MiR profiling of apoptotic murine neurons and their supernatent
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus synthetic construct
PROVIDER: GSE165263 | GEO | 2021/11/29
REPOSITORIES: GEO
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Project description:Identifying the physiological functions of microRNAs (miRNAs) is often challenging because miRNAs commonly impact gene expression under specific physiological conditions through complex miRNA::mRNA interaction networks and in coordination with other means of gene regulation, such as transcriptional regulation and protein degradation. Such complexity creates difficulties in dissecting miRNA functions through traditional genetic methods using individual miRNA mutations. To investigate the physiological functions of miRNAs in neurons, we combined a genetic M-bM-^@M-^\enhancerM-bM-^@M-^] approach complemented by biochemical analysis of neuronal miRNA-induced silencing complexes (miRISCs) in C. elegans. Total miRNA function can be compromised by mutating one of the two GW182 proteins (AIN-1), an important component of miRISC. We found that combining an ain-1 mutation with a mutation in unc-3, a neuronal transcription factor, resulted in an inappropriate entrance into the stress-induced, alternative larval stage known as dauer, indicating a role of miRNAs in preventing aberrant dauer formation. Analysis of this genetic interaction suggests that neuronal miRNAs perform such a role partly by regulating endogenous cyclic guanosine monophosphate (cGMP) signaling, potentially influencing two other dauer-regulating pathways. Through tissue-specific immunoprecipitations of miRISC, we identified miRNAs and their likely target mRNAs within neuronal tissue. We verified the biological relevance of several of these miRNAs and found that many miRNAs likely regulate dauer formation through multiple dauer-related targets. Further analysis of target mRNAs suggests potential miRNA involvement in various neuronal processes, but the importance of these miRNA::mRNA interactions remains unclear. Finally, we found that neuronal genes may be more highly regulated by miRNAs than intestinal genes. Overall, our study identifies miRNAs and their targets, and a physiological function of these miRNAs in neurons. It also suggests that compromising other aspects of gene expression, along with miRISC, can be an effective approach to reveal miRNA functions in specific tissues under specific physiological conditions. Each array was used for one biological replicate, where the red channel is used for IP RNA and the green channel is used for Total RNA. IP RNA indicates the transcripts associated with a neuronally expressed, GFP-tagged, miRISC. Five biological replicates were done on asynchronous worms. Please note the two different microarray platforms that were used.
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Project description:Over the last decade, small noncoding RNA molecules such as microRNAs (miRNAs) have emerged as critical regulators in the expression and function of eukaryotic genomes. It has been suggested that viral infections and neurological disease outcome may also be shaped by the influence of small RNAs. This has prompted us to suggest that HIV infection alters the endogenous miRNA expression patterns, thereby contributing to neuronal deregulation and AIDS dementia. Therefore, using primary cultures and neuronal cell lines, we examined the impact of a viral protein (HIV-1 Tat) on the expression of miRNAs due to its characteristic features such as release from the infected cells and taken up by noninfected cells. Using microRNA array assay, we demonstrated that Tat deregulates the levels of several miRNAs. Interestingly, miR-34a was among the most highly induced miRNAs in Tat-treated neurons. Tat also decreases the levels of miR-34a target genes such as CREB protein as shown by real time PCR. The effect of Tat was neutralized in the presence of anti-miR-34a. Using in situ hybridization assay, we found that the levels of miR-34a increase in Tat transgenic mice when compared with the parental mice. Therefore, we conclude that deregulation of neuronal functions by HIV-1 Tat protein is miRNA-dependent. Human neurons SH-SY5Y were chosen to examine the impact of HIV-1 Tat protein on gene expression
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Project description:Identifying the physiological functions of microRNAs (miRNAs) is often challenging because miRNAs commonly impact gene expression under specific physiological conditions through complex miRNA::mRNA interaction networks and in coordination with other means of gene regulation, such as transcriptional regulation and protein degradation. Such complexity creates difficulties in dissecting miRNA functions through traditional genetic methods using individual miRNA mutations. To investigate the physiological functions of miRNAs in neurons, we combined a genetic “enhancer” approach complemented by biochemical analysis of neuronal miRNA-induced silencing complexes (miRISCs) in C. elegans. Total miRNA function can be compromised by mutating one of the two GW182 proteins (AIN-1), an important component of miRISC. We found that combining an ain-1 mutation with a mutation in unc-3, a neuronal transcription factor, resulted in an inappropriate entrance into the stress-induced, alternative larval stage known as dauer, indicating a role of miRNAs in preventing aberrant dauer formation. Analysis of this genetic interaction suggests that neuronal miRNAs perform such a role partly by regulating endogenous cyclic guanosine monophosphate (cGMP) signaling, potentially influencing two other dauer-regulating pathways. Through tissue-specific immunoprecipitations of miRISC, we identified miRNAs and their likely target mRNAs within neuronal tissue. We verified the biological relevance of several of these miRNAs and found that many miRNAs likely regulate dauer formation through multiple dauer-related targets. Further analysis of target mRNAs suggests potential miRNA involvement in various neuronal processes, but the importance of these miRNA::mRNA interactions remains unclear. Finally, we found that neuronal genes may be more highly regulated by miRNAs than intestinal genes. Overall, our study identifies miRNAs and their targets, and a physiological function of these miRNAs in neurons. It also suggests that compromising other aspects of gene expression, along with miRISC, can be an effective approach to reveal miRNA functions in specific tissues under specific physiological conditions.
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