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Neural circular RNAs are derived from synaptic genes and regulated by development and plasticity.

You X, Vlatkovic I, Babic A, Will T, Epstein I, Tushev G, Akbalik G, Wang M, Glock C, Quedenau C, Wang X, Hou J, Liu H, Sun W, Sambandan S, Chen T, Schuman EM, Chen W - Nat. Neurosci. (2015)

Bottom Line: Using high-resolution in situ hybridization, we visualized circRNA punctae in the dendrites of neurons.In addition, following a homeostatic downscaling of neuronal activity many circRNAs exhibited substantial up- or downregulation.Together, our data indicate that brain circRNAs are positioned to respond to and regulate synaptic function.

View Article: PubMed Central - PubMed

Affiliation: Berlin Institute for Medical Systems Biology, Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany.

ABSTRACT
Circular RNAs (circRNAs) have re-emerged as an interesting RNA species. Using deep RNA profiling in different mouse tissues, we observed that circRNAs were substantially enriched in brain and a disproportionate fraction of them were derived from host genes that encode synaptic proteins. Moreover, on the basis of separate profiling of the RNAs localized in neuronal cell bodies and neuropil, circRNAs were, on average, more enriched in the neuropil than their host gene mRNA isoforms. Using high-resolution in situ hybridization, we visualized circRNA punctae in the dendrites of neurons. Consistent with the idea that circRNAs might regulate synaptic function during development, many circRNAs changed their abundance abruptly at a time corresponding to synaptogenesis. In addition, following a homeostatic downscaling of neuronal activity many circRNAs exhibited substantial up- or downregulation. Together, our data indicate that brain circRNAs are positioned to respond to and regulate synaptic function.

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Regulated expression of brain circRNAs during developmentA. Heatmap of circRNA expression across four different developmental stages showing the regulation of several circRNA clusters between P0 and P10- the time at which synapses typically form. The abundance of circRNAs across four developmental stages is depicted on a scale from red (low) to yellow (high). A developmentally down-regulated cluster consisting of 43 circRNAs exhibited an early peak expression at E18 or P0 and then declined over subsequent developmental time points. A developmentally up-regulated cluster, consisting of 181 circRNAs, exhibited increasing expression that peaked at P10 or P30. B. The significantly enriched GO terms (p-value < 0.05 in either cluster). The host genes with circRNAs that exhibited peak expression associated with the time of synapse formation were enriched for synaptic function whereas the other group (down-regulated) did not exhibit significant enrichment of any GO terms. C. Fold change of both circRNA abundance (Y-axis) and the total transcriptional output (TTO) of their gene loci (X-axis) between stage E18 and P30. Each dot represents one circRNA. Dots in red and yellow highlight circRNAs that changed significantly whilst the total transcriptional output of their host loci was not substantially altered. Inset shows that while most circRNA-hosting genes do not change much in abundance compared to all genes (two-sided unpaired Student-t test, ns for p = 0.09709), circRNAs are significantly up-regulated (two-sided unpaired Student’s-t test, *** p < 2.2E-16). Six or seven mice were pooled in each of two replicates of E18. Three or four mice were pooled in each of two replicates of P30. D. The expression change for both circRNA and mRNA was validated using quantitative PCR for 13 circRNAs including Homer1, Dlgap1, Rmst, Myst4 and Ezh2. Error bars represent standard deviation. E–F. Validation of circRNA expression changes over developmental stages using high resolution in situ hybridization for circKlhl2 (green) at two time points -days in culture 4 (n = 26) or 21 (n = 24). circKlhl2 expression was significantly up-regulated between these developmental stages (two-sided unpaired Student’s t-test with Welch’s correction, *** p < 0.0001). The outline of the neuronal somata was identified using an anti-MAP2 antibody (red). Scale bar = 10 microns.
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Figure 3: Regulated expression of brain circRNAs during developmentA. Heatmap of circRNA expression across four different developmental stages showing the regulation of several circRNA clusters between P0 and P10- the time at which synapses typically form. The abundance of circRNAs across four developmental stages is depicted on a scale from red (low) to yellow (high). A developmentally down-regulated cluster consisting of 43 circRNAs exhibited an early peak expression at E18 or P0 and then declined over subsequent developmental time points. A developmentally up-regulated cluster, consisting of 181 circRNAs, exhibited increasing expression that peaked at P10 or P30. B. The significantly enriched GO terms (p-value < 0.05 in either cluster). The host genes with circRNAs that exhibited peak expression associated with the time of synapse formation were enriched for synaptic function whereas the other group (down-regulated) did not exhibit significant enrichment of any GO terms. C. Fold change of both circRNA abundance (Y-axis) and the total transcriptional output (TTO) of their gene loci (X-axis) between stage E18 and P30. Each dot represents one circRNA. Dots in red and yellow highlight circRNAs that changed significantly whilst the total transcriptional output of their host loci was not substantially altered. Inset shows that while most circRNA-hosting genes do not change much in abundance compared to all genes (two-sided unpaired Student-t test, ns for p = 0.09709), circRNAs are significantly up-regulated (two-sided unpaired Student’s-t test, *** p < 2.2E-16). Six or seven mice were pooled in each of two replicates of E18. Three or four mice were pooled in each of two replicates of P30. D. The expression change for both circRNA and mRNA was validated using quantitative PCR for 13 circRNAs including Homer1, Dlgap1, Rmst, Myst4 and Ezh2. Error bars represent standard deviation. E–F. Validation of circRNA expression changes over developmental stages using high resolution in situ hybridization for circKlhl2 (green) at two time points -days in culture 4 (n = 26) or 21 (n = 24). circKlhl2 expression was significantly up-regulated between these developmental stages (two-sided unpaired Student’s t-test with Welch’s correction, *** p < 0.0001). The outline of the neuronal somata was identified using an anti-MAP2 antibody (red). Scale bar = 10 microns.

Mentions: To determine whether the expression of circRNAs is developmentally regulated in brain we profiled the circRNA population in the hippocampus over several stages: embryonic (E18), early postnatal (P1), postnatal at the beginning of synapse formation (P10) and late postnatal hippocampus following the establishment of mature neural circuits (P30) (Figure S7). As shown in Figure 3A, there was a clear shift in the circRNA expression pattern associated with the onset of synaptogenesis at P10. Interestingly, the circRNAs that were consistently up-regulated during hippocampal development were produced from the gene loci that also code for proteins enriched with synapse-related functions (Figure 3B). In contrast, no enrichment of any functional categories could be observed for the gene loci showing the opposite (down-regulated) circRNA dynamic expression pattern. We next examined the relationship between the expression of a circRNA and its linear host comparing the earliest (E18) and latest (P30) developmental stages. We found that many circRNAs change their expression independent of their host transcripts during synaptogenesis (Figure 3C). Thirteen circRNA/mRNA pairs with different expression patterns were validated using quantitative PCR (Figure 3D). Dlgap1, whose protein product is a core component of postsynaptic density (PSD), showed an over 20-fold increase in circRNA expression at P30 when compared with E18, whilst the mRNA expression increased only less than 4-fold. Genes such as Myst4, Klhl2, and Aagab dramatically increased their circRNA expression over development while their mRNA expression significantly decreased. In contrast, Cacna1c showed substantial decreases in circRNA expression along developmental stages, while the mRNA remained unchanged. Using high-resolution in situ hybridization in cultured hippocampal neurons, we further validated the developmental regulation of a circRNA that exhibited strong up-regulation during development circKlhl2 (Figure 3E). Analysis of the average fluorescence intensity at an early and late developmental stage (neurons cultured beginning at P1, days in vitro = 4 or 21) revealed a significant enhancement of the circKlhl2 expression levels (Figure 3F). Thus, taken together the data from high-throughput sequencing, quantitative PCR and in situ hybridization, indicate that the expression of circRNAs is developmentally regulated in neurons and that many circRNAs change their expression independent of their host linear transcripts.


Neural circular RNAs are derived from synaptic genes and regulated by development and plasticity.

You X, Vlatkovic I, Babic A, Will T, Epstein I, Tushev G, Akbalik G, Wang M, Glock C, Quedenau C, Wang X, Hou J, Liu H, Sun W, Sambandan S, Chen T, Schuman EM, Chen W - Nat. Neurosci. (2015)

Regulated expression of brain circRNAs during developmentA. Heatmap of circRNA expression across four different developmental stages showing the regulation of several circRNA clusters between P0 and P10- the time at which synapses typically form. The abundance of circRNAs across four developmental stages is depicted on a scale from red (low) to yellow (high). A developmentally down-regulated cluster consisting of 43 circRNAs exhibited an early peak expression at E18 or P0 and then declined over subsequent developmental time points. A developmentally up-regulated cluster, consisting of 181 circRNAs, exhibited increasing expression that peaked at P10 or P30. B. The significantly enriched GO terms (p-value < 0.05 in either cluster). The host genes with circRNAs that exhibited peak expression associated with the time of synapse formation were enriched for synaptic function whereas the other group (down-regulated) did not exhibit significant enrichment of any GO terms. C. Fold change of both circRNA abundance (Y-axis) and the total transcriptional output (TTO) of their gene loci (X-axis) between stage E18 and P30. Each dot represents one circRNA. Dots in red and yellow highlight circRNAs that changed significantly whilst the total transcriptional output of their host loci was not substantially altered. Inset shows that while most circRNA-hosting genes do not change much in abundance compared to all genes (two-sided unpaired Student-t test, ns for p = 0.09709), circRNAs are significantly up-regulated (two-sided unpaired Student’s-t test, *** p < 2.2E-16). Six or seven mice were pooled in each of two replicates of E18. Three or four mice were pooled in each of two replicates of P30. D. The expression change for both circRNA and mRNA was validated using quantitative PCR for 13 circRNAs including Homer1, Dlgap1, Rmst, Myst4 and Ezh2. Error bars represent standard deviation. E–F. Validation of circRNA expression changes over developmental stages using high resolution in situ hybridization for circKlhl2 (green) at two time points -days in culture 4 (n = 26) or 21 (n = 24). circKlhl2 expression was significantly up-regulated between these developmental stages (two-sided unpaired Student’s t-test with Welch’s correction, *** p < 0.0001). The outline of the neuronal somata was identified using an anti-MAP2 antibody (red). Scale bar = 10 microns.
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Figure 3: Regulated expression of brain circRNAs during developmentA. Heatmap of circRNA expression across four different developmental stages showing the regulation of several circRNA clusters between P0 and P10- the time at which synapses typically form. The abundance of circRNAs across four developmental stages is depicted on a scale from red (low) to yellow (high). A developmentally down-regulated cluster consisting of 43 circRNAs exhibited an early peak expression at E18 or P0 and then declined over subsequent developmental time points. A developmentally up-regulated cluster, consisting of 181 circRNAs, exhibited increasing expression that peaked at P10 or P30. B. The significantly enriched GO terms (p-value < 0.05 in either cluster). The host genes with circRNAs that exhibited peak expression associated with the time of synapse formation were enriched for synaptic function whereas the other group (down-regulated) did not exhibit significant enrichment of any GO terms. C. Fold change of both circRNA abundance (Y-axis) and the total transcriptional output (TTO) of their gene loci (X-axis) between stage E18 and P30. Each dot represents one circRNA. Dots in red and yellow highlight circRNAs that changed significantly whilst the total transcriptional output of their host loci was not substantially altered. Inset shows that while most circRNA-hosting genes do not change much in abundance compared to all genes (two-sided unpaired Student-t test, ns for p = 0.09709), circRNAs are significantly up-regulated (two-sided unpaired Student’s-t test, *** p < 2.2E-16). Six or seven mice were pooled in each of two replicates of E18. Three or four mice were pooled in each of two replicates of P30. D. The expression change for both circRNA and mRNA was validated using quantitative PCR for 13 circRNAs including Homer1, Dlgap1, Rmst, Myst4 and Ezh2. Error bars represent standard deviation. E–F. Validation of circRNA expression changes over developmental stages using high resolution in situ hybridization for circKlhl2 (green) at two time points -days in culture 4 (n = 26) or 21 (n = 24). circKlhl2 expression was significantly up-regulated between these developmental stages (two-sided unpaired Student’s t-test with Welch’s correction, *** p < 0.0001). The outline of the neuronal somata was identified using an anti-MAP2 antibody (red). Scale bar = 10 microns.
Mentions: To determine whether the expression of circRNAs is developmentally regulated in brain we profiled the circRNA population in the hippocampus over several stages: embryonic (E18), early postnatal (P1), postnatal at the beginning of synapse formation (P10) and late postnatal hippocampus following the establishment of mature neural circuits (P30) (Figure S7). As shown in Figure 3A, there was a clear shift in the circRNA expression pattern associated with the onset of synaptogenesis at P10. Interestingly, the circRNAs that were consistently up-regulated during hippocampal development were produced from the gene loci that also code for proteins enriched with synapse-related functions (Figure 3B). In contrast, no enrichment of any functional categories could be observed for the gene loci showing the opposite (down-regulated) circRNA dynamic expression pattern. We next examined the relationship between the expression of a circRNA and its linear host comparing the earliest (E18) and latest (P30) developmental stages. We found that many circRNAs change their expression independent of their host transcripts during synaptogenesis (Figure 3C). Thirteen circRNA/mRNA pairs with different expression patterns were validated using quantitative PCR (Figure 3D). Dlgap1, whose protein product is a core component of postsynaptic density (PSD), showed an over 20-fold increase in circRNA expression at P30 when compared with E18, whilst the mRNA expression increased only less than 4-fold. Genes such as Myst4, Klhl2, and Aagab dramatically increased their circRNA expression over development while their mRNA expression significantly decreased. In contrast, Cacna1c showed substantial decreases in circRNA expression along developmental stages, while the mRNA remained unchanged. Using high-resolution in situ hybridization in cultured hippocampal neurons, we further validated the developmental regulation of a circRNA that exhibited strong up-regulation during development circKlhl2 (Figure 3E). Analysis of the average fluorescence intensity at an early and late developmental stage (neurons cultured beginning at P1, days in vitro = 4 or 21) revealed a significant enhancement of the circKlhl2 expression levels (Figure 3F). Thus, taken together the data from high-throughput sequencing, quantitative PCR and in situ hybridization, indicate that the expression of circRNAs is developmentally regulated in neurons and that many circRNAs change their expression independent of their host linear transcripts.

Bottom Line: Using high-resolution in situ hybridization, we visualized circRNA punctae in the dendrites of neurons.In addition, following a homeostatic downscaling of neuronal activity many circRNAs exhibited substantial up- or downregulation.Together, our data indicate that brain circRNAs are positioned to respond to and regulate synaptic function.

View Article: PubMed Central - PubMed

Affiliation: Berlin Institute for Medical Systems Biology, Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany.

ABSTRACT
Circular RNAs (circRNAs) have re-emerged as an interesting RNA species. Using deep RNA profiling in different mouse tissues, we observed that circRNAs were substantially enriched in brain and a disproportionate fraction of them were derived from host genes that encode synaptic proteins. Moreover, on the basis of separate profiling of the RNAs localized in neuronal cell bodies and neuropil, circRNAs were, on average, more enriched in the neuropil than their host gene mRNA isoforms. Using high-resolution in situ hybridization, we visualized circRNA punctae in the dendrites of neurons. Consistent with the idea that circRNAs might regulate synaptic function during development, many circRNAs changed their abundance abruptly at a time corresponding to synaptogenesis. In addition, following a homeostatic downscaling of neuronal activity many circRNAs exhibited substantial up- or downregulation. Together, our data indicate that brain circRNAs are positioned to respond to and regulate synaptic function.

Show MeSH