A cAMP-response element binding protein-induced microRNA regulates neuronal morphogenesis.
Journal: 2005/December - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
Abstract:
MicroRNAs (miRNAs) regulate cellular fate by controlling the stability or translation of mRNA transcripts. Although the spatial and temporal patterning of miRNA expression is tightly controlled, little is known about signals that induce their expression nor mechanisms of their transcriptional regulation. Furthermore, few miRNA targets have been validated experimentally. The miRNA, miR132, was identified through a genome-wide screen as a target of the transcription factor, cAMP-response element binding protein (CREB). miR132 is enriched in neurons and, like many neuronal CREB targets, is highly induced by neurotrophins. Expression of miR132 in cortical neurons induced neurite outgrowth. Conversely, inhibition of miR132 function attenuated neuronal outgrowth. We provide evidence that miR132 regulates neuronal morphogenesis by decreasing levels of the GTPase-activating protein, p250GAP. These data reveal that a CREB-regulated miRNA regulates neuronal morphogenesis by responding to extrinsic trophic cues.
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Proc Natl Acad Sci U S A 102(45): 16426-16431

A cAMP-response element binding protein-induced microRNA regulates neuronal morphogenesis

Vollum Institute, Oregon Health & Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97239; Reed College, 3203 SE Woodstock Boulevard, Portland, OR 97202; and Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan
To whom correspondence may be addressed: E-mail: ude.usho@rnamdoog or ude.usho@syepmi.
N.V. and M.E.K. contributed equally to this work.
Contributed by Richard H. Goodman, September 27, 2005
Contributed by Richard H. Goodman, September 27, 2005

Abstract

MicroRNAs (miRNAs) regulate cellular fate by controlling the stability or translation of mRNA transcripts. Although the spatial and temporal patterning of miRNA expression is tightly controlled, little is known about signals that induce their expression nor mechanisms of their transcriptional regulation. Furthermore, few miRNA targets have been validated experimentally. The miRNA, miR132, was identified through a genome-wide screen as a target of the transcription factor, cAMP-response element binding protein (CREB). miR132 is enriched in neurons and, like many neuronal CREB targets, is highly induced by neurotrophins. Expression of miR132 in cortical neurons induced neurite outgrowth. Conversely, inhibition of miR132 function attenuated neuronal outgrowth. We provide evidence that miR132 regulates neuronal morphogenesis by decreasing levels of the GTPase-activating protein, p250GAP. These data reveal that a CREB-regulated miRNA regulates neuronal morphogenesis by responding to extrinsic trophic cues.

Keywords: neurite, neurotrophin, plasticity, microRNA, transcription
Abstract

Hormones, growth factors, and electrical activity regulate proliferation, differentiation, survival, and plasticity by triggering programs of gene expression. Although many transcription factors respond to environmental cues, the basic leucine zipper transcription factor, cAMP-response element binding protein (CREB), is considered prototypical because it was among the first identified, is expressed widely, and regulates many rapid-response genes (1). Phosphorylation of a conserved residue in its activation domain is required for CREB function (2). Although CREB was identified by virtue of its responsiveness to cAMP signaling, it is activated by an array of other cellular signals, including, but not limited to, neurotrophic factors, cytokines, and neuronal activity. The ability of CREB to recruit the coactivator, CREB binding protein (CBP) (3), in a phosphorylation-dependent manner underlies its capacity to mediate stimulus-induced transcription. CBP recruitment is believed to trigger transcriptional activation via intrinsic or associated acetylase activities and/or by interacting with general transcription factors (4). Recently, signal-dependent nuclear translocation of the coactivator transducer of regulated CREB has also been implicated in CREB activation (5).

The role of CREB in cellular adaptive responses has been studied most extensively in the nervous system. Early experiments revealed that stimuli known to effect neuronal maturation and plasticity, including cAMP signaling, membrane depolarization, and neurotrophins, were robust activators of CREB-dependent gene expression. These observations inspired other studies suggesting that CREB is a central regulator of memory formation and other forms of behavioral adaptation believed to require programs of de novo gene expression (6). Subsequent studies supported a role for CREB as a key regulator of developmental plasticity, addiction, and circadian rhythmicity (1).

A prominent role for CREB in neuronal survival has hampered analysis of its actions in regulating neuronal maturation and function. Nevertheless, a recent study provided strong evidence that CREB mediates neurotrophin-dependent morphogenesis of peripheral neurons (7). Additional studies have suggested a role for CREB in CNS morphogenesis (8, 9). The set of genes that directly mediate these effects has not been defined, however. To gain insight into the molecular mechanisms underlying CREB-regulated plasticity, we developed a genome-wide screen, termed Serial Analysis of Chromatin Occupancy that can profile transcription factor binding sites in an unbiased manner (10). This screen identified hundreds of CREB-binding sites that are tightly associated with noncoding transcripts and microRNAs (miRNAs) (S.I. and R.H.G., unpublished data). In this study, we characterize one of these unique transcripts.

miRNAs were first described in Caenorhabditis elegans (11, 12). Subsequent work demonstrated that these molecules encode 19- to 24-bp double-stranded RNAs that mediate gene silencing (13-15). miRNAs have since been identified in vertebrates and have been proposed to regulate a significant fraction of cellular mRNAs. miRNAs regulate a diverse set of biological functions, including regulation of developmental timing and neuronal asymmetry in C. elegans (11, 12, 16, 17); cell proliferation, suppression of apoptosis (18), and fat metabolism (19) in Drosophila; and hematopoetic and adipocyte differentiation (20, 21), insulin secretion (22), cardiomyocyte development (23), oncogenesis (24-27), and viral defense in mammals (28-30). Mature miRNAs silence gene expression by binding to the 3′UTRs of target mRNAs and promote translational repression or mRNA degradation (31).

Significant strides have been made in deciphering the stepwise processing of miRNAs from their larger precursors (32). The relatively few primary transcripts thus far studied suggest that miRNAs are derived from spliced coding or noncoding transcripts (33-35). Because many miRNA genes are near complex transcriptional loci, the regulatory elements responsible for miRNA biogenesis have been difficult to determine. Most studies indicate that miRNAs are derived from polyadenylated RNA polymerase II transcripts, but polymerase III-dependent mechanisms have also been suggested (36).

By analogy with protein-coding genes, identification of the pathways that control miRNA transcription should provide insight into the biological functions of these molecules. We focused on miRNA 132 (miR132) because it was tightly associated with a CREB-binding site and was highly responsive to neurotrophin signaling, suggesting a role in neuronal differentiation. Overexpression of this miRNA in cortical neurons dramatically increased the sprouting of neuronal processes. Conversely, inactivation of this miRNA reversed this process. We used bioinformatic databases to identify putative targets of miR132 and found that a highly conserved putative target, p250 GTPase-activating protein (GAP), had previously been linked to neuronal differentiation. We confirmed that p250GAP protein levels were controlled by miR132 in cortical neurons and showed that the ability of miR132 to regulate neuronal morphogenesis depends, at least in part, on its ability to target p250GAP.

Acknowledgments

We thank Gail Mandel and Karl Obrietan for their comments on the manuscript. This work was supported by National Institutes of Health Grants NS047176 (to S.I.) and DK45423 (to R.H.G.) and a grant from the Rett Syndrome Research Foundation (to R.H.G.).

Acknowledgments

Notes

Author contributions: R.H.G. and S.I. designed research; N.V., M.E.K., O.V., D.M.K., and S.I. performed research; T.Y. contributed new reagents/analytical tools; N.V., M.E.K., O.V., D.M.K., and S.I. analyzed data; and N.V., M.E.K., R.H.G., and S.I. wrote the paper.

Conflict of interest statement: No conflicts declared.

Abbreviations: CREB, cAMP-response element binding protein; miRNA, microRNA; GAP, GTPase-activating protein; BDNF, brain-derived neurotrophic factor; shRNA, short hairpin RNA; CBP, CREB binding protein; premiR, pre-miRNA.

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. DQ223059).

Notes
Author contributions: R.H.G. and S.I. designed research; N.V., M.E.K., O.V., D.M.K., and S.I. performed research; T.Y. contributed new reagents/analytical tools; N.V., M.E.K., O.V., D.M.K., and S.I. analyzed data; and N.V., M.E.K., R.H.G., and S.I. wrote the paper.
Conflict of interest statement: No conflicts declared.
Abbreviations: CREB, cAMP-response element binding protein; miRNA, microRNA; GAP, GTPase-activating protein; BDNF, brain-derived neurotrophic factor; shRNA, short hairpin RNA; CBP, CREB binding protein; premiR, pre-miRNA.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. DQ223059).

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