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At least 271 records · Page 15Linked to original sources
PubMed2024

Transcriptome-wide N6-methyladenosine modification profiling of long non-coding RNAs in patients with recurrent implantation failure.

N6-methyladenosine (m6A) is involved in most biological processes and actively participates in the regulation of reproduction. According to recent research, long non-coding RNAs (lncRNAs) and their m6A modifications are involved in reproductive diseases. In the present study, using m6A-modified RNA immunoprecipitation sequencing (m6A-seq), we established the m6A methylation transcription profiles in patients with recurrent implantation failure (RIF) for the first time. There were 1443 significantly upregulated m6A peaks and 425 significantly downregulated m6A peaks in RIF. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that genes associated with differentially methylated lncRNAs are involved in the p53 signalling pathway and amino acid metabolism. The competing endogenous RNA network revealed a regulatory relationship between lncRNAs, microRNAs and messenger RNAs. We verified the m6A methylation abundances of lncRNAs by using m6A-RNA immunoprecipitation (MeRIP)-real-time polymerase chain reaction. This study lays a foundation for further exploration of the potential role of m6A modification in the pathogenesis of RIF.

Humans↗
PubMed2025

LncRNA Profiling and ceRNA Network Construction of Intrauterine Exosomes in Goats During Embryo Implantation.

Exosomes have been shown to play an important role in embryo implantation, but the mechanism is still unclear. This study aimed to investigate the functional roles of lncRNAs in intrauterine exosomes in goat pregnancy. We used RNA-seq to identify the lncRNA profiles of exosomes obtained from goat uterine rinsing fluid at 5, 15, and 18 days of gestation. In addition, we performed weighted gene co-expression network analysis based on differentially expressed mRNAs (DEMs) and lncRNAs (DELs). Functional enrichment analyses of gene modules were conducted using Gene Ontology classification (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. A lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) regulatory network was constructed based on predictive interaction derived from miRTarBase, miRDB and RNAhybrid databases. Altogether, 831 DELs were identified. GO and KEGG analysis showed that the target genes were enriched in processes associated with embryo implantation, such as signaling receptor activity, binding and immune response. Nine functional co-expression modules were enriched in various biological processes, such as metabolic pathways, protein transport, cell cycle and VEGF signaling pathway. Additionally, 12 lncRNA-mediated ceRNA networks were constructed. Our results demonstrate that exosomal lncRNAs in uterine flushing fluid exhibit dynamic changes across gestational stages and play an important role in regulating the uterine microenvironment during embryo implantation. These findings provide a foundational basis for screening exosome-derived lncRNAs that influence embryo implantation and contribute to elucidating the mechanistic roles of lncRNAs in exosome-mediated processes during early pregnancy.

embryo implantation↗
PubMed2002

Engineering pathogen resistance in crop plants.

As the world population continues to increase, food supplies must also grow to meet nutritional requirements. One means of ensuring the stability and plentitude of the food supply is to mitigate crop loss caused by plant pathogens. Strategies for combating disease include traditional technologies such as plant breeding and chemical applications; current technologies such as generating transgenic plants that express components of known defense signaling pathways; and the adaptation of newer technologies such as RNA silencing of pathogen and plant transcripts. Breeding has been used to pyramid resistance (R) genes into many different plants including rice. Chemical strategies include application of salicylic acid (SA) analogs to stimulate systemic acquired resistance (SAR) responses. Genetic screens in Arabidopsis have identified genes controlling SAR and these genes have been manipulated and used to engineer crop plants. The diseases caused by plant viruses are being thwarted through the initiation of endogenous RNA silencing mechanisms. Many of these strategies show great promise, some limitations, and exciting opportunities to develop many new tools for combating plant pests.

Crops, Agricultural↗
PubMed2026

lncRNA JPX promotes radioresistance in nasopharyngeal carcinoma via the miR-1301-3p/PIK3R2-mediated autophagy pathway.

BACKGROUND: Nasopharyngeal carcinoma (NPC) represents an aggressive head and neck malignancy with high metastatic potential. Radioresistance remains a major therapeutic obstacle associated with poor prognosis. Although the long non-coding RNA (lncRNA) JPX has been implicated in various cancers, its specific role in NPC radioresistance requires further elucidation. This study aimed to investigate whether JPX modulates radiosensitivity through autophagy regulation and to delineate the underlying molecular mechanisms. METHODS: JPX expression was analyzed in NPC cell lines and The Cancer Genome Atlas (TCGA) datasets, with subcellular localization determined through cellular fractionation. Functional characterization was performed using short hairpin RNA (shRNA)-mediated knockdown in CNE-2 and HONE-1 cell lines. Radiosensitivity was evaluated by clonogenic survival assays at a clinically relevant radiation dose, with cell viability assessed by MTT as a screening measure. while autophagy activity was assessed through Western blot analysis of LC3-II and p62. Molecular interactions were validated using dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. RESULTS: JPX was significantly upregulated in head and neck squamous cell carcinoma (HNSCC) tissues and NPC cell lines, showing predominant cytoplasmic localization. Clinical association analysis in the TCGA-HNSCC cohort revealed that elevated JPX expression correlated with advanced tumor stage and poor overall survival, although NPC-specific clinical validation remains to be established. Genetic silencing of JPX attenuated autophagic flux and enhanced radiosensitivity. Mechanistic investigations revealed that JPX functions as a competitive endogenous RNA (ceRNA) functionally associating with miR-1301-3p, thereby alleviating miR-1301-3p-mediated repression of PIK3R2 and subsequently activating pro-survival autophagy pathways. CONCLUSIONS: The findings demonstrate that JPX promotes radioresistance in NPC through a ceRNA mechanism involving the miR-1301-3p/PIK3R2/autophagy regulatory axis. The JPX/miR-1301-3p/PIK3R2 axis thus emerges as a potential mechanistic candidate for radiosensitization; however, this notion remains strictly provisional and requires rigorous validation in authenticated NPC models, in vivo systems, and patient-derived samples before any translational consideration can be justified. Despite the cell line limitations acknowledged herein, our findings provide a mechanistic framework for understanding JPX-mediated radioresistance that warrants further investigation in more physiologically relevant models.

JPX↗
PubMed1993

TFIIIA and DNA supercoiling: toward resolving a controversy.

DNA supercoiling in eukaryotes is mediated by the process of chromatin assembly. This process couples the binding and wrapping of DNA around nucleosomal core particles and the enzymatic activity of DNA topoisomerase(s). Kmiec and Worcel (1985) reported that the binding of an RNA polymerase III transcription factor (TFIIIA) could accelerate DNA supercoiling catalysed by a Xenopus laevis cell-free extract. Although the reaction was repeatedly reproduced in the parent laboratory by numerous workers, another laboratory, interested in the molecular biology of TFIIIA, failed to reproduce TFIIIA-mediated supercoiling or gyration. In this review, an attempt is made to transcend personal beliefs and describe the experimental approaches used to clarify this issue. The original variability lay in the amount of endogenous RNA within the cell free extract and the concentrations of MgCl2 used to conduct the experiments.

Adenosine Triphosphate↗
PubMed1995

Isolation and characterization of an RNA-dependent RNA polymerase from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus.

A template-bound RNA polymerase was isolated from Nicotiana clevelandii plants infected with red clover necrotic mosaic dianthovirus (RCNMV) by differential centrifugation, solubilization with dodecyl beta-D-maltopyranoside, and chromatography on columns of Sephacryl S-400 and Q-Sepharose. Analysis of the purified polymerase by SDS-polyacrylamide gel electrophoresis, followed by silver staining or immunoblotting, showed that it contained virus-encoded proteins of molecular masses 27 kDa and 88 kDa together with several minor proteins possibly of host origin. After removal of endogenous RNA with micrococcal nuclease, the polymerase became template-dependent. It was also template-specific, being able to utilize as templates RNA of two strains of RCNMV, but not RNAs of three viruses in different taxonomic groups, namely cucumber mosaic cucumovirus, tomato bushy stunt tombusvirus and tomato mosaic tobamovirus. The products of RNA polymerase reactions were double-stranded RNAs corresponding to RCNMV RNAs 1 and 2. The ability of the template-dependent RNA polymerase to synthesize RNA was completely inhibited by antibodies to a peptide containing the GDD motif, whereas the activity of the template-bound enzyme was unaffected by these antibodies.

Amino Acid Sequence↗
PubMed1989

Inhibition of in vivo and in vitro transcription by monoclonal antibodies prepared against wheat germ RNA polymerase II that react with the heptapeptide repeat of eukaryotic RNA polymerase II.

Wheat germ RNA polymerase II was used to raise monoclonal antibodies (mAbs) that cross-react with the largest subunit of calf thymus RNA polymerase II. Most of these mAbs were of the IgM isotype and were shown to react with a synthetic peptide containing the consensus sequence for the C-terminal heptapeptide repeat that has been found on the largest subunit of RNA polymerase II from a variety of eukaryotic organisms. A representative mAb (3WG2) was tested for its effect on transcription in both in vitro and in vivo systems. Antibody 3WG2 did not affect the transcription (elongation) of wheat germ RNA polymerase II on denatured calf thymus DNA. When HeLa cell nuclear extracts were preincubated with the mAb, run-off transcription from a promoter that contains a TATA box (the adenovirus-2 major late promoter) and from a promoter that does not contain a TATA box (the murine dihydrofolate reductase gene promoter = dhfr) was inhibited. Transcription from these promoters was also inhibited by the synthetic peptide containing the consensus sequence when it was conjugated to bovine serum albumin. HeLa cell nuclear extract in which the endogenous RNA polymerase II had been inhibited by the specific mAb was used to examine the ability of added mammalian RNA polymerase II that lacks the C-terminal domain to accurately transcribe specific genes. When calf thymus RNA polymerase II that lacked the C-terminal domain was added back to the inhibited extract, a discrete transcript that was initiated correctly was obtained with the adenovirus-2 major late promoter; however, no discrete transcript was observed from the mouse dhfr gene promoter. When injected into Xenopus laevis oocytes, antibody 3WG2 inhibited transcription of the human histone H2b gene (contains a TATA box) and the human U1 small nuclear RNA gene (does not contain a TATA box), but did not inhibit transcription from RNA polymerase I or RNA polymerase III promoters. These results indicate that the C-terminal heptapeptide repeat plays a critical role in promoter-directed transcription, although enzyme that lacks this domain can initiate from some promoters in vitro.

Adenoviridae↗
PubMed1982

Isolation and characterization of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-resistant mutants of the Chinese hamster ovary cell line.

Mutants resistant to the RNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofurano-sylbenzimidazole (DRB) have been isolated in the Chinese hamster ovary cell line CHO-K1. Three independently isolated mutants, DRB6 DRB10, and DRB13, were 3-, 5-, and 3.5-fold, respectively, more resistant to DRB than the parental cell line WTCHO. The DRB-resistant mutations were expressed codominantly in somatic cell hybrids of DRB-resistant and DRB-sensitive cell lines. In vivo treatment of CHO-K1 cells with DRB resulted in specific inhibition of endogenous RNA polymerase II activity in cell lysates. Whereas DRB inhibited RNA polymerase II activity in WTCHO cells by a maximum of 60% at concentrations as low as 60 microM, 300 microM DRB was required to inhibit 60% of the RNA polymerase II activity in DRB10 cells. However, the inhibition of the DRB-sensitive RNA polymerase II activity in DRB10 was biphasic. About half (53 to 56%) of this activity was inhibited by 90 microM DRB and thus showed a DRB sensitivity similar to the wild-type RNA polymerase II activity; the remaining DRB-sensitive RNA polymerase II activity was maximally inhibited by 300 microM DRB. These results indicated that there were two copies of the drbR locus (drb+ and drbR-10) in DRB10 and confirmed that the drbR-10 mutation was expressed codominantly. Somatic cell hybrids of DRB-resistant and alpha-amanitin-resistant cell lines grew in medium containing both DRB and alpha-amanitin, demonstrating that the drbR and amaR mutations were not in the same gene. Thus, the drbR mutations may define an additional component of the RNA polymerase II transcriptional complex in mammalian cells.

Amanitins↗
PubMed1999

Nuclear c-Abl is a COOH-terminal repeated domain (CTD)-tyrosine (CTD)-tyrosine kinase-specific for the mammalian RNA polymerase II: possible role in transcription elongation.

The c-Abl tyrosine kinase has been shown to interact with the COOH-terminal repeated domain (CTD) of mammalian RNA polymerase II and can phosphorylate the tyrosine residues in the CTD. Interestingly, the Drosophila or the yeast CTD were not efficiently phosphorylated by the mammalian c-Abl. This species-specificity was found to be determined by the extreme COOH-terminal CTD sequences that are not conserved through evolution. In vitro, COOH-terminal-truncated CTD could neither bind to, nor be phosphorylated by, c-Abl. In vivo, coexpression of a full length CTD prevents c-Abl from inducing the tyrosine phosphorylation of endogenous RNA polymerase II, and such inhibitory effect was not observed with the coexpression of COOH-terminal-truncated CTD. Serine/threonine phosphorylation of the CTD has been linked to the regulation of transcription elongation. Transcription from the human immunodeficiency virus type 1 (HIV-1) promoter requires CTD-phosphorylation, which is stimulated by the viral Tat protein through the recruitment of cellular Ser/Thr CTD kinases. In transient cotransfection experiments, the c-Abl kinase was found to activate the HIV promoter in the absence of Tat. The activation of the HIV promoter required the nuclear localization of c-Abl and could be correlated with increased tyrosine phosphorylation of RNA polymerase II. These observations suggest that tyrosine phosphorylation of the CTD may be functionally equivalent to its serine/threonine phosphorylation in stimulating transcription elongation.

Animals↗
PubMed1990

Most of the homeobox-containing Xhox 36 transcripts in early Xenopus embryos cannot encode a homeodomain protein.

Multiple Xhox 36 transcripts accumulate in Xenopus embryos from gastrula to early tadpole stages. The transcripts were characterized by sequencing cDNA clones and by S1 protection and Northern (RNA) blotting of embryonic RNA with probes derived from the cDNAs. The Xhox 36 RNAs included unspliced precursor transcripts that accumulated in the embryonic nuclei, spliced transcripts that contained multiple stop codons in frame with the homeobox, and less abundant coding mRNAs. These transcripts were generated either by alternative splicing or multiple initiations from a single Xhox 36 gene. The sequence of a cDNA clone of the unspliced transcript showed that the intron contained a noncanonical 3' splice site. However, the intron was spliced efficiently when expressed from a plasmid injected into Xenopus embryos, suggesting that the inefficient splicing of the endogenous RNA is not due to the unusual 3' splice site. The accumulation of noncoding and unspliced transcripts suggests multiple levels of regulation in the embryonic expression of the Xhox 36 gene.

Animals↗
PubMed2026

Sodium Overload-Related Molecular Subtypes and a Four-Gene Prognostic Signature Predict Survival, Immune Landscape, and Therapeutic Response in Acute Myeloid Leukemia.

Sodium overload has recently emerged as a critical metabolic stressor involved in cancer progression; however, its molecular characteristics and clinical relevance in acute myeloid leukemia (AML) remain unexplored. RNA-seq data sets, clinical annotations, and mutational profiles of AML patients were annotations from The Cancer Genome Atlas and integrated with Genotype-Tissue Expression normal samples. Sodium overload-related genes (SORGs) were obtained from GeneCards. Differentially expressed SORGs (DESORGs) screened by applying the limma statistical model, followed by univariate Cox proportional hazards regression, consensus clustering, functional enrichment, immune infiltration analysis, and pathway evaluation. A prognostic signature was developed through least absolute shrinkage and selection operator regression followed by multivariate Cox modeling. The model's performance was further verified in two external GEO data sets (GSE71014 and GSE37642). Nomogram construction, subgroup analysis, tumor mutational burden (TMB) assessment, drug sensitivity prediction, transcription factor (TF) analysis, and competing endogenous RNA (ceRNA) network analyses were also performed. A total of 57 DESORGs were identified, and 2 sodium overload-related molecular subtypes exhibited distinct survival, immune infiltration, and inflammatory pathway activation. A robust four-gene signature (DOCK1, GABRE, HTR7, ACSM1) stratified patients into high- and low-risk categories with significantly different survival across training and validation cohorts. High-risk patients displayed increased immune infiltration, higher TMB, reduced sensitivity to multiple chemotherapeutic drugs, and inferior predicted response to PD-L1 blockade. TF and ceRNA networks revealed multilayered transcriptional and post-transcriptional regulation of the signature genes. This study identifies sodium overload-related molecular heterogeneity in AML and establishes a validated four-gene prognostic signature that integrates genomic, immunologic, and therapeutic features, offering potential utility for personalized risk assessment and treatment optimization.

Humans↗
PubMed1979

Effect of starvation on the genetic activity of nucleus and nucleolar organizer.

The investigations of root meristems and hypocotyls of Lupinus albus L. starved, and then fed with 2% sucrose were carried out and several variations in nuclear and nucleolar dimensions, their ultrastructure, template activity of DNA, activities of RNA polymerases and transcriptional activity, were found. As a result of starvation, the surface area of nuclei and nucleoli decreases several times; after 24 hours, in the presence of sucrose, it grows again, but the control state is not achieved. Moreover, in a starved material the area of condensed chromatin in nucleus is increased by 1/3; after feeding, its partial recovery to the initial state is observed. The intensity of binding of 3H AMD in a fed material is increased by 1/3 as compared with the starved one. Transcriptional activity, estimated on the basis of 3H uridine incorporation is decreased in a starved material, especially in the meristematic tissue; feeding intensificates the transcriptional activity whereas the activity of endogenous RNA polymerase, investigated in hypocotyl, is drastically lowered in a starved material. Sucrose feeding does not restore the control state, though the per cent of nuclei and nucleoli revealing the activity of RNA polymerase is much higher in a fed material than in a starved one.

Cell Nucleolus↗
PubMed1999

Cyclin C/CDK8 and cyclin H/CDK7/p36 are biochemically distinct CTD kinases.

Phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II is important for basal transcriptional processes in vivo and for cell viability. Several kinases, including certain cyclin-dependent kinases, can phosphorylate this substrate in vitro. It has been proposed that differential CTD phosphorylation by different kinases may regulate distinct transcriptional processes. We have found that two of these kinases, cyclin C/CDK8 and cyclin H/CDK7/p36, can specifically phosphorylate distinct residues in recombinant CTD substrates. This difference in specificity may be largely due to their varying ability to phosphorylate lysine-substituted heptapeptide repeats within the CTD, since they phosphorylate the same residue in CTD consensus heptapeptide repeats. Furthermore, this substrate specificity is reflected in vivo where cyclin C/ CDK8 and cyclin H/CDK7/p36 can differentially phosphorylate an endogenous RNA polymerase II substrate. Several small-molecule kinase inhibitors have different specificities for these related kinases, indicating that these enzymes have diverse active-site conformations. These results suggest that cyclin C/CDK8 and cyclin H/CDK7/p36 are physically distinct enzymes that may have unique roles in transcriptional regulation mediated by their phosphorylation of specific sites on RNA polymerase II.

Cyclin C↗
PubMed2003

Viroid RNA systemic spread may depend on the interaction of a 71-nucleotide bulged hairpin with the host protein VirP1.

Viroids are noncoding circular single-stranded RNAs that are propagated systemically in plants. VirP1 is a protein from tomato, which is an excellent host for potato spindle tuber viroid (PSTVd), and it has been isolated by virtue of its specific in vitro binding to PSTVd RNA. We report on the specific in vivo interaction of VirP1 with full-length viroid RNA as well as with subfragments in the three-hybrid system. The terminal right domain (TR) of PSTVd was identified as a strong interacting partner for VirP1. A weaker partner is provided by a right-hand subfragment of hop stunt viroid (HSVd), a viroid that infects tomato poorly. We present a sequence and structural motif of the VirP1-interacting subfragments. The motif is disturbed in the replicative but nonspreading R+ mutant of the TR. According to our in vivo and in vitro binding assays, the interaction of this mutant with VirP1 is compromised. We propose that the AGG/CCUUC motif bolsters recognition of the TR by VirP1 to achieve access of the viroid to pathways that propagate endogenous RNA systemic signals in plants. Systemic trafficking has been suggested for miRNA precursors, of which the TR, as a stable bulged hairpin 71 nt long, is quite reminiscent.

Base Sequence↗
PubMed2003

Dynamic association of RNA-editing enzymes with the nucleolus.

ADAR1 and ADAR2 are editing enzymes that deaminate adenosine to inosine in long double stranded RNA duplexes and specific pre-mRNA transcripts. Here, we show that full-length and N-terminally truncated forms of ADAR1 are simultaneously expressed in HeLa and COS7 cells owing to the usage of alternative starting methionines. Because the N-terminus of ADAR1 contains a nuclear export signal, the full-length protein localizes predominantly in the cytoplasm, whereas the N-terminally truncated forms are exclusively nuclear and accumulate in the nucleolus. ADAR2, which lacks a region homologous to the N-terminal domain of ADAR1, localizes exclusively to the nucleus and similarly accumulates in the nucleolus. Within the nucleolus, ADAR1 and ADAR2 co-localize in a novel compartment. Photobleaching experiments demonstrate that, in live cells, ADAR1 and ADAR2 are in constant flux in and out of the nucleolus. When cells express the editing-competent glutamate receptor GluR-B RNA, endogenous ADAR1 and ADAR2 de-localize from the nucleolus and accumulate at sites where the substrate transcripts accumulate. This suggests that ADAR1 and ADAR2 are constantly moving through the nucleolus and might be recruited onto specific editing substrates present elsewhere in the cell.

Adenosine Deaminase↗
PubMed1978

Template-specific requirements for DNA synthesis by the Mason-Pfizer monkey virus DNA polymerase: unique aspects.

The biochemical properties of DNA polymerase purified from Mason-Pfizer monkey virus were studied, with respect to synthetic and natural template-primer utilization. Thes studies revealed the following new information about the Mason-Pfizer monkey virus enzyme: (a) Mason-Pfizer monkey virus polymerase was found to prefer template: primer molar nucleotide ratios of 2.5-5: 1 for optimal rates of synthesis with poly(C) .(dG)12-18 as template-primer. (b) Poly(A)-directed synthesis was stimulated by the addition of low concentrations of inorganic phosphate to the reaction mixture. (c) Poly(2' -O-methyl-cytidylate), poly(rCm), was the only template studied for which Mn2+ proved the preferred divalent cation. Combinations of divalent cations stimulated rather than inhibited poly(rCm)-directed poly(dG) synthesis by the Mason-Pfizer monkey virus enzyme. (d) Heteropolymeric regions of rabbit globin mRNA and avian myeloblastosis virus 70 S RNA could be copied by the Mason-Pfizer monkey virus polymerase with oligo(dT), oligo(U) or in the case of avian myeloblastosis virus RNA, endogenous primers. In all such studies, Mg2+ was the preferred divalent cation and a distinct preference for the DNA primer in the reverse transcription of natural RNAs was observed. These new findings necessitated comparative studies with the DNA polymerases from Rauscher murine leukemia virus and murine mammary tumor virus, as representative type C and type B retroviruses. Although the Mason-Pfizer monkey virus enzyme was found to share some properties in common with both type C and type B mammalian viral enzymes, certain of the above properties rendered it unique among the polymerases examined.

Animals↗
PubMed2017

Circular RNA profiling reveals an abundant circLMO7 that regulates myoblasts differentiation and survival by sponging miR-378a-3p.

Circular RNAs (circRNAs) have been identified from various tissues and species, but their regulatory functions during developmental processes are not well understood. We examined circRNA expression profiles of two developmental stages of bovine skeletal muscle (embryonic and adult musculus longissimus) to provide first insights into their potential involvement in bovine myogenesis. We identified 12 981 circRNAs and annotated them to the Bos taurus reference genome, including 530 circular intronic RNAs (ciRNAs). One parental gene could generate multiple circRNA isoforms, with only one or two isoforms being expressed at higher expression levels. Also, several host genes produced different isoforms when comparing development stages. Most circRNA candidates contained two to seven exons, and genomic distances to back-splicing sites were usually less than 50 kb. The length of upstream or downstream flanking introns was usually less than 105 nt (mean≈11 000 nt). Several circRNAs differed in abundance between developmental stages, and real-time quantitative PCR (qPCR) analysis largely confirmed differential expression of the 17 circRNAs included in this analysis. The second part of our study characterized the role of circLMO7-one of the most down-regulated circRNAs when comparing adult to embryonic muscle tissue-in bovine muscle development. Overexpression of circLMO7 inhibited the differentiation of primary bovine myoblasts, and it appears to function as a competing endogenous RNA for miR-378a-3p, whose involvement in bovine muscle development has been characterized beforehand. Congruent with our interpretation, circLMO7 increased the number of myoblasts in the S-phase of the cell cycle and decreased the proportion of cells in the G0/G1 phase. Moreover, it promoted the proliferation of myoblasts and protected them from apoptosis. Our study provides novel insights into the regulatory mechanisms underlying skeletal muscle development and identifies a number of circRNAs whose regulatory potential will need to be explored in the future.

Animals↗
PubMed1998

Competitive reverse transcription polymerase chain reaction for quantifying pre-mRNA and mRNA of major acute phase proteins.

Competitive reverse transcription polymerase chain reaction (RT-PCR) is an increasingly used method for quantifying RNA. The technique involves co-amplification from test RNA with an internal standard using common primers in a single reaction. The standard competes for primers and enzyme and it is therefore referred to as a competitor. A RT-PCR polycompetitor for use in quantifying acute phase serum amyloid A, constitutive serum amyloid A, serum amyloid P component, C-reactive protein, apolipoprotein A1, apolipoprotein A2, glyceraldehyde-3-phosphate dehydrogenase and beta-actin mRNAs has been generated and used in quantitative PCR. The polycompetitor was synthesised from ten oligonucleotides of 77-90 bases using primer extension and contains sequences which permit amplification using priming sites that are present in both hnRNA (pre-mRNA) and mRNA. The polycompetitor was cloned into the expression vector pSP64(polyA) and a polycompetitor transcript with a poly(A)-tail sequence at the 3'-end was generated by in vitro transcription. The poly(A)-tail sequence allows the option of performing reverse transcription using oligo(dT) in addition to directed reverse transcription using the specific 3'-reverse PCR primers. cDNA products generated from amplification of the internal polycompetitor standard and endogenous RNA species were separated by capillary electrophoresis and quantified by UV absorbance at 254 nm. Reproducibility was determined to be very high when starting ratios of internal standard and target mRNA are at an approximate equivalence point. Relative standard deviations were less than 5% between independent RT-PCR reactions with the same sample mix of internal standard and total RNA. Applying the method to total RNA samples harvested at various timepoints following cytokine induction of acute phase mRNAs in endothelial cells demonstrated that quantitative readout from the RT-PCR method correlates well with that obtained from Northern-blotting and is at least 100-fold more sensitive. This method will be useful for studying regulation of acute phase proteins in in vitro tissue culture experiments and may also be applied to clinical tissue samples from patients with inflammatory diseases.

Acute-Phase Proteins↗