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PubMed2026

Screening and identification of the ncRNA-mRNA regulatory network associated with DNA methylation in goose embryonic myoblasts.

BACKGROUND: Local goose breeds Shitou and Wuzong exhibit distinct growth rates, implying divergent embryonic muscle development. This study used embryonic myoblasts from the Magang goose, an established model with superior growth traits, to explore the underlying common regulatory mechanisms. Extending our previous findings that 5-AZA (DNA methylation inhibitor) and BC339 (DNA hydroxylation inhibitor) oppositely affect myoblast proliferation and differentiation, we performed whole-transcriptome sequencing on inhibitor-treated goose embryonic myoblasts. This aimed to identify DNA methylation-mediated ncRNA-mRNA networks governing myoblast fate, with key interactions being functionally validated. RESULT: 5-AZA significantly promotes cell proliferation and differentiation by inhibiting DNA methyltransferase activity and reducing DNA methylation levels, whereas BC339 significantly suppresses cell proliferation and differentiation by inhibiting demethylation and increasing DNA methylation levels. Specifically, we identified 6,309 mRNAs, 579 lncRNAs, 194 miRNAs, and 825 circRNAs that were differentially expressed in response to 5-AZA and BC339 treatment. Based on GO and KEGG enrichment analyses, differentially expressed genes related to muscle development were selected to construct a ceRNA network. This network comprises 5 differentially expressed lncRNAs (DELs: MSTRG.17572.1, XR_001211738.1, MSTRG.1886.1, XR_001212555.1, MSTRG.8995.2), 2 differentially expressed circRNAs (DECs: novel_circ_029953, novel_circ_017636), 11 differentially expressed miRNAs (DEMs: miR-383-x, miR-10174-y, miR-191-x, miR-24-x, miR-9619-y, novel-m0303-5p, novel-m0105-3p, miR-204-x, miR-211-z, novel-m0075, miR-26-y), 5 differentially expressed genes (DEGs: KIF3A, CCND1, PPM1A, Table 2, TGFBR1), forming a total of 24 interactions. This study identified miR-9619-y as a critical negative regulator of goose embryonic myoblast development through targeted inhibition of CCND1. Dual-luciferase reporter assays confirmed the direct binding of miR-9619-y to the 3'-untranslated region of CCND1. Functional experiments demonstrated that overexpression of miR-9619-y significantly reduced the EdU-positive cell ratio and myotube area percentage, accompanied by cell cycle arrest at the G0/G1 phase. Conversely, inhibition of miR-9619-y promoted myoblast proliferation and differentiation while decreasing the proportion of cells in G0/G1 phase. During the proliferation stage, miR-9619-y overexpression significantly suppressed CCND1 expression at both mRNA and protein levels, down-regulated MyoD expression, and reduced Myf5 mRNA abundance; whereas miR-9619-y inhibition up-regulated these genes and their corresponding proteins. During the differentiation stage, overexpression of miR-9619-y similarly decreased the mRNA levels of CCND1, Myh1, and MyoG, as well as the protein levels of MyHC and CCND1, with inhibition producing the opposite effects. CONCLUSION: In this study, we predicted a ceRNA network based on bioinformatics analysis governing goose embryonic myoblast development, identifying key molecular components including mRNAs, miRNAs, lncRNAs, and circRNAs, along with 24 regulatory axes. Functional experiments further demonstrated that miR-9619-y arrests cell cycle progression and negatively regulates the proliferation and differentiation of goose embryonic myoblasts, as evidenced by its impact on both the mRNA and protein expression of key myogenic factors through targeted inhibition of CCND1. These findings, together with the bioinformatically predicted ceRNA network, suggest potential complex post-transcriptional regulatory mechanisms underlying myogenesis in geese and offer candidate molecular targets for genetic improvement of meat production performance in waterfowl breeding programs.

Animals↗
PubMed2004

A new role for expressed pseudogenes as ncRNA: regulation of mRNA stability of its homologous coding gene.

We have earlier generated a mutant mouse in a course of making a transgenic line that exhibited interesting heterozygote phenotypes, which exhibited failure to thrive, severe bone deformities, and polycystic kidneys. This mutant mouse provided a clue to uncover a unique role of expressed pseudogenes. In this mutant the transgene was integrated into the vicinity of the expressing pseudogene of Makorin1 called Makorin1-p1. This insertion reduced transcription of the Makorin1-p1, resulting in destabilization of the Makorin1 mRNA in trans via a cis-acting RNA decay element within the 5' region of Makorin1 that is homologous between Makorin1 and Makorin1-p1. These findings demonstrate a novel and specific regulatory role of an expressed pseudogene as well as functional significance for noncoding RNAs. Next, we developed an original algorithm to determine how many pseudogenes are expressed. Based on our examination 2-3% of human processed pseudogenes are expressed using the most strict criteria. Interestingly, the mouse has a much smaller proportion of expressed pseudogenes (0.5-1%). Pseudogenes are functionally less constrained, and have accumulated more mutations than translated genes. If they have some functions in gene regulation, this property would allow more rapid functional diversification than protein-coding genes. In addition, some genetic phenomena that exhibit incomplete penetrance might be attributed to "mutation" or "variation" of pseudogenes.

Animals↗
PubMed2006

Profiling Caenorhabditis elegans non-coding RNA expression with a combined microarray.

Small non-coding RNAs (ncRNAs) are encoded by genes that function at the RNA level, and several hundred ncRNAs have been identified in various organisms. Here we describe an analysis of the small non-coding transcriptome of Caenorhabditis elegans, microRNAs excepted. As a substantial fraction of the ncRNAs is located in introns of protein-coding genes in C.elegans, we also analysed the relationship between ncRNA and host gene expression. To this end, we designed a combined microarray, which included probes against ncRNA as well as host gene mRNA transcripts. The microarray revealed pronounced differences in expression profiles, even among ncRNAs with housekeeping functions (e.g. snRNAs and snoRNAs), indicating distinct developmental regulation and stage-specific functions of a number of novel transcripts. Analysis of ncRNA-host mRNA relations showed that the expression of intronic ncRNA loci with conserved upstream motifs was not correlated to (and much higher than) expression levels of their host genes. Even promoter-less intronic ncRNA loci, though showing a clear correlation to host gene expression, appeared to have a surprising amount of 'expressional freedom', depending on host gene function. Taken together, our microarray analysis presents a more complete and detailed picture of a non-coding transcriptome than hitherto has been presented for any other multicellular organism.

Animals↗
PubMed2004

Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs.

The bacterial Sm-like protein Hfq facilitates RNA-RNA interactions involved in post-transcriptional regulation of the stress response. Specifically, Hfq helps pair noncoding RNAs (ncRNAs) with complementary regions of target mRNAs. To probe the mechanism of this pairing, we generated a series of Hfq mutants and measured their affinity for RNAs like those with which Hfq must associate in vivo. We tested the mutants' DsrA-dependent activation of rpoS, and their ability to stabilize DsrA ncRNA against degradation in vivo. Our results suggest that Hfq has two independent RNA-binding surfaces. In addition to a well-known site around the core of the Hfq hexamer, we observe interactions with the distal face of Hfq, a new locus with which mRNAs and poly(A) sequences associate. Our model explains how Hfq can simultaneously bind a ncRNA and its mRNA target to facilitate the strand displacement reaction required for Hfq-dependent translational regulation.

Amino Acid Sequence↗
PubMed2026

Comprehensive analysis of differentially expressed mRNAs, lncRNAs, and miRNAs involved in ovarian differentiation and development in Qihe gibel carp (Carassius gibelio var. Qihe).

Qihe gibel carp (Carassius gibelio var. Qihe) exhibits diverse reproductive modes including gynogenesis and sexual reproduction, yet the molecular mechanisms of ovarian differentiation remain poorly understood. Ovarian tissues at 20, 30, and 60 days after hatching (dah), representing key stages covering early ovarian differentiation and primary oocyte growth, were subjected to whole-transcriptome sequencing. A total of 27,259 mRNAs, 2622 lncRNAs, and 2467 miRNAs were differentially expressed. Cell cycle, transcription, translation, and DNA replication pathways were significantly upregulated from 20 to 60 dah. Oocyte meiosis was enriched from 20 and 30 dah, whereas metabolic pathways (lipid, carbohydrate, and nucleotide metabolism) were enriched from 30 to 60 dah, indicating sequential progression from meiosis initiation to primary oocyte growth with nutrient synthesis. Hub lncRNAs and key ceRNA networks (e.g., MSTRG.28669.5-miR-221-ccnb2) were identified. This study provides the first comprehensive characterization of ncRNA-mediated regulation and ceRNA networks during ovarian development in Qihe gibel carp, establishing a foundation for understanding ovarian differentiation in this species.

Animals↗
PubMed2005

New perspectives on neoplasia and the RNA world.

Key tenets of modern biology are the central place of protein in cell regulation and the flow of genetic information from DNA to RNA to protein. However, it is becoming increasingly apparent that genomes are much more complex than hitherto thought with remarkably complex regulatory systems. The notion that the fraction of the genome involved in coding protein is all that matters is increasingly being questioned as the roles of non-coding RNA (ncRNA) in cellular systems becomes recognised. The RNA world, including microRNA (miRNA), small inhibitory RNA (siRNA) and other RNA species, are now recognised as being crucial for the regulation of chromatin structure, gene expression, mRNA processing and splicing, mRNA stability and translational control. Furthermore such ncRNA systems may be perturbed in disease states and most notably in neoplasia, including in haematological malignancies. Here the burgeoning evidence for a role of miRNA in neoplasia is reviewed and the importance of understanding the RNA world emphasised.

Alternative Splicing↗
PubMed2006

Prediction of non-coding and antisense RNA genes in Escherichia coli with Gapped Markov Model.

A new mathematical index was developed to identify and characterize non-coding RNA (ncRNA) genes encoded within the Escherichia coli (E. coli) genome. It was designated the GMMI (Gapped Markov Model Index) and used to evaluate sequence patterns located at the separate positions of consensus sequences, codon biases and/or possible RNA structures on the basis of the Markov model. The GMMI was able to separate a set of known mRNA sequences from a mixture of ncRNAs including tRNAs and rRNAs. Consequently, the GMMI was employed to predict novel ncRNA candidates. At the beginning, possible transcription units were extracted from the E. coli genome using consensus sequences for the sigma70 promoter and the rho-independent terminator. Then, these units were evaluated by using the GMMI. This identified 133 candidate ncRNAs, which contain 29 previously annotated small RNA genes and 46 possible antisense ncRNAs. Furthermore 12 transcripts (including five antisense RNAs) were confirmed according to the expression analysis. These data suggests that the expression of small antisense RNAs might be more common than previously thought in the E. coli genome.

Computational Biology↗
PubMed2003

Challenging the dogma: the hidden layer of non-protein-coding RNAs in complex organisms.

The central dogma of biology holds that genetic information normally flows from DNA to RNA to protein. As a consequence it has been generally assumed that genes generally code for proteins, and that proteins fulfil not only most structural and catalytic but also most regulatory functions, in all cells, from microbes to mammals. However, the latter may not be the case in complex organisms. A number of startling observations about the extent of non-protein-coding RNA (ncRNA) transcription in the higher eukaryotes and the range of genetic and epigenetic phenomena that are RNA-directed suggests that the traditional view of the structure of genetic regulatory systems in animals and plants may be incorrect. ncRNA dominates the genomic output of the higher organisms and has been shown to control chromosome architecture, mRNA turnover and the developmental timing of protein expression, and may also regulate transcription and alternative splicing. This paper re-examines the available evidence and suggests a new framework for considering and understanding the genomic programming of biological complexity, autopoietic development and phenotypic variation.

Alternative Splicing↗
PubMed2005

Experimental validation of the regulated expression of large numbers of non-coding RNAs from the mouse genome.

Recent large-scale analyses of mainly full-length cDNA libraries generated from a variety of mouse tissues indicated that almost half of all representative cloned sequences did not contain an apparent protein-coding sequence, and were putatively derived from non-protein-coding RNA (ncRNA) genes. However, many of these clones were singletons and the majority were unspliced, raising the possibility that they may be derived from genomic DNA or unprocessed pre-mRNA contamination during library construction, or alternatively represent nonspecific "transcriptional noise." Here we show, using reverse transcriptase-dependent PCR, microarray, and Northern blot analyses, that many of these clones were derived from genuine transcripts of unknown function whose expression appears to be regulated. The ncRNA transcripts have larger exons and fewer introns than protein-coding transcripts. Analysis of the genomic landscape around these sequences indicates that some cDNA clones were produced not from terminal poly(A) tracts but internal priming sites within longer transcripts, only a minority of which is encompassed by known genes. A significant proportion of these transcripts exhibit tissue-specific expression patterns, as well as dynamic changes in their expression in macrophages following lipopolysaccharide stimulation. Taken together, the data provide strong support for the conclusion that ncRNAs are an important, regulated component of the mammalian transcriptome.

Animals↗
PubMed2024

Whole transcriptome sequencing analyses of islets reveal ncRNA regulatory networks underlying impaired insulin secretion and increased β-cell mass in high fat diet-induced diabetes mellitus.

AIM: Our study aims to identify novel non-coding RNA-mRNA regulatory networks associated with β-cell dysfunction and compensatory responses in obesity-related diabetes. METHODS: Glucose metabolism, islet architecture and secretion, and insulin sensitivity were characterized in C57BL/6J mice fed on a 60% high-fat diet (HFD) or control for 24 weeks. Islets were isolated for whole transcriptome sequencing to identify differentially expressed (DE) mRNAs, miRNAs, IncRNAs, and circRNAs. Regulatory networks involving miRNA-mRNA, lncRNA-mRNA, and lncRNA-miRNA-mRNA were constructed and functions were assessed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. RESULTS: Despite compensatory hyperinsulinemia and a significant increase in β-cell mass with a slow rate of proliferation, HFD mice exhibited impaired glucose tolerance. In isolated islets, insulin secretion in response to glucose and palmitic acid deteriorated after 24 weeks of HFD. Whole transcriptomic sequencing identified a total of 1324 DE mRNAs, 14 DE miRNAs, 179 DE lncRNAs, and 680 DE circRNAs. Our transcriptomic dataset unveiled several core regulatory axes involved in the impaired insulin secretion in HFD mice, such as miR-6948-5p/Cacna1c, miR-6964-3p/Cacna1b, miR-3572-5p/Hk2, miR-3572-5p/Cckar and miR-677-5p/Camk2d. Additionally, proliferative and apoptotic targets, including miR-216a-3p/FKBP5, miR-670-3p/Foxo3, miR-677-5p/RIPK1, miR-802-3p/Smad2 and ENSMUST00000176781/Caspase9 possibly contribute to the increased β-cell mass in HFD islets. Furthermore, competing endogenous RNAs (ceRNA) regulatory network involving 7 DE miRNAs, 15 DE lncRNAs and 38 DE mRNAs might also participate in the development of HFD-induced diabetes. CONCLUSIONS: The comprehensive whole transcriptomic sequencing revealed novel non-coding RNA-mRNA regulatory networks associated with impaired insulin secretion and increased β-cell mass in obesity-related diabetes.

Mice↗
PubMed2006

A large noncoding RNA is a marker for murine hepatocellular carcinomas and a spectrum of human carcinomas.

Tumor markers can facilitate understanding molecular cell biology of neoplasia and provide potential targets for the diagnosis and insight for intervention. We here identify a novel murine gene, hepcarcin (hcn), encoding a 7-kb mRNA-like transcript. The gene appears to be the murine ortholog of the human alpha gene, that is, MALAT-1. The gene and homologs lack credible open reading frames, consistent with a highly conserved large noncoding RNA (ncRNA). In all nodules of procarcinogen-induced murine hepatocellular carcinomas (HCCs) and human HCCs, expression was markedly elevated compared to the uninvolved liver. Quantitative analyses indicated a 6-7-fold increased RNA level in HCCs versus uninvolved liver, advancing this as a molecule of interest. This ncRNA was overexpressed in all five non-hepatic human carcinomas analysed, consistent with a potential marker for neoplastic cells and potential participant in the molecular cell biology of neoplasia.

Amino Acid Sequence↗
PubMed2006

Non-coding-RNA regulators of RNA polymerase II transcription.

Several non-coding RNAs (ncRNAs) that regulate eukaryotic mRNA transcription have recently been discovered. Their mechanisms of action and biological roles are extremely diverse, which indicates that, so far, we have only had a glimpse of this new class of regulatory factor. Many surprises are likely to be revealed as further ncRNA transcriptional regulators are identified and characterized.

Aptamers, Nucleotide↗
PubMed2006

RNAdb 2.0--an expanded database of mammalian non-coding RNAs.

RNAdb is a comprehensive database of mammalian non-protein-coding RNAs (ncRNAs). There is increasing recognition that ncRNAs play important regulatory roles in multicellular organisms, and there is an expanding rate of discovery of novel ncRNAs as well as an increasing allocation of function. In this update to RNAdb, we provide nucleotide sequences and annotations for tens of thousands of non-housekeeping ncRNAs, including a wide range of mammalian microRNAs, small nucleolar RNAs and larger mRNA-like ncRNAs. Some of these have documented functions and/or expression patterns, but the majority remain of unclear significance, and include PIWI-interacting RNAs, ncRNAs identified from the latest rounds of large-scale cDNA sequencing projects, putative antisense transcripts, as well as ncRNAs predicted on the basis of structural features and alignments. Improvements to the database comprise not only new and updated ncRNA datasets, but also provision of microarray-based expression data and closer interface with more specialized ncRNA resources such as miRBase and snoRNA-LBME-db. To access RNAdb, visit http://research.imb.uq.edu.au/RNAdb.

Animals↗
PubMed2005

Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus.

By generating a specialized cDNA library from the archaeon Sulfolobus solfataricus, we have identified 57 novel small non-coding RNA (ncRNA) candidates and confirmed their expression by Northern blot analysis. The majority was found to belong to one of two classes, either antisense or antisense-box RNAs, where the latter only exhibit partial complementarity to RNA targets. The most prominent group of antisense RNAs is transcribed in the opposite orientation to the transposase genes, encoded by insertion elements (transposons). Thus, these antisense RNAs may regulate transposition of insertion elements by inhibiting expression of the transposase mRNA. Surprisingly, the class of antisense RNAs also contained RNAs complementary to tRNAs or sRNAs (small-nucleolar-like RNAs). For the antisense-box ncRNAs, the majority could be assigned to the class of C/D sRNAs, which specify 2'-O-methylation sites on rRNAs or tRNAs. Five C/D sRNAs of this group are predicted to target methylation at six sites in 13 different tRNAs, thus pointing to the widespread role of these sRNA species in tRNA modification in Archaea. Another group of antisense-box RNAs, lacking typical C/D sRNA motifs, was predicted to target the 3'-untranslated regions of certain mRNAs. Furthermore, one of the ncRNAs that does not show antisense elements is transcribed from a repeat unit of a cluster of small regularly spaced repeats in S. solfataricus which is potentially involved in replicon partitioning. In conclusion, this is the first report of stably expressed antisense RNAs in an archaeal species and it raises the prospect that antisense-based mechanisms are also used widely in Archaea to regulate gene expression.

Base Sequence↗
PubMed2006

Escherichia coli Hfq binds A18 and DsrA domain II with similar 2:1 Hfq6/RNA stoichiometry using different surface sites.

Hfq is a RNA-binding protein in Escherichia coli that plays an essential role in post-transcriptional regulation of mRNAs by facilitating pairing of noncoding RNAs (ncRNAs) to mRNA target sites. Recent work has provided evidence that E. coli Hfq has two distinct RNA-binding surfaces. In this study, a comparative sequence-structure analysis of hfq genes in bacterial genomes was employed to identify conserved residues that may be involved in binding RNA. A covariance of residue properties at neighboring positions 12 and 39 and conserved surface residues with high propensities at binding sites of RNA-binding proteins suggested several sites for Hfq-RNA interactions. On the basis of these predictions, eight mutant Hfq proteins were produced and their interactions were examined with the 38 nucleotide (nt) domain II of DsrA ncRNA (DsrA(DII)) and A(18) by a gel-mobility shift assay, fluorescence anisotropy, and fluorescence quenching. Mutations on the proximal surface of Hfq had a small affect on Hfq binding to A(18) (<or=2-fold), while the mutations Y25A and K31A on the distal surface decreased affinity to A(18) by 100-fold in solution. Mutations F39A and R16A on the proximal surface reduced affinity to DsrA(DII) by 6-8-fold, while other mutations on the distal or proximal surfaces affected affinity to DsrA(DII) by <or=2-fold using the gel-mobility shift assay. The F39A/L12F double mutation partially regained the affinity for DsrA(DII) lost by the F39A mutation. The latter observation is consistent with the implied importance of an aromatic residue at position 12 or 39 suggested by the sequence covariance. Titration experiments indicate a 2:1 Hfq(6)/RNA stoichiometry for the strong binding complexes of Hfq with either A(18) or DsrA(DII) and suggests that RNA-induced dimer formation of Hfq(6) is a common feature of Hfq-RNA interactions.

Amino Acid Sequence↗