Letizia Pitto

MicroRNAs couple cell fate and developmental timing in retina

Cell identity is acquired in different brain structures according to a stereotyped timing schedule, by accommodating the proliferation of multipotent progenitor cells and the generation of distinct types of mature nerve cells at precise times. However, the molecular mechanisms coupling the identity of a specific neuron and its birth date are poorly understood. In the neural retina, only late progenitor cells that divide slowly can become bipolar neurons, by the activation of otx2 and vsx1 genes. In Xenopus, we found that Xotx2 and Xvsx1 translation is inhibited in early progenitor cells that divide rapidly by a set of cell cycle-related microRNAs (miRNAs). Through expression and functional screenings, we selected 4 miRNAs—mir-129, mir-155, mir-214, and mir-222—that are highly expressed at early developmental stages in the embryonic retina and bind to the 3′ UTR of Xotx2 and Xvsx1 mRNAs inhibiting their translation. The functional inactivation of these miRNAs in vivo releases the inhibition, supporting the generation of additional bipolar cells. We propose a model in which the proliferation rate and the age of a retinal progenitor are linked to each other and determine the progenitor fate through the activity of a set of miRNAs.

The Proto-Oncogene LRF Is under Post-Transcriptional Control of MiR-20a: Implications for Senescence

MicroRNAs (miRNAs) are short 20–22 nucleotide RNA molecules that act as negative regulators of gene expression via translational repression: they have been shown to play a role in development, proliferation, stress response, and apoptosis. The transcriptional regulator LRF (Leukemia/lymphoma Related Factor) has been shown to prevent p19ARF transcription and consequently to inhibit senescence in mouse embryonic fibroblasts (MEF). Here we report, for the first time, that LRF is post-transcriptionally regulated by miR-20a. Using a gene reporter assay, direct interaction of miR-20a with the LRF 3′UTR is demonstrated. To validate the interaction miR-20a/3′UTR LRF miR-20a was over-expressed, either by transient transfection or retroviral infection, in wild type mouse embryo fibroblasts and in LRF-null MEF derived from LRF knock-out mice. We observed LRF decrease, p19ARF increase, inhibition of cell proliferation and induction of senescence. The comparison of miR-20a activity in wt and LRF-null MEF indicates that LRF is the main mediator of the miR-20a-induced senescence and that other targets are cooperating. As LRF down-regulation/p19ARF induction is always accompanied by E2F1 down-regulation and increase of p16, we propose that all these events act in synergy to accomplish miR-20a-induced senescence in MEF. Senescence has been recently revaluated as a tumor suppressor mechanism, alternative to apoptosis; from this point of view the discovery of new physiological “senescence inducer” appears to be promising as this molecule could be used as anticancer drug.

Triiodothyronine Prevents Cardiac Ischemia/Reperfusion Mitochondrial Impairment and Cell Loss by Regulating miR30a/p53 Axis

Mitochondrial dysfunctions critically affect cardiomyocyte survival during ischemia/reperfusion (I/R) injury. In this scenario p53 activates multiple signaling pathways that impair cardiac mitochondria and promote cell death. p53 is a validated target of miR-30 whose levels fall under ischemic conditions. Although triiodothyronine (T3) rescues post-ischemic mitochondrial activity and cell viability, no data are available on its role in the modulation of p53 signaling in I/R. Here we test the hypothesis that early T3 supplementation in rats inhibits the post I/R activation of p53 pro-death cascade through the maintenance of miRNA 30a expression. In our model, T3 infusion improves the recovery of post-ischemic cardiac performance. At the molecular level, the beneficial effect of T3 is associated with restored levels of miR-30a expression in the area at risk (AAR) that correspond to p53 mRNA downregulation. The concomitant decrease in p53 protein content reduces Bax expression and limits mitochondrial membrane depolarization resulting in preserved mitochondrial function and decreased apoptosis and necrosis extent in the AAR. Also in primary cardiomyocyte culture of neonatal rats, T3 prevents both miR-30a downregulation and p53 raise induced by hypoxia. The regulatory effect of T3 is greatly suppressed by miR-30a knockdown. Overall these data suggest a new mechanism of T3-mediated cardioprotection that is targeted to mitochondria and acts, at least in part, through the regulation of miR-30a/p53 axis.

MicroRNA (miRNA)-mediated Interaction between Leukemia/Lymphoma-related Factor (LRF) and Alternative Splicing Factor/Splicing Factor 2 (ASF/SF2) Affects Mouse Embryonic Fibroblast Senescence and Apoptosis

Leukemia/lymphoma-related factor (LRF) is a transcriptional repressor, which by recruiting histone deacetylases specifically represses p19/ARF expression, thus behaving as an oncogene. Conversely, in mouse embryonic fibroblasts (MEF), LRF inhibition causes aberrant p19ARF up-regulation resulting in proliferative defects and premature senescence. We have recently shown that LRF is controlled by microRNAs. Here we show that LRF acts on MEF proliferation and senescence/apoptosis by repressing miR-28 and miR-505, revealing a regulatory circuit where microRNAs (miRNAs) work both upstream and downstream of LRF. By analyzing miRNA expression profiles of MEF transfected with LRF-specific short interfering RNAs, we found that miR-28 and miR-505 are modulated by LRF. Both miRNAs are predicted to target alternative splicing factor/splicing factor 2 (ASF/SF2), a serine/arginine protein essential for cell viability. In vertebrates, loss or inactivation of ASF/SF2 may result in genomic instability and induce G2 cell cycle arrest and apoptosis. We showed that miR-28 and miR-505 modulate ASF/SF2 by directly binding ASF/SF2 3′-UTR. Decrease in LRF causes a decrease in ASF/SF2, which depends on up-regulation of miR-28 and miR-505. Alteration of each of the members of the LRF/miR-28/miR-505/ASF/SF2 axis affects MEF proliferation and the number of senescent and apoptotic cells. Consistently, the axis is coordinately modulated as cell senescence increases with passages in MEF culture. In conclusion, we show that LRF-dependent miRNAs miR-28 and miR-505 control MEF proliferation and survival by targeting ASF/SF2 and suggest a central role of LRF-related miRNAs, in addition to the role of LRF-dependent p53 control, in cellular homeostasis.

MicroRNA 218 Mediates the Effects of Tbx5a Over-Expression on Zebrafish Heart Development

tbx5, a member of the T-box gene family, encodes one of the key transcription factors mediating vertebrate heart development. Tbx5 function in heart development appears to be exquisitely sensitive to gene dosage, since both haploinsufficiency and gene duplication generate the cardiac abnormalities associated with Holt−Oram syndrome (HOS), a highly penetrant autosomal dominant disease characterized by congenital heart defects of varying severity and upper limb malformation. It is suggested that tight integration of microRNAs and transcription factors into the cardiac genetic circuitry provides a rich and robust array of regulatory interactions to control cardiac gene expression. Based on these considerations, we performed an in silico screening to identify microRNAs embedded in genes highly sensitive to Tbx5 dosage. Among the identified microRNAs, we focused our attention on miR-218-1 that, together with its host gene, slit2, is involved in heart development. We found correlated expression of tbx5 and miR-218 during cardiomyocyte differentiation of mouse P19CL6 cells. In zebrafish embryos, we show that both Tbx5 and miR-218 dysregulation have a severe impact on heart development, affecting early heart morphogenesis. Interestingly, down-regulation of miR-218 is able to rescue the heart defects generated by tbx5 over-expression supporting the notion that miR-218 is a crucial mediator of Tbx5 in heart development and suggesting its possible involvement in the onset of heart malformations.

miR-290 acts as a physiological effector of senescence in mouse embryo fibroblasts

The culture-induced senescence of mouse embryo fibroblasts (MEF) correlates with reduction of cell proliferation. In this work we found that the accumulation of cells with 4C DNA content and the transcriptional change of several microRNAs (miRNAs or miRs) are relevant events in culture senescence. By comparing the miRNA expression profiles of physiologically senescent MEF and that of senescent MEF induced by the downregulation of leukemia-related factor, we identified miR-290 as a common upregulated miRNA. When miR-290 was transfected in presenescent MEF, SA-beta-gal(+) cells and p16, two markers of culture senescence, increased compared with control, indicating that miR-290 is causally involved in senescence. Interestingly, nocodazole (NCZ), which induces G2/M block, increased the percentage of senescent cells as well as the expression of miR-290 and of the tumor suppressor p16, thus mimicking culture senescence. As miR-290 was overexpressed in NCZ-treated cells and it was able to induce senescence in proliferating MEF, we investigated whether miR-290 and NCZ could share common mechanisms of culture senescence. Whereas the induction of SA-beta-gal(+) by miR-290 was not strengthened by coupling its transfection with NCZ treatment, the transfection of the antagomir 290 (d-290) plus NCZ treatment, while blocking cells at G2/M, suppressed SA-beta-gal(+) and p16 induction. On the basis of these findings we conclude that miR-290 might act as a physiological effector of NCZ induced as well as culture senescence via p16 regulation expanding the role of this miRNA from embryonic stem to differentiated cells.

miR-20a and miR-290, multi-faceted players with a role in tumourigenesis and senescence.

Expression of microRNAs changes markedly in tumours and evidence indicates that they are causatively related to tumourigenesis, behaving as tumour suppressor microRNAs or onco microRNAs; in some cases they can behave as both depending on the type of cancer. Some tumour suppressor microRNAs appear to be an integral part of the p53 and Retinoblastoma (RB) network, the main regulatory pathways controlling senescence, a major tumour suppressor mechanism. The INK4a/ARF locus which codifies for two proteins, p19ARF and p16INK4a, plays a central role in senescence by controlling both p53 and RB. Recent evidence shows that the proto‐oncogene leukaemia/lymphoma related factor, a p19ARF specific repressor, is controlled by miRNAs and that miRNAs, in particular miR‐20a and miR‐290, are causatively involved in mouse embryo fibroblasts (MEF) senescence in culture. Intriguingly, both miR‐20a, member of the oncogenic miR‐17–92 cluster, and miR‐290, belonging to the miR‐290–295 cluster, are highly expressed in embryonic stem (ES) cells. The pro‐senescence role of miR‐20a and miR‐290 in MEF is apparently in contrast with their proliferative role in tumour and ES cells. We propose that miRNAs may exert opposing functions depending on the miRNAs repertoire as well as target/s level/s present in different cellular contexts, suggesting the importance of evaluating miRNAs activity in diverse genetic settings before their therapeutic use as tumour suppressors.

microRNA(interference) networks are embedded in the gene regulatory networks

microRNAs (miRNAs) are a class of endogenous 22-25nt single-stranded RNA molecules that regulate gene expression post-transcriptionally. They are highly conserved among species with distinct temporal and spatial patterns of expression, each of them potentially interacting with hundreds of messenger RNAs. Since miRNAs, like transcription factors (TFs), are trans-acting factors that interact with cis-regulatory elements, they potentially generate a complex combinatorial code. Moreover, as TFs and genes containing binding sites for TFs have a high probability of being targeted by miRNAs, the basic interplay miRNA/TF renders miRNAs key components of gene regulatory networks. Several biological processes, including diseases such as cancer, have been causatively associated to disturbances of miRNAs/TF interplay both in vitro and in vivo. These aspects, cumulatively, indicate that miRNAs and transcription factors have a crucial role in determining cellular behaviour, highlighting the role of small RNA molecules in regulatory mechanisms and indicating other routes in the evolutionary path of gene expression.

Characterization of carrot nuclear proteins that exhibit specific binding affinity towards conventional and non-conventional DNA methylation.

DNA methylation is associated with transcriptional silencing in vertebrates and plants. In mammals, the effects of methylation are mediated by a family of methyl-CpG-binding proteins. In plants the mechanisms by which methylation represses transcription are still not clear. In this paper we describe protein factors in carrot nuclear extracts exhibiting specific affinities for conventional or non-conventional methylation acceptor sites. We characterized two classes of proteins: the first, dcMBP1 (Daucus carota methylated DNA-binding protein 1), shows high affinity for sequences containing 5-methylcytosine; the second, dcMBP2 (Daucus carota methylated DNA-binding protein 2), efficiently complexes sequences containing 5-methylcytosine in both CpXpX and CpXpG trinucleotides and shows much lower affinity for 5-methyl CpG dinucleotides. Both dcMBP1 and dcMBP2 are abundant proteins differing in molecular weight and binding features. Their activities are modulated during carrot vegetative cell growth and somatic embryo development. This is the first time that, in either plants or mammals, proteins exhibiting specific binding affinities for conventional or non-conventional DNA methylation have been shown. Based on these results, the possibility that both the extent and the context of the methylation might contribute to modulate gene expression is discussed.

Holt-Oram syndrome with intermediate atrioventricular canal defect, and aortic coarctation: Functional characterization of a de novo TBX5 mutation

Holt–Oram syndrome (HOS) is a rare autosomal dominant disorder characterized by upper limb defects and congenital heart defects (CHD), which are often simple septal and conduction defects, less frequently complex CHDs. We report on a 9 year‐old boy with clinical and radiologic features of HOS consisting of bilateral asymmetric hypoplastic thumbs, generalized brachydactyly, limited supination due to radioulnar synostosis, and sloping shoulders, and intermediate atrioventricular canal defect (AVCD) with aortic coarctation. A de novo, previously described mutation, (Arg279ter) was identified in the TBX5 gene. Molecular characterization of this mutation was carried out due to the atypical CHD. In order to investigate whether the mutated transcript of TBX5 was able to escape the post‐transcriptional surveillance mechanism and to produce a truncated TBX5 protein, we analyzed the TBX5 transcript, and protein pattern in HOS, and WT cardiac tissues. Our results demonstrate that the mutant TBX5 transcript is cleared by the cellular mechanism of surveillance. This data provides some support for the hypothesis that a dominant negative mutation, which strongly impairs the WT allele, might be too hazardous to be maintained. The literature suggests that HOS is relatively common among syndromes associated with AVCD.