42 research outputs found
Circular RNAs expression and function in myogenesis
No full textSkeletal myogenesis is a well-characterized biological process driving the formation of the adult muscle tissue from mesodermal progenitors. The key molecular and cellular steps of such process can be replicated in vitro, by culturing muscle precursor cells called myoblasts, which can be induced to differentiate into mature myotubes. We characterized the role of a long non-coding RNA, linc-MD1, in regulating in vitro differentiation of murine myoblasts by acting as a molecular decoy for two microRNAs, miR-133 and miR-135, and we further dissected the regulation of linc-MD1 biosynthesis by the RNA binding protein HuR. Searching for new non-canonical RNA species involved in myogenesis, we directed our attention to circular RNAs (circRNAs). Circular RNAs are produced by the spliceosome via a particular reaction called back-splicing, which links a donor splice site to an upstream acceptor site, thereby generating a covalently closed RNA molecule. We performed high-throughput expression profiling of circRNAs in murine and human cells and studied the principles of their regulation during myogenesis. We then applied a high-content functional genomic screen of conserved circRNAs and found that these molecules are actively involved in the control of myoblasts differentiation. Among them, we further characterized circ-ZNF609 and found that it is able to control myoblast proliferation, providing the first example of a circular RNA involved in a relevant biological process. Moreover, sequence and biochemical analyses suggested that circ-ZNF609 might be translated in a functional protein, representing a possible molecular mechanism for circRNA function
Biogenesis and function of non-coding RNAs in muscle differentiation and in Duchenne muscular dystrophy
No full textIt is now becoming largely accepted that the non-coding portion of the genome, rather than its coding counterpart, is likely to account for the greater complexity of higher eukaryotes. Moreover, non-coding RNAs have been demonstrated to participate in regulatory circuitries that are crucial for development and differentiation. Whereas the biogenesis and function of small non-coding RNAs, particularly miRNAs (microRNAs), has been extensively clarified in many eukaryotic systems, very little is known about the long non-coding counterpart of the transcriptome. In the present review, we revise the current knowledge of how small non-coding RNAs and IncRNAs (long non-coding RNAs) impinge on circuitries controlling proper muscle differentiation and homoeostasis and how their biogenesis is regulated. Moreover, we provide new insights into an additional mechanism of post-transcriptional regulation mediated by IncRNAs, which, acting as miRNA 'sponges', have an impact on the distribution of miRNA molecules on their targets with features similar to those described for ceRNAs (competing endogenous RNAs).It is now becoming largely accepted that the non-coding portion of the genome, rather than its coding counterpart, is likely to account for the greater complexity of higher eukaryotes. Moreover, non-coding RNAs have been demonstrated to participate in regulatory circuitries that are crucial for development and differentiation. Whereas the biogenesis and function of small non-coding RNAs, particularly miRNAs (microRNAs), has been extensively clarified in many eukaryotic systems, very little is known about the long non-coding counterpart of the transcriptome. In the present review, we revise the current knowledge of how small non-coding RNAs and lncRNAs (long non-coding RNAs) impinge on circuitries controlling proper muscle differentiation and homoeostasis and how their biogenesis is regulated. Moreover, we provide new insights into an additional mechanism of post-transcriptional regulation mediated by lncRNAs, which, acting as miRNA 'sponges', have an impact on the distribution of miRN
Exon skipping and duchenne muscular dystrophy therapy: selection of the most active U1 snRNA antisense able to induce dystrophin exon 51 skipping.
No full textOne promising approach for the gene therapy of Duchenne muscular dystrophy (DMD) is exon skipping. When thinking of possible intervention on human, it is very crucial to identify the most appropriate antisense sequences able to provide the highest possible skipping efficiency. In this article, we compared the exon 51 skipping activity of 10 different antisense molecules, raised against splice junctions and/or exonic splicing enhancers (ESEs), expressed as part of the U1 small nuclear RNA (snRNA). The effectiveness of each construct was tested in human DMD myoblasts carrying the deletion of exons 48-50, which can be treated with skipping of exon 51. Our results show that the highest skipping activity and dystrophin rescue is achieved upon expression of a U1 snRNA-derived antisense molecule targeting exon 51 splice sites in combination with an internal exon sequence. The efficacy of this molecule was further proven on an exon 45-50 deletion background, utilizing patient's fibroblasts transdifferentiated into myoblasts. In this system, we showed that the selected antisense was able to produce 50% skipping of exon 51
Multifunctional system-on-glass for lab-on-chip applications
No full textLab-on-Chip are miniaturized systems able to perform biomolecular analysis in shorter time and with lower reagent consumption than a standard laboratory. Their miniaturization interferes with the multiple functions that the biochemical procedures require. In order to address this issue, our paper presents, for the first time, the integration on a single glass substrate of different thin film technologies in order to develop a multifunctional platform suitable for on-chip thermal treatments and on-chip detection of biomolecules. The proposed System on-Glass hosts thin metal films acting as heating sources; hydrogenated amorphous silicon diodes acting both as temperature sensors to monitor the temperature distribution and photosensors for the on-chip detection and a ground plane ensuring that the heater operation does not affect the photodiode currents. The sequence of the technological steps, the deposition temperatures of the thin films and the parameters of the photolithographic processes have been optimized in order to overcome all the issues of the technological integration. The device has been designed, fabricated and tested for the implementation of DNA amplification through the Polymerase Chain Reaction (PCR) with thermal cycling among three different temperatures on a single site. The glass has been connected to an electronic system that drives the heaters and controls the temperature and light sensors. It has been optically and thermally coupled with another glass hosting a microfluidic network made in polydimethylsiloxane that includes thermally actuated microvalves and a PCR process chamber. The successful DNA amplification has been verified off-chip by using a standard fluorometer
The miR-223 host non-coding transcript linc-223 induces IRF4 expression in acute myeloid leukemia by acting as a competing endogenous RNA
No full textAlterations in genetic programs required for terminal myeloid differentiation and aberrant proliferation characterize acute myeloid leukemia (AML) cells. Here, we identify the host transcript of miR-223, linc-223, as a novel functional long non-coding RNA (lncRNA) in AML. We show that from the primary nuclear transcript, the alternative production of miR-223 and linc-223 is finely regulated during monocytic differentiation. Moreover, linc-223 expression inhibits cell cycle progression and promotes monocytic differentiation of AML cells. We also demonstrate that endogenous linc-223 localizes in the cytoplasm and acts as a competing endogenous RNA for miR-125-5p, an oncogenic microRNA in leukemia. In particular, we show that linc-223 directly binds to miR-125-5p and that its knockdown increases the repressing activity of miR-125-5p resulting in the downregulation of its target interferon regulatory factor 4 (IRF4), which it was previously shown to inhibit the oncogenic activity of miR-125-5p in vivo. Furthermore, data from primary AML samples show significant downregulation of linc-223 in different AML subtypes. Therein, these findings indicate that the newly identified lncRNA linc-223 may have an important role in myeloid differentiation and leukemogenesis, at least in part, by cross-talking with IRF4 mRNA
Circ-ZNF609 regulates G1-S progression in rhabdomyosarcoma
No full textCircular RNAs (circRNAs) represent a class of covalently closed RNAs, derived from non-canonical splicing events, which are expressed in all eukaryotes and often conserved among different species. We previously showed that the circRNA originating from the ZNF609 locus (circ-ZNF609) acts as a crucial regulator of human primary myoblast growth: indeed, the downregulation of the circRNA, and not of its linear counterpart, strongly reduced the proliferation rate of in vitro cultured myoblasts. To deepen our knowledge about circ-ZNF609 role in cell cycle regulation, we studied its expression and function in rhabdomyosarcoma (RMS), a pediatric skeletal muscle malignancy. We found that circ-ZNF609 is upregulated in biopsies from the two major RMS subtypes, embryonal (ERMS) and alveolar (ARMS). Moreover, we discovered that in an ERMS-derived cell line circ-ZNF609 knock-down induced a specific block at the G1-S transition, a strong decrease of p-Akt protein level and an alteration of the pRb/Rb ratio. Regarding p-Akt, we were able to show that circ-ZNF609 acts by counteracting p-Akt proteasome-dependent degradation, thus working as a new regulator of cell proliferation-related pathways. As opposed to ERMS-derived cells, the circRNA depletion had no cell cycle effects in ARMS-derived cells. Since in these cells the p53 gene resulted downregulated, with a concomitant upregulation of its cell cycle-related target genes, we suggest that this could account for the lack of circ-ZNF609 effect in ARMS. © 2019, The Author(s)
A Long Noncoding RNA Controls Muscle Differentiation by Functioning as a Competing Endogenous RNA
No full textRecently, a new regulatory circuitry has been identified in which RNAs can crosstalk with each other by competing for shared microRNAs. Such competing endogenous RNAs (ceRNAs) regulate the distribution of miRNA molecules on their targets and thereby impose an additional level of post-transcriptional regulation. Here we identify a muscle-specific long noncoding RNA, linc-MD1, which governs the time of muscle differentiation by acting as a ceRNA in mouse and human myoblasts. Downregulation or overexpression of linc-MD1 correlate with retardation or anticipation of the muscle differentiation program, respectively. We show that linc-MD1 "sponges" miR-133 and miR-135 to regulate the expression of MAML1 and MEF2C, transcription factors that activate muscle-specific gene expression. Finally, we demonstrate that linc-MD1 exerts the same control over differentiation timing in human myoblasts, and that its levels are strongly reduced in Duchenne muscle cells. We conclude that the ceRNA network plays an important role in muscle differentiation
The lack of the Celf2a splicing factor converts a Duchenne genotype into a Becker phenotype
No full textSubstitutions, deletions and duplications in the dystrophin gene lead to either the severe Duchenne muscular dystrophy (DMD) or mild Becker muscular dystrophy depending on whether out-of-frame or in-frame transcripts are produced. We identified a DMD case (GSΔ44) where the correlation between genotype and phenotype is not respected, even if carrying a typical Duchenne mutation (exon 44 deletion) a Becker-like phenotype was observed. Here we report that in this patient, partial restoration of an in-frame transcript occurs by natural skipping of exon 45 and that this is due to the lack of Celf2a, a splicing factor that interacts with exon 45 in the dystrophin pre-mRNA. Several experiments are presented that demonstrate the central role of Celf2a in controlling exon 45 splicing; our data point to this factor as a potential target for the improvement of those DMD therapeutic treatments, which requires exon 45 skipping
C/EBPα-p30 protein induces expression of the oncogenic long non-coding RNA UCA1 in acute myeloid leukemia
No full textAccumulating evidences indicate that different long non-coding RNAs (lncRNAs) might play a relevant role in tumorigenesis, with their expression and function already associated to cancer development and progression. CCAAT/enhancer-binding protein-α (CEBPA) is a critical regulator of myeloid differentiation whose inactivation contributes to the development of acute myeloid leukemia (AML). Mutations in C/EBPα occur in around 10% of AML cases, leading to the expression of a 30-kDa dominant negative isoform (C/EBPα-p30). In this study, we identified the oncogenic urothelial carcinoma associated 1 (UCA1) lncRNA as a novel target of the C/EBPα-p30. We show that wild-type C/EBPα and C/EBPα-p30 isoform can bind the UCA1 promoter but have opposite effects on UCA1 expression. While wild-type C/EBPα represses, C/EBPα-p30 can induce UCA1 transcription. Notably, we also show that UCA1 expression increases in cytogenetically normal AML cases carrying biallelic CEBPA mutations. Furthermore, we demonstrate that UCA1 sustains proliferation of AML cells by repressing the expression of the cell cycle regulator p27kip1. Thus, we identified, for the first time, an oncogenic lncRNA functioning in concert with the dominant negative isoform of C/EBPα in AML
Linc-NeD125 establishes a ceRNA network in Group 4 Medulloblastoma
No full textLong noncoding RNAs (lncRNAs) are regarded as crucial regulators of
cellular processes in Eukaryotes. About 40% of currently characterized
lncRNAs are specifically expressed in central nervous system,
where they are involved in critical neural functions. Consistently,
lncRNA aberrant expression is associated to neurological disorders.
We recently identified a novel human lncRNA, linc-NeD125,
that is induced in response to neuronal differentiation stimulus
both in tumour cell lines and in embryonic stem cells. Notably,
linc-Ned125 is significantly upregulated in a specific and still
largely uncharacterized subgroup (Group 4) of Medulloblastoma
(MB), the most common malignant paediatric brain tumour.
Combining mechanistic and functional studies, we unveiled a novel
lncRNA-mediated miRNA sponge regulatory network, in which the
cross-talk among linc-NeD125, microRNAs and four Group 4 MB
driver gene transcripts may significantly contribute to Group 4 MB
cancerogenesis
