InteractiveFly: GeneBrief

mir-277 stem loop: Biological Overview | References

BIOLOGICAL OVERVIEW

Fragile X-associated tremor/ataxia syndrome (FXTAS), a late-onset neurodegenerative disorder, has been recognized in older male fragile X premutation carriers and is uncoupled from fragile X syndrome. Using a Drosophila model of FXTAS, it has been shown that transcribed premutation repeats alone are sufficient to cause neurodegeneration. miRNAs are sequence-specific regulators of post-transcriptional gene expression. To determine the role of miRNAs in rCGG repeat-mediated neurodegeneration, miRNA expression was profiled, and selective miRNAs were identified, including miR-277, that are altered specifically in Drosophila brains expressing rCGG repeats. Their genetic interactions with rCGG repeats were tested, and it was found that miR-277 can modulate rCGG repeat-mediated neurodegeneration. Furthermore, Drep-2 and Vimar were identified as functional targets of miR-277 that could modulate rCGG repeat-mediated neurodegeneration. Finally, it was found that hnRNP A2/B1, an rCGG repeat-binding protein, can directly regulate the expression of miR-277. These results suggest that sequestration of specific rCGG repeat-binding proteins could lead to aberrant expression of selective miRNAs, which may modulate the pathogenesis of FXTAS by post-transcriptionally regulating the expression of specific mRNAs involved in FXTAS (Tan, 2012).

Fragile X syndrome (FXS), the most common form of inherited mental retardation, is caused by expansion of the rCGG trinucleotide repeat in the 5' untranslated region (5' UTR) of the fragile X mental retardation 1 (FMR1) gene, which leads to silencing of its transcript and the loss of the encoded fragile X mental retardation protein (FMRP). Most affected individuals have more than 200 rCGG repeats, referred to as full mutation alleles. Fragile X syndrome carriers have FMR1 alleles, called premutations, with an intermediate number of rCGG repeats between patients (>200 repeats) and normal individuals (<60 repeats). Recently, the discovery was made that male and, to a lesser degree, female premutation carriers are at greater risk of developing an age-dependent progressive intention tremor and ataxia syndrome, which is uncoupled from fragile X syndrome and known as fragile X-associated tremor/ataxia syndrome (FXTAS). This is combined with cognitive decline associated with the accumulation of ubiquitin-positive intranuclear inclusions broadly distributed throughout the brain in neurons, astrocytes, and in the spinal column (Tan, 2012).

At the molecular level, the premutation is different from either the normal or full mutation alleles. Based on the observation of significantly elevated levels of rCGG-containing FMR1 mRNA, along with either no detectable change in FMRP or slightly reduced FMRP levels in premutation carriers, an RNA-mediated gain-of-function toxicity model has been proposed for FXTAS. Several lines of evidence in mouse and Drosophila models further support the notion that transcription of the CGG repeats leads to this RNA-mediated neurodegenerative disease (Greco, 2006; Jin, 2003; Willemsen, 2003; Arocena, 2005). The hypothesis is that specific RNA-binding proteins may be sequestered by overproduced rCGG repeats in FXTAS and become functionally limited, thereby contributing to the pathogenesis of this disorder (Jin, 2003; Willemsen, 2003; Arocena, 2005; Tassone, 2004). There are three RNA-binding proteins found to modulate rCGG-mediated neuronal toxicity: Pur α, hnRNP A2/B1, and CUGBP1, which bind rCGG repeats either directly (Pur α and hnRNP A2/B1) or indirectly (CUGBP1, through the interaction with hnRNP A2/B1) (Tan, 2012).

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression at the post-transcriptional level by targeting mRNAs, leading to translational inhibition, cleavage of the target mRNAs or mRNA decapping/deadenylation. Mounting evidence suggests that miRNAs play essential functions in multiple biological pathways and diseases, from developmental timing, fate determination, apoptosis, and metabolism to immune response and tumorigenesis. Recent studies have shown that miRNAs are highly expressed in the central nervous system (CNS), and some miRNAs have been implicated in neurogenesis and brain development (Tan, 2012).

Interest in the functions of miRNAs in the CNS has recently expanded to encompass their roles in neurodegeneration. Investigators have begun to reveal the influence of miRNAs on both neuronal survival and the accumulation of toxic proteins that are associated with neurodegeneration, and are uncovering clues as to how these toxic proteins can influence miRNA expression. For example, miR-133b is found to regulate the maturation and function of midbrain dopaminergic neurons (DNs) within a negative feedback circuit that includes the homeodomain transcription factor Pitx3 in Parkinson's disease. In addition, reduced miR-29a/b-1-mediated suppression of BACE1 protein expression contributes to Aβ accumulation and Alzheimer's disease pathology. Moreover, the miRNA bantam is found to be a potent modulator of poly-Q- and tau-associated degeneration in Drosophila. Other specific miRNAs have also been linked to other neurodegenerative disorders, such as spinocerebellar ataxia type 1 (SCA1) and Huntington's disease (HD). Therefore, miRNA-mediated gene regulation could be a novel mechanism, adding a new dimension to the pathogenesis of neurodegenerative disorders (Tan, 2012).

This study shows that fragile X premutation rCGG repeats can alter the expression of specific miRNAs, including miR-277, in a FXTAS Drosophila model. miR-277 modulates rCGG-mediated neurodegeneration. Furthermore, Drep-2, which is associated with the chromatin condensation and DNA fragmentation events of apoptosis, and Vimar, a modulator of mitochondrial function, were identified two of the mRNA targets regulated by miR-277. Functionally, Drep-2 and Vimar could modulate the rCGG-mediated neurodegeneration, as well. Finally, hnRNP A2/B1, an rCGG repeat-binding protein, can directly regulate the expression of miR-277. These data suggest that hnRNP A2/B1 could be involved in the transcriptional regulation of selective miRNAs, and fragile X premutation rCGG repeats could alter the expression of specific miRNAs, potentially contributing to the molecular pathogenesis of FXTAS (Tan, 2012).

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder that afflicts fragile X syndrome premutation carriers, with earlier studies pointing to FXTAS as an RNA-mediated neurodegenerative disease. Several lines of evidence suggest that rCGG premutation repeats may sequester specific RNA-binding proteins, namely Pur α, hnRNP A2/B1, and CUGBP1, and reduce their ability to perform their normal cellular functions, thereby contributing significantly to the pathology of this disorder. The miRNA pathway has been implicated in the regulation of neuronal development and neurogenesis. A growing body of evidence has now revealed the role of the miRNA pathway in the molecular pathogenesis of neurodegenerative disorders. This study demonstrates that specific miRNAs can contribute to fragile X rCGG repeat-mediated neurodegeneration by post-transcriptionally regulating target mRNAs that are involved in FXTAS. miR-277 plays a significant role in modulating rCGG repeat-mediated neurodegeneration. Overexpression of miR-277 enhances rCGG repeat-induced neuronal toxicity, whereas blocking miR-277 activity could suppress rCGG repeat-mediated neurodegeneration. Furthermore, Drep-2 and Vimar were identified as the functional miR-277 targets that could modulate rCGG repeat-induced neurodegeneration. Finally, hnRNP A2/B1, an rCGG repeat-binding protein, can directly regulate the expression of miR-277. These biochemical and genetic studies demonstrate a novel miRNA-mediated mechanism involving miR-277, Drep-2, and Vimar in the regulation of neuronal survival in FXTAS (Tan, 2012).

Several lines of evidence from studies in mouse and Drosophila models strongly support FXTAS as an RNA-mediated neurodegenerative disorder caused by excessive rCGG repeats. The current working model is that specific RNA-binding proteins could be sequestered by overproduced rCGG repeats in FXTAS and become functionally limited, thereby contributing to the pathogenesis of this disorder. Three RNA-binding proteins are known to modulate rCGG-mediated neuronal toxicity: Pur α, hnRNP A2/B1, and CUGBP1, which bind rCGG repeats either directly (Pur α and hnRNP A2/B1) or indirectly (CUGBP1, through the interaction with hnRNP A2/B1); how the depletion of these RNA-binding proteins could alter RNA metabolism and contribute to FXTAS pathogenesis has thus become the focus in the quest to understand the molecular pathogenesis of this disorder. Nevertheless, the data presented in this study suggest that the depletion of hnRNP A2/B1 could also directly impact the transcriptional regulation of specific loci, such as miR-277. It is known that hnRNPs can interact with HP1 to bind to genomic DNA and modulate heterochromatin formation. The results indicate that hnRNP A2/B1 could participate in the transcriptional regulation of miR-277; however, it remains to be determined whether other loci could be directly regulated by hnRNP A2/B1, as well. Identifying those loci will be important to better understand how the depletion of rCGG repeat-binding proteins could lead to neuronal apoptosis (Tan, 2012).

In recent years, several classes of small regulatory RNAs have been identified in a range of tissues and in many species. In particular, miRNAs have been linked to a host of human diseases. Some evidence suggests the involvement of miRNAs in the emergence or progression of neurodegenerative diseases. For example, accumulation of nuclear aggregates that are toxic to neurons have been linked to many neurodegenerative diseases, and miRNAs are known to modulate the accumulation of the toxic proteins by regulating either their mRNAs or the mRNAs of proteins that affect their expression. Moreover, miRNAs might contribute to the pathogenesis of neurodegenerative disease downstream of the accumulation of toxic proteins by altering the expression of other proteins that promote or inhibit cell survival. The current genetic modifier screen revealed that miR-277 could modulate rCGG repeat-mediated neurodegeneration. By combining genetic screen and reporter assays, Drep-2 and Vimar were identified as the functional targets of miR-277 that could modulate rCGG-mediated neurodegeneration. The closest ortholog of miR-277 in human is miR-597 based on the seed sequence. It would be interesting to further examine the role of miR-597 in FXTAS using mammalian model systems (Tan, 2012).

Drep-2 is associated with the chromatin condensation and DNA fragmentation events of apoptosis (Inohara, 1998; Inohara, 1999). Drep-2 is one of four Drosophila DFF (DNA fragmentation factor)-related proteins. While Drep-1 is a Drosophila homolog of DFF45 that can inhibit CIDE-A mediated apoptosis. Drep-2 has been shown to interact with Drep-1 and to regulate its anti-apoptotic activity (Inohara, 1999). Vimar is a Ral GTPase-binding protein that has been shown to regulate mitochondrial function via an increase in citrate synthase activity (Chen, 2000). In the presence of fragile X premutation rCGG repeats, overexpression of miR-277 will suppress the expression of both Drep-2 and Vimar, thereby altering anti-apoptotic activity as well as mitochondrial functions, which have been linked to neuronal cell death associated with neurodegenerative disorders in general. Interestingly, a significant reduction of Drep-2 mRNA was seen in the flies expressing rCGG repeats, while Vimar mRNA levels remained similar to control flies. This observed difference may be due to the fact that miRNA could be involved in different modes of action, including mRNA cleavage, translational inhibition and mRNA decapping/deadenylation its target mRNAs (Tan, 2012).

In summary, this study provides both biochemical and genetic evidence to support a role for miRNA and its selective mRNA targets in rCGG-mediated neurodegeneration. The results suggest that sequestration of specific rCGG repeat-binding proteins can lead to aberrant expression of selective miRNAs that could modulate the pathogenesis of FXTAS by post-transcriptionally regulating the expression of specific mRNAs involved in this disorder. Identification of these miRNAs and their targets could reveal potential new targets for therapeutic interventions to treat FXTAS, as well as other neurodegenerative disorders (Tan, 2012).

The full-length transcripts and promoter analysis of intergenic microRNAs in Drosophila melanogaster

MicroRNA (miRNA) transcription is poorly understood until now. To increase miRNA abundance, miRNA transcription was stimulated with CuSO(4) and Drosha enzyme was knocked down using dsRNA in Drosophila S2 cells. The full length transcripts of bantam, miR-276a and miR-277, the 5'-end of miR-8, the 3'-end of miR-2b and miR-10 were obtained. A series of miRNA promoter analyses was conducted to prove the reliability of RACE results. Luciferase-reporter assays proved that both bantam and miR-276a promoters successfully drove the expressions of downstream luciferase genes. The promoter activities were impaired by introducing one or multiple mutations at predicted transcription factor binding sites. Chromatin immunoprecipitation analysis confirmed that hypophosphorylated RNA polymerase II and transcription factor c-Myc physically bind at miRNA promoters. RNA interference of transcription factors Mad and Prd led to down-expression of bantam, miR-277 and miR-2b but not miR-276a, whereas RNAi of Dorsal had the opposite effect (Qian, 2011).

Search PubMed for articles about Drosophila mir-127

Arocena, D. G., Iwahashi, C. K., Won, N., Beilina, A., Ludwig, A. L., Tassone, F., Schwartz, P. H. and Hagerman, P. J. (2005). Induction of inclusion formation and disruption of lamin A/C structure by premutation CGG-repeat RNA in human cultured neural cells. Hum Mol Genet 14: 3661-3671. PubMed ID:16239243

Chen, J., Shi, X., Padmanabhan, R., Wang, Q., Wu, Z., Stevenson, S. C., Hild, M., Garza, D. and Li, H. (2008). Identification of novel modulators of mitochondrial function by a genome-wide RNAi screen in Drosophila melanogaster. Genome Res 18: 123-136. PubMed ID:18042644

Greco, C. M., Berman, R. F., Martin, R. M., Tassone, F., Schwartz, P. H., Chang, A., Trapp, B. D., Iwahashi, C., Brunberg, J., Grigsby, J., Hessl, D., Becker, E. J., Papazian, J., Leehey, M. A., Hagerman, R. J. and Hagerman, P. J. (2006). Neuropathology of fragile X-associated tremor/ataxia syndrome (FXTAS). Brain 129: 243-255. PubMed ID:16332642

Inohara, N., Koseki, T., Chen, S., Wu, X. and Nunez, G. (1998). CIDE, a novel family of cell death activators with homology to the 45 kDa subunit of the DNA fragmentation factor. EMBO J 17: 2526-2533. PubMed ID:9564035

Inohara, N. and Nunez, G. (1999). Genes with homology to DFF/CIDEs found in Drosophila melanogaster. Cell Death Differ 6: 823-824. PubMed ID:10627165

Jin, P., Zarnescu, D. C., Zhang, F., Pearson, C. E., Lucchesi, J. C., Moses, K. and Warren, S. T. (2003). RNA-mediated neurodegeneration caused by the fragile X premutation rCGG repeats in Drosophila. Neuron 39: 739-747. PubMed ID:12948442

Qian, J., Zhang, Z., Liang, J., Ge, Q., Duan, X., Ma, F. and Li, F. (2011). The full-length transcripts and promoter analysis of intergenic microRNAs in Drosophila melanogaster. Genomics 97: 294-303. PubMed ID:21333734

Tan, H., Poidevin, M., Li, H., Chen, D. and Jin, P. (2012). MicroRNA-277 modulates the neurodegeneration caused by Fragile X premutation rCGG repeats. PLoS Genet 8: e1002681. PubMed ID:22570635

Tassone, F., Iwahashi, C., Hagerman, P. J. (2004). FMR1 RNA within the intranuclear inclusions of fragile X-associated tremor/ataxia syndrome (FXTAS). RNA Biology 1: 103-105. PubMed ID: 17179750

Willemsen, R., Hoogeveen-Westerveld, M., Reis, S., Holstege, J., Severijnen, L. A., Nieuwenhuizen, I. M., Schrier, M., van Unen, L., Tassone, F., Hoogeveen, A. T., Hagerman, P. J., Mientjes, E. J. and Oostra, B. A. (2003). The FMR1 CGG repeat mouse displays ubiquitin-positive intranuclear neuronal inclusions; implications for the cerebellar tremor/ataxia syndrome. Hum Mol Genet 12: 949-959. PubMed ID:12700164

date revised: 28 January 2013

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