Gama:Projects

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=== RNA regulation in the somite clock ===
=== RNA regulation in the somite clock ===
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The formation of somites, during embryonic development, is highly regulated both temporally and spatially. The main effectors of this regulation are a set of cyclic genes, that display an oscillating pattern of expression, and a morphogenetic gradient, which constitutes the wavefront of differentiation. Wilts the oscillating behavior of the cyclic genes requires a certain instability at the mRNA level, the wavefront gradient is formed by the slow degradation of mRNA. Our aim is the study and characterization of the mechanisms that regulate mRNA stability in this context. Using Zebrafish as a model organism, we are combining bioinformatic and biochemical approaches with ''in vivo'' studies.
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The formation of somites, during embryonic development, is highly regulated both temporally and spatially. The main effectors of this regulation are a set of cyclic genes, that display an oscillating pattern of expression, and a morphogenetic gradient, which constitutes the wavefront of differentiation. Wilts the oscillating behavior of the cyclic genes requires a certain instability at the mRNA level, the wavefront gradient is formed by the slow degradation of mRNA. Our aim is the study and characterization of the mechanisms that regulate mRNA stability in this context. Using Zebrafish as a model organism, we are combining bioinformatic and biochemical approaches with ''in vivo'' studies.
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This project is a collaboration with Dr. Leonor Saúde, Vertebrate Developmental Biology Unit, Institute of Molecular Medicine.
[[Image:Zbf2.png|thumb|center|800px|Zebrafish embryos (left to right) injected with fluorescein, under normal light and injected with Cherry RNA]]
[[Image:Zbf2.png|thumb|center|800px|Zebrafish embryos (left to right) injected with fluorescein, under normal light and injected with Cherry RNA]]

Revision as of 14:15, 15 April 2011

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Contents

Ongoing projects

Post transcriptional control of SMN2 expression

Spinal muscular atrophy (SMA), a leading cause of inherited infant mortality, is a severe neuromuscular disorder caused by loss of function of the SMN1 gene. In spite of significant research efforts undertaken since the identification of the disease gene, effective therapies for SMA are still not available.

SMA is unlike most loss of function diseases in that a potentially effective therapeutic target has been clearly identified. Indeed, the human genome harbors a copy of the SMN1 gene (termed SMN2), resulting from an inverted duplication within chromosome 5. However, due to a non-coding substitution that alters pre-mRNA splicing, only 25% of the mRNAs produced from SMN2 encode full-length SMN protein. Genetic characterization of mild patients and healthy carriers of the SMA mutation has revealed that the presence of extra copies of SMN2 can reduce and even eliminate the disease phenotype. Thus, therapeutic strategies aiming at increasing the output of SMN protein from the SMN2 gene are likely to provide significant benefits for SMA patients.

Current ongoing research efforts are aimed at regulating SMN2 alternative splicing in order to increase the production of full length SMN protein. Alternatively, large scale drug screens to identify compounds that modulate SMN2 transcription or overall protein levels have been performed with some promising results. However, ideal therapeutic approaches should target specifically the gene of interest to avoid unintended secondary effects.

In the past few years, an impressive body of evidence has demonstrated that a significant part of gene expression control is taking place at the post-transcriptional level, determining the stability and translation efficiency of mRNA molecules. These regulatory switches are under the control of RNA binding proteins and microRNAs that associate predominantly with the 5’ and 3’ UTRs of the mRNA molecule. Blocking the binding sites of these regulators can be achieved by the design of complementary modified small oligonucleotides, which are highly specific for the target gene. This approach has been shown to result in significant changes in protein synthesis and is a promising candidate for therapy. Its application to SMA is however hampered by the lack of studies on similar mechanisms acting on the SMN2 gene.

The aim of this project is to obtain a detailed characterization of post-transcriptional regulatory mechanisms acting on the SMN2 mRNA through the UTR sequence elements, with the long term aim of developing therapeutic approaches for increasing the production of functional SMN protein in SMA patients. Our approach focuese on the identification of both regulatory sequence elements and of the trans-acting factors that bind to them and characterize their functional effects. The knowledge derived from these studies may then be applied in the design of highly specific probes that can enhance SMN protein production.

This project is being developed in collaboration with the group of Dr. Luis Ferreira Moita, at the Institute of Molecular Medicine, University of Lisbon and is supported by a grant from SMA-Europe.

Role of the U2AF65 splicing factor in mRNA regulatory networks

RNA regulation in the somite clock

The formation of somites, during embryonic development, is highly regulated both temporally and spatially. The main effectors of this regulation are a set of cyclic genes, that display an oscillating pattern of expression, and a morphogenetic gradient, which constitutes the wavefront of differentiation. Wilts the oscillating behavior of the cyclic genes requires a certain instability at the mRNA level, the wavefront gradient is formed by the slow degradation of mRNA. Our aim is the study and characterization of the mechanisms that regulate mRNA stability in this context. Using Zebrafish as a model organism, we are combining bioinformatic and biochemical approaches with in vivo studies. This project is a collaboration with Dr. Leonor Saúde, Vertebrate Developmental Biology Unit, Institute of Molecular Medicine.

Zebrafish embryos (left to right) injected with fluorescein, under normal light and injected with Cherry RNA
Zebrafish embryos (left to right) injected with fluorescein, under normal light and injected with Cherry RNA

small RNAs in HIV infection

HIV infection occurs following a series of virus-host interactions that culminate in the integration of viral DNA into the host cell genome. This process results in the generation of a pool of latently infected CD4 T cells, resistant to therapies that target replicating viruses, and constitutes a major viral reservoir. It is hoped that drugs that prevent the establishment of this pool, such as Raltegravir, may help to address this problem. Regulatory small non-coding RNAs are likely to impact on the molecular processes of the HIV life-cycle particularly through the regulation of the cellular pathways of T cell activation. With this reasoning we have performed a deep-sequencing analysis of the profile of small RNA expression in primary human naïve CD4 T cells, and upon T cell activation and HIV infection. Our preliminary data showed a differential expression of miRNAs upon cell activation, and/or HIV infection, which is modulated by Raltegravir. Our results also supported the hypothesis that some miRNAs are distinctly modulated by HIV-1 and HIV-2 and may potentially be implicated in their distinct levels of replication and/or latency. Lastly, our preliminary results show that HIV-1 and HIV-2 infection induces significant changes of other small RNAs, which we are currently investigating. This project is being developed in collaboration with the Clinical Immunology Unit (PI: Ana E. Sousa) and the Retroviruses and Antiviral Research Unit (PI: João Gonçalves) at the Institute of Molecular Medicine, University of Lisbon. This project is being supported by Merck Investigator-Initiated Studies Program (IISP).


microRNA regulation of cardiac stem cells

The mammalian heart has been traditionally considered to be a post-mitotic organ because cardiomyocytes were never seen to divide. This paradigm was challenged by the isolation adult heart cells expressing stemness markers with the ability to proliferate and differentiate into myocardium, endothelial and smooth muscle vascular cells, identifying them as true cardiac stem cells (CSCs. In terms of phenotype, CSCs are undifferentiated, keep quiescent until induced to proliferate and may differentiate into one of the cardiac cell lineages. It is expected that their profile of expressed genes and active control mechanisms should reflect both of these properties: stemness and differentiation The paradigm shift described above is changing our current biological, physiopathological and possibly therapeutic approaches to heart function and disease. We must now consider the mechanisms involved in cardiac organ homeostasis that depend on CSCs and try to understand how specific diseases may affect them. These findings may translate into novel therapies based on CSCs to repair heart damage. However, to fully benefit from their potential, a more in depth understanding of the mechanisms controlling maintenance, proliferation and differentiation of CSCs is needed. Thus, the identification of key regulators of gene expression programs is an essential step for the development of this promising field. During the last decade, microRNAs (miRs) have emerged as important regulators of cell proliferation, differentiation and response to stress. miRs act to regulate gene expression at the post-transcriptional level by influencing the translation and turnover of target mRNA molecules. Through their simultaneous interactions with several mRNAs, miRs seem to constitute a major driving force behind the establishment of cellular phenotypes and responses to external stimuli. In this project we have been working on the identification of microRNAs expressed in adult Cardiac Stem Cells and relate them to the regulation of key aspects of their biology. In particular, we are trying to understand to what degree miRs influence the commitment of these cells to different cardiac lineages and which role do they play in the tight control of their proliferation and differentiation. This project is a collaboration with Luis Brás-Rosário, Cardiac Regeneration Group, Gulbenkian Institute of Science, Oeiras, Portugal.

Collaborations

Ana Espada Sousa, MD/PhD - Clinical Imunology Unit, Institute of Molecular Medicine, University of Lisbon

João Gonçalves, PhD - Retrovirus Unit, Institute of Molecular Medicine, University of Lisbon

Leonor Saúde, PhD - Vertebrate Developmental Biology Unit, Institute of Molecular Medicine, University of Lisbon

Luis Ferreira Moita, MD/PhD - Cell Biology of the Immune System Unit, Institute of Molecular Medicine, University of Lisbon


Luis Brás-Rosário, MD/PhD, Cardiac Regeneration Group, Gulbenkian Institute of Science



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