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Mechanism and physiological significance of the rapid microRNA catabolism in retinal and non-retinal neurons

Applicant Filipowicz Witold
Number 129941
Funding scheme Project funding (Div. I-III)
Research institution Friedrich Miescher Institute for Biomedical Research
Institution of higher education Institute Friedrich Miescher - FMI
Main discipline Molecular Biology
Start/End 01.04.2010 - 31.03.2012
Approved amount 200'000.00
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All Disciplines (2)

Discipline
Molecular Biology
Neurophysiology and Brain Research

Keywords (12)

microRNA; neuron; retina; RNA metabolism; translational regulation; gene expression; vision; dendrites; synapses; RNA turover; nucleases; deep sequencing

Lay Summary (English)

Lead
Lay summary
Background: MicroRNAs (miRNAs) represent a new class of small non-coding RNA regulators which repress protein synthesis by either inhibiting translation or causing mRNA destabilization. Particularly many miRNAs are specifically expressed in neuronal cells, including retinal neurons, consistent with an evidence of importance of miRNAs for brain development and function. In neurons, miRNAs are implicated in the regulation of translation at synaptic connections in dendrites in response to synaptic stimulation but mechanistic aspects of this regulation are not understood. We have recently found that miRNAs in retinal neurons turnover much faster than miRNAs in non-neuronal cells. The fast miRNA turnover is also characteristic of hippocampal and cortical neurons, and neurons differentiated from mouse embryonic stem cells (ESCs). Importantly, blocking action potentials or glutamate receptors with specific inhibitors reduced miRNA turnover, indicating that active miRNA metabolism may be important for the function of both retinal and non-retinal neurons. Project and its possible outcome: The aim of the project is to dissect a molecular mechanism of the activity-regulated miRNA turnover and to gain insight to its physiological role in neurons. We will investigate whether rapid miRNA turnover occurs in both soma and neuronal processes, or in only one of these compartments. We will use purified populations of specific neuronal cells isolated from mouse retinas subjected to different physiological and pharmacological conditions. For non-retinal studies, we will use mouse glutamatergic ESCs-derived neurons and primary dissociated hippocampal neurons. Multi-electrode array recordings and other assays will be used to correlate electrophysiological properties and miRNA turnover. Deep sequencing will provide information about intermediates in miRNA degradation in neurons and will help to identify cellular enzymes involved.The proposed research will determine whether the activity-regulated miRNA turnover applies to all types of neurons and all neuronal miRNAs. It will help to understand how modulation of neuronal activity by pharmacological agents or light affects miRNA decay and miRNA-mediated repression. The integrative approach involving research on different classes of neurons will help to establish general principles of miRNA metabolism and function, and their role in synaptic plasticity in neurons. Our findings may have implications for designing future therapeutic interventions in neuropathies or malfunction of the visual system.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Regulation of miRNA Biogenesis, Function and Decay
Krol Jacek Loedige Inga Filipowicz Witold (2010), Regulation of miRNA Biogenesis, Function and Decay, in Nature Reviews Genetics, 11, 597-615.

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Keystone Symposium "Protein-RNA interactions in Biology & Disease" 04.03.2012 Santa Fe, USA
FEBS Congress 16.06.2011 Torina, Italy
EMBO Symposium "The Non-coding Genome" 16.06.2010 Heidelberg, Germany


Awards

Title Year
"Ambizione" SNF Grant/Award 2011
Life Achievement in Science Award of RNA Society 2011

Associated projects

Number Title Start Funding scheme
136744 Regulatory function of microRNAs in mouse retinal neurons 01.01.2012 Ambizione

Abstract

SUMMARYMicroRNAs (miRNAs) are a class of post-transcriptional regulators of gene expression which repress protein synthesis by either inhibiting translation of mRNAs or causing their destabilization. Hundreds of different miRNAs have been identified in mammals. They are implicated in control of all fundamental cellular processes and most of them are expressed in a development- or tissue-specific manner. Particularly many miRNAs are specifically expressed or enriched in neuronal cells, including retinal neurons, consistent with a growing evidence of importance of miRNAs for brain development and function. Although details of miRNA biogenesis and its regulation are quite well established, little is known about catabolism of mature miRNAs. miRNAs are generally assumed to have a very long half-life, corresponding to many hours or even days. However, such slow turnover may not be a universal feature of miRNAs since they often play a role in developmental transitions or act as “on” and “off” switches, conditions which call for a more active miRNA metabolism. In neurons, miRNAs are implicated in the regulation of translation at dendritic spines in response to synaptic stimulation that may also require local rapid turnover of miRNAs.We have recently identified miRNAs which are reversibly up- and down-regulated in vivo in the mouse retina during dark-light adaptation, independent of the circadian rhythm. The sensory neuron-specific miR-182/183/96 cluster, and a small number of other miRNAs, was down-regulated during dark adaptation and up-regulated in light, with rapid miRNA decay and increased transcription being responsible for the changes. We found that miRNAs in retinal neurons, both light-regulated and constitutively expressed, turnover much faster (T1/2 of 1 h or less) than miRNAs in non-neuronal cells. We further demonstrated that the fast turnover of miRNAs is also characteristic of hippocampal and cortical neurons, and neurons differentiated from mouse embryonic stem (ES) cells in vitro. Importantly, blocking action potentials with tetrodotoxin (TTX) reduced miRNA turnover, indicating that active miRNA metabolism may be important for the function of neurons. Collectively, these results indicated that metabolism of miRNAs in neurons is higher than in most other cells and that a link exists between neuronal activity and turnover of miRNAs.We intend to dissect a molecular mechanism of the activity-regulated miRNA turnover and gain some insight to its physiological role in retinal and non-retinal neurons. We will investigate miRNA turnover in isolated cell-body and synaptic retinal layers and in purified populations of specific neuronal cells isolated from retinas subjected to different physiological and pharmacological conditions in vivo. For non-retinal studies, we will use mouse glutamatergic neurons differentiated in vitro from ES cells and also primary dissociated hippocampal neurons and cultured hippocampal brain slices. We will search for agents other than TTX which modify neuronal activity and modulate miRNA turnover. Multi-electrode array (MEA) recordings and other assays will be used to correlate electrophysiological properties and miRNA turnover. Deep sequencing of small RNAs will provide information about directionality of miRNA degradation and intermediates of miRNA catabolism in different types of neurons. The nucleases involved will be studied in ES cell-derived neurons expressing specific knock-down hairpins and by in vitro approaches. Finally, we will use cell lines expressing different classes of mRNA reporters targeted by neuronal miRNAs to investigate the mechanism of miRNA repression in neurons and the effect of neuronal activity on miRNA-mediated silencing.
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