Knockdown of plakophilin 2 downregulates MIR-184 through CpG hypermethylation and suppression of the E2F1 pathway and leads to enhanced adipogenesis in vitro

Rationale: PKP2, encoding plakophilin 2 (PKP2), is the most common causal gene for arrhythmogenic cardiomyopathy. Objective: To characterize miRNA expression profile in PKP2-deficient cells. Methods and results: Control and PKP2-knockdown HL-1 (HL-1(Pkp2-shRNA)) cells were screened for 750 miRNAs using low-density microfluidic panels. Fifty-nine miRNAs were differentially expressed. MiR-184 was the most downregulated miRNA. Expression of miR-184 in the heart and cardiac myocyte was developmentally downregulated and was low in mature myocytes. MicroRNA-184 was predominantly expressed in cardiac mesenchymal progenitor cells. Knockdown of Pkp2 in cardiac mesenchymal progenitor cells also reduced miR-184 levels. Expression of miR-184 was transcriptionally regulated by the E2F1 pathway, which was suppressed in PKP2-deficient cells. Activation of E2F1, on overexpression of its activator CCND1 (cyclin D1) or knockdown of its inhibitor retinoblastoma 1, partially rescued miR-184 levels. In addition, DNA methyltransferase-1 was recruited to the promoter region of miR-184, and the CpG sites at the upstream region of miR-184 were hypermethylated. Treatment with 5-aza-2'-deoxycytidine, a demethylation agent, and knockdown of DNA methyltransferase-1 partially rescued miR-184 level. Pathway analysis of paired miR-184:mRNA targets identified cell proliferation, differentiation, and death as the main affected biological processes. Knockdown of miR-184 in HL-1 cells and mesenchymal progenitor cells induced and, conversely, its overexpression attenuated adipogenesis. Conclusions: PKP2 deficiency leads to suppression of the E2F1 pathway and hypermethylation of the CpG sites at miR-184 promoter, resulting in downregulation of miR-184 levels. Suppression of miR-184 enhances and its activation attenuates adipogenesis in vitro. Thus, miR-184 contributes to the pathogenesis of adipogenesis in PKP2-deficient cells

Role of forefinger and thumb loops in production of Ψ54 and Ψ55 in tRNAs by archaeal Pus10

Pseudouridines (Ψ) are found in structurally and functionally important regions of RNAs. Six families of Ψ synthases, TruA, TruB, TruD, RsuA, RluA, and Pus10 have been identified. Pus10 is present in Archaea and Eukarya. While most archaeal Pus10 produce both tRNA Ψ54 and Ψ55, some produce only Ψ55. Interestingly, human PUS10 has been implicated in apoptosis and Crohn\u27s and Celiac diseases. Homology models of archaeal Pus10 proteins based on the crystal structure of human PUS10 reveal that there are subtle structural differences in all of these Pus10 proteins. These observations suggest that structural changes in homologous proteins may lead to loss, gain, or change of their functions, warranting the need to study the structure-function relationship of these proteins. Using comparison of structural models and a series of mutations, we identified forefinger loop (reminiscent of that of RluA) and an Arg and a Tyr residue of archaeal Pus10 as critical determinants for its Ψ54, but not for its Ψ55 activity. We also found that a Leu residue, in addition to the catalytic Asp, is essential for both activities. Since forefinger loop is needed for both rRNA and tRNA Ψ synthase activities of RluA, but only for tRNA Ψ54 activity of Pus10, archaeal Pus10 proteins must use a different mechanism of recognition for Ψ55 activity. We propose that archaeal Pus10 uses two distinct mechanisms for substrate uridine recognition and binding. However, since we did not observe any mutation that affected only Ψ55 activity, both mechanisms for archaeal Pus10 activities must share some common features

BET bromodomain inhibition attenuates cardiac phenotype in myocyte-specific lamin A/C-deficient mice

Mutation in the LMNA gene, encoding lamin A/C, causes a diverse group of diseases called laminopathies. Cardiac involvement is the major cause of death and manifests as dilated cardiomyopathy, heart failure, arrhythmias, and sudden death. There is no specific therapy for LMNA-associated cardiomyopathy. We report that deletion of Lmna in cardiomyocytes in mice leads to severe cardiac dysfunction, conduction defect, ventricular arrhythmias, fibrosis, apoptosis, and premature death within 4 weeks. The phenotype is similar to LMNA-associated cardiomyopathy in humans. RNA sequencing, performed before the onset of cardiac dysfunction, led to identification of 2338 differentially expressed genes (DEGs) in Lmna-deleted cardiomyocytes. DEGs predicted activation of bromodomain-containing protein 4 (BRD4), a regulator of chromatin-associated proteins and transcription factors, which was confirmed by complementary approaches, including chromatin immunoprecipitation sequencing. Daily injection of JQ1, a specific BET bromodomain inhibitor, partially reversed the DEGs, including those encoding secretome; improved cardiac function; abrogated cardiac arrhythmias, fibrosis, and apoptosis; and prolonged the median survival time 2-fold in the myocyte-specific Lmna-deleted mice. The findings highlight the important role of LMNA in cardiomyocytes and identify BET bromodomain inhibition as a potential therapeutic target in LMNA-associated cardiomyopathy, for which there is no specific effective therapy

Identification of Genes and Pathways Regulated by Lamin A in Heart

Background Mutations in the LMNA gene, encoding LMNA (lamin A/C), causes distinct disorders, including dilated cardiomyopathies, collectively referred to as laminopathies. The genes (coding and noncoding) and regulatory pathways controlled by LMNA in the heart are not completely defined. Methods and Results We analyzed cardiac transcriptome from wild-type, loss-of-function (Lmna-/-), and gain-of-function (Lmna-/- injected with adeno-associated virus serotype 9 expressing LMNA) mice with normal cardiac function. Deletion of Lmna (Lmna-/-) led to differential expression of 2193 coding and 629 long noncoding RNA genes in the heart (q<0.05). Re-expression of LMNA in the Lmna-/- mouse heart, completely rescued 501 coding and 208 non-coding and partially rescued 1862 coding and 607 lncRNA genes. Pathway analysis of differentially expressed genes predicted activation of transcriptional regulators lysine-specific demethylase 5A, lysine-specific demethylase 5B, tumor protein 53, and suppression of retinoblastoma 1, paired-like homeodomain 2, and melanocyte-inducing transcription factor, which were completely or partially rescued upon reexpression of LMNA. Furthermore, lysine-specific demethylase 5A and 5B protein levels were increased in the Lmna-/- hearts and were partially rescued upon LMNA reexpression. Analysis of biological function for rescued genes identified activation of tumor necrosis factor-α, epithelial to mesenchymal transition, and suppression of the oxidative phosphorylation pathway upon Lmna deletion and their restoration upon LMNA reintroduction in the heart. Restoration of the gene expression and transcriptional regulators in the heart was associated with improved cardiac function and increased survival of the Lmna-/- mice. Conclusions The findings identify LMNA-regulated cardiac genes and their upstream transcriptional regulators in the heart and implicate lysine-specific demethylase 5A and B as epigenetic regulators of a subset of the dysregulated genes in laminopathies

Role of forefinger and thumb loops in production of 54 and 55 in tRNAs by archaeal Pus10

Joardar A, Jana S, Fitzek E, et al. Role of forefinger and thumb loops in production of  54 and  55 in tRNAs by archaeal Pus10. RNA. 2013;19(9):1279-1294

RNA sequencing-based transcriptome profiling of cardiac tissue Implicados novela putative disease mechanisms in FLNC-associated arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) encompasses a group of inherited cardiomyopathies including arrhythmogenic right ventricular cardiomyopathy (ARVC) whose molecular disease mechanism is associated with dysregulation of the canonical WNT signalling pathway. Recent evidence indicates that ARVC and ACM caused by pathogenic variants in the FLNC gene encoding filamin C, a major cardiac structural protein, may have different molecular mechanisms of pathogenesis. We sought to identify dysregulated biological pathways in FLNC-associated ACM. RNA was extracted from seven paraffin-embedded left ventricular tissue samples from deceased ACM patients carrying FLNC variants and sequenced. Transcript levels of 623 genes were upregulated and 486 genes were reduced in ACM in comparison to control samples. The cell adhesion pathway and ILK signalling were among the prominent dysregulated pathways in ACM. Consistent with these findings, transcript levels of cell adhesion genes JAM2, NEO1, VCAM1 and PTPRC were upregulated in ACM samples. Moreover, several actin-associated genes, including FLNC, VCL, PARVB and MYL7, were suppressed, suggesting dysregulation of the actin cytoskeleton. Analysis of the transcriptome for biological pathways predicted activation of inflammation and apoptosis and suppression of oxidative phosphorylation and MTORC1 signalling in ACM. Our data suggests dysregulated cell adhesion and ILK signalling as novel putative pathogenic mechanisms of ACM caused by FLNC variants which are distinct from the postulated disease mechanism of classic ARVC caused by desmosomal gene mutations. This knowledge could help in the design of future gene therapy strategies which would target specific components of these pathways and potentially lead to novel treatments for ACM

Combined in silico and experimental identification of the Pyrococcus abyssi H/ACA sRNAs and their target sites in ribosomal RNAs

How far do H/ACA sRNPs contribute to rRNA pseudouridylation in Archaea was still an open question. Hence here, by computational search in three Pyrococcus genomes, we identified seven H/ACA sRNAs and predicted their target sites in rRNAs. In parallel, we experimentally identified 17 Ψ residues in P. abyssi rRNAs. By in vitro reconstitution of H/ACA sRNPs, we assigned 15 out of the 17 Ψ residues to the 7 identified H/ACA sRNAs: one H/ACA motif can guide up to three distinct pseudouridylations. Interestingly, by using a 23S rRNA fragment as the substrate, one of the two remaining Ψ residues could be formed in vitro by the aCBF5/aNOP10/aGAR1 complex without guide sRNA. Our results shed light on structural constraints in archaeal H/ACA sRNPs: the length of helix H2 is of 5 or 6 bps, the distance between the ANA motif and the targeted U residue is of 14 or 15 nts, and the stability of the interaction formed by the substrate rRNA and the 3′-guide sequence is more important than that formed with the 5′-guide sequence. Surprisingly, we showed that a sRNA–rRNA interaction with the targeted uridine in a single-stranded 5′-UNN-3′ trinucleotide instead of the canonical 5′-UN-3′ dinucleotide is functional

Pseudouridine at position 55 in tRNA controls the contents of other modified nucleotides for low-temperature adaptation in the extreme-thermophilic eubacterium Thermus thermophilus

Pseudouridine at position 55 (Ψ55) in eubacterial tRNA is produced by TruB. To clarify the role of the Ψ55 modification, we constructed a truB gene disruptant (ΔtruB) strain of Thermus thermophilus which is an extreme-thermophilic eubacterium. Unexpectedly, the ΔtruB strain exhibited severe growth retardation at 50°C. We assumed that these phenomena might be caused by lack of RNA chaperone activity of TruB, which was previously hypothetically proposed by others. To confirm this idea, we replaced the truB gene in the genome with mutant genes, which express TruB proteins with very weak or no enzymatic activity. However the growth retardation at 50°C was not rescued by these mutant proteins. Nucleoside analysis revealed that Gm18, m5s2U54 and m1A58 in tRNA from the ΔtruB strain were abnormally increased. An in vitro assay using purified tRNA modification enzymes demonstrated that the Ψ55 modification has a negative effect on Gm18 formation by TrmH. These experimental results show that the Ψ55 modification is required for low-temperature adaptation to control other modified. 35S-Met incorporation analysis showed that the protein synthesis activity of the ΔtruB strain was inferior to that of the wild-type strain and that the cold-shock proteins were absence in the ΔtruB cells at 50°C

Small noncoding differentially methylated copy-number variants, including lncRNA genes, cause a lethal lung developmental disorder

An unanticipated and tremendous amount of the noncoding sequence of the human genome is transcribed. Long noncoding RNAs (lncRNAs) constitute a significant fraction of non-protein-coding transcripts; however, their functions remain enigmatic. We demonstrate that deletions of a small noncoding differentially methylated region at 16q24.1, including lncRNA genes, cause a lethal lung developmental disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV), with parent-of-origin effects. We identify overlapping deletions 250 kb upstream of FOXF1 in nine patients with ACD/MPV that arose de novo specifically on the maternally inherited chromosome and delete lung-specific lncRNA genes. These deletions define a distant cis-regulatory region that harbors, besides lncRNA genes, also a differentially methylated CpG island, binds GLI2 depending on the methylation status of this CpG island, and physically interacts with and up-regulates the FOXF1 promoter. We suggest that lung-transcribed 16q24.1 lncRNAs may contribute to long-range regulation of FOXF1 by GLI2 and other transcription factors. Perturbation of lncRNA-mediated chromatin interactions may, in general, be responsible for position effect phenomena and potentially cause many disorders of human development