopathy.

The aim of the study was to demonstrate how transthyretin (TTR) could affect long non-coding RNA (lncRNA) of maternally expressed gene 3 (MEG3) and play important roles in diabetic retinopathy (DR). A DR model in C57BL/6 mice was established after intraperitoneal injection of streptozotocin (STZ). After intravitreal injection with TTR pAAV vector, MEG3 short hairpin RNA (shRNA), scrambled shRNA, or MEG3, retinal imaging, retinal trypsin digestion, and fundus vascular permeability tests were performed. Cell counting kit-8 (CCK8), transwell, and Matrigel assays were employed to detect the proliferation and migration of human retinal microvascular endothelial cells (hRECs). The binding between long non-coding RNA of maternally expressed gene 3 (lncRNA-MEG3) and microRNA-223-3p (miR-223-3p) was observed by using luciferase reporter assays, while co-immunoprecipitation (co-IP) was employed to confirm the interaction between TTR and the target. In the DR mice model, retinal vascular leakage and angiogenesis were repressed by overexpressing TTR. In vitro, the added TTR promoted the level of lncRNA-MEG3 by interacting with poly (A) binding protein cytoplasmic 1 (PABPC1), and then repressed proliferation and angiogenesis of hRECs. In vivo, silencing or overexpressing lncRNA-MEG3 significantly affected retinal vascular phenotypes. Additionally, the interaction between lncRNA-MEG3 and miR-223-3p was confirmed, and silencing of miR-223-3p revealed similar effects on hRECs as overexpression of lncRNA-MEG3. In summary, in the DR environment, TTR might affect the lncRNA MEG3/miR-223-3p axis by the direct binding with PABPC1, and finally repress retinal vessel proliferation.

Cytokine genes as potential biomarkers for muscle weakness in OPMD.

Molecular biomarkers emerge as an accurate diagnostic tool, but are scarce for myopathies. Lack of outcome measures sensitive to disease onset and symptom severity hamper evaluation of therapeutic developments. Cytokines are circulating immunogenic molecules, and their potential as biomarkers has been exploited in the last decade. Cytokines are released from many tissues, including skeletal muscles, but their application to monitor muscle pathology is sparse. We report that the cytokine functional group is altered in the transcriptome of oculopharyngeal muscular dystrophy (OPMD). OPMD is a dominant, late-onset myopathy, caused by an alanine-expansion mutation in the gene encoding for poly(A) binding protein nuclear 1 (expPABPN1). Here, we investigated the hypothesis that cytokines could mark OPMD disease state. We determined cytokines levels the vastus lateralis muscle from genetically confirmed expPABPN1 carriers at a symptomatic or a presymptomatic stage. We identified cytokine-related genes candidates from a transcriptome study in a mouse overexpressing exp PABPN1 Six cytokines were found to be consistently down-regulated in OPMD vastus lateralis muscles. Expression levels of these cytokines were highly correlated in controls, but this correlation pattern was disrupted in OPMD. The levels of these 6 cytokines were not altered in expPABPN1 carriers at a pre-symptomatic stage, suggesting that this group of cytokines is a potential biomarker for muscle weakness in OPMD. Correlation pattern of expression levels could be a novel measurer for disease state.

PABPN1-Dependent mRNA Processing Induces Muscle Wasting.

Poly(A) Binding Protein Nuclear 1 (PABPN1) is a multifunctional regulator of mRNA processing, and its expression levels specifically decline in aging muscles. An expansion mutation in PABPN1 is the genetic cause of oculopharyngeal muscle dystrophy (OPMD), a late onset and rare myopathy. Moreover, reduced PABPN1 expression correlates with symptom manifestation in OPMD. PABPN1 regulates alternative polyadenylation site (PAS) utilization. However, the impact of PAS utilization on cell and tissue function is poorly understood. We hypothesized that altered PABPN1 expression levels is an underlying cause of muscle wasting. To test this, we stably down-regulated PABPN1 in mouse tibialis anterior (TA) muscles by localized injection of adeno-associated viruses expressing shRNA to PABPN1 (shPab). We found that a mild reduction in PABPN1 levels causes muscle pathology including myofiber atrophy, thickening of extracellular matrix and myofiber-type transition. Moreover, reduced PABPN1 levels caused a consistent decline in distal PAS utilization in the 3'-UTR of a subset of OPMD-dysregulated genes. This alternative PAS utilization led to up-regulation of Atrogin-1, a key muscle atrophy regulator, but down regulation of proteasomal genes. Additionally reduced PABPN1 levels caused a reduction in proteasomal activity, and transition in MyHC isotope expression pattern in myofibers. We suggest that PABPN1-mediated alternative PAS utilization plays a central role in aging-associated muscle wasting.

Systematic profiling of poly(A)+ transcripts modulated by core 3' end processing and splicing factors reveals regulatory rules of alternative cleavage and polyadenylation.

Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3' untranslated regions (3'UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3'UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5' end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors.

Mitochondrial dysfunction reveals the role of mRNA poly(A) tail regulation in oculopharyngeal muscular dystrophy pathogenesis.

Oculopharyngeal muscular dystrophy (OPMD), a late-onset disorder characterized by progressive degeneration of specific muscles, results from the extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice are established, the molecular mechanisms behind OPMD remain undetermined. Here, we show, using Drosophila and mouse models, that OPMD pathogenesis depends on affected poly(A) tail lengths of specific mRNAs. We identify a set of mRNAs encoding mitochondrial proteins that are down-regulated starting at the earliest stages of OPMD progression. The down-regulation of these mRNAs correlates with their shortened poly(A) tails and partial rescue of their levels when deadenylation is genetically reduced improves muscle function. Genetic analysis of candidate genes encoding RNA binding proteins using the Drosophila OPMD model uncovers a potential role of a number of them. We focus on the deadenylation regulator Smaug and show that it is expressed in adult muscles and specifically binds to the down-regulated mRNAs. In addition, the first step of the cleavage and polyadenylation reaction, mRNA cleavage, is affected in muscles expressing alanine-expanded PABPN1. We propose that impaired cleavage during nuclear cleavage/polyadenylation is an early defect in OPMD. This defect followed by active deadenylation of specific mRNAs, involving Smaug and the CCR4-NOT deadenylation complex, leads to their destabilization and mitochondrial dysfunction. These results broaden our understanding of the role of mRNA regulation in pathologies and might help to understand the molecular mechanisms underlying neurodegenerative disorders that involve mitochondrial dysfunction.

Epab and Pabpc1 are differentially expressed in the postnatal mouse ovaries.

PURPOSE: Embryonic poly(A)-binding protein (EPAB) and poly(A)-binding protein, cytoplasmic 1 (PABPC1) bind poly(A) tails of mRNAs and mediate their translational regulation in germ cells and early preimplantation embryos. Although expression patterns and possible functions of the Epab and Pabpc1 genes have been examined in vertebrate germ cells and early embryos, their expression levels and cellular localizations in the postnatal mouse ovaries remained elusive. METHODS: In the present study, we first aimed to characterize expression levels of the Epab and Pabpc1 genes in the prepubertal (1-, 2-, and 3-week old), pubertal (4-, 5-, and 6-week old), postpubertal (16-week and 18-week old), and aged (52-, 60-, and 72-week old) mouse ovaries by using quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Epab mRNA was predominantly expressed in the prepubertal ovaries when compared to later developmental periods. However, Pabpc1 transcript was highly generated in the prepubertal and pubertal mouse ovaries except for 1-week old ovary than those of other developmental terms. In the prepubertal mouse ovaries, RNA in situ hybridization localized both Epab and Pabpc1 transcripts in the cytoplasm of oocytes and granulosa cells of all follicular stages. Consistently, Epab and Pabpc1 gene expression were detected in the cumulus cells and MII oocytes obtained from cumulus oocyte complexes (COCs). Ovarian follicle counting in the postnatal ovaries revealed that total number of follicles was higher in the prepubertal ovaries in comparison with later stages of development. CONCLUSION: As a result, Epab and Pabpc1 expression exhibit differences at postnatal ovary development stages and both genes are transcribed in the granulosa cells and oocytes. These findings suggest that EPAB may predominantly play roles in translational regulation of the mRNAs during early oogenesis and folliculogenesis, but PABPC1 most likely perform these roles in the later terms of ovarian development along with EPAB protein.

Epab and Pabpc1 are differentially expressed during male germ cell development.

Modification of poly(A) tail length constitutes the main posttranscriptional mechanism by which gene expression is regulated during spermatogenesis. Embryonic poly(A)-binding protein (EPAB) and somatic cytoplasmic poly(A)-binding protein (PABPC1) are the 2 key proteins implicated in this pathway. In this study we characterized the temporal and spatial expression of Epab and Pabpc1 in immature (D6-D32) and mature (D88) mouse testis and in isolated spermatogenic cells. Both Epab and Pabpc1 expression increased during early postnatal life and reached their peak at D32 testis. This was due to an increase in both spermatogonia (SG) and spermatocytes. In the mature testis, the highest levels of Epab were detected in SG, followed by round spermatids (RSs), while the most prominent Pabpc1 expression was detected in spermatocytes and RSs. Our findings suggest that PABPC1 may play a role in translational regulation of gene expression by cytoplasmic polyadenylation, which occurs in spermatocytes, while both EPAB and PABPC1 may help stabilize stored polyadenylated messenger RNAs in RSs.

Transgenic expression of an expanded (GCG)13 repeat PABPN1 leads to weakness and coordination defects in mice.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder caused by a (GCG)n trinucleotide repeat expansion in the poly(A) binding protein nuclear-1 (PABPN1) gene, which in turn leads to an expanded polyalanine tract in the protein. We generated transgenic mice expressing either the wild type or the expanded form of human PABPN1, and transgenic animals with the expanded form showed clear signs of abnormal limb clasping, muscle weakness, coordination deficits, and peripheral nerves alterations. Analysis of mitotic and postmitotic tissues in those transgenic animals revealed ubiquitinated PABPN1-positive intranuclear inclusions (INIs) in neuronal cells. This latter observation led us to test and confirm the presence of similar INIs in postmortem brain sections from an OPMD patient. Our results indicate that expanded PABPN1, presumably via the toxic effects of its polyalanine tract, can lead to inclusion formation and neurodegeneration in both the mouse and the human.

PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function.