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MDRL lncRNA regulates the processing of miR-484 primary transcript by targeting miR-361.

Wang K, Sun T, Li N, Wang Y, Wang JX, Zhou LY, Long B, Liu CY, Liu F, Li PF - PLoS Genet. (2014)

Bottom Line: The results showed that miR-361 that predominantly located in nucleus can directly bind to primary transcript of miR-484 (pri-miR-484) and prevent its processing by Drosha into pre-miR-484. miR-361 is able to regulate mitochondrial fission and apoptosis by regulating miR-484 levels.Our present study reveals a novel regulating model of mitochondrial fission program which is composed of MDRL, miR-361 and miR-484.Our work not only expands the function of the lncRNA pathway in gene regulation but also establishes a new mechanism for controlling miRNA expression.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular Research, State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Long noncoding RNAs (lncRNAs) are emerging as new players in gene regulation, but whether lncRNAs operate in the processing of miRNA primary transcript is unclear. Also, whether lncRNAs are involved in the regulation of the mitochondrial network remains to be elucidated. Here, we report that a long noncoding RNA, named mitochondrial dynamic related lncRNA (MDRL), affects the processing of miR-484 primary transcript in nucleus and regulates the mitochondrial network by targeting miR-361 and miR-484. The results showed that miR-361 that predominantly located in nucleus can directly bind to primary transcript of miR-484 (pri-miR-484) and prevent its processing by Drosha into pre-miR-484. miR-361 is able to regulate mitochondrial fission and apoptosis by regulating miR-484 levels. In exploring the underlying molecular mechanism by which miR-361 is regulated, we identified MDRL and demonstrated that it could directly bind to miR-361 and downregulate its expression levels, which promotes the processing of pri-miR-484. MDRL inhibits mitochondrial fission and apoptosis by downregulating miR-361, which in turn relieves inhibition of miR-484 processing by miR-361. Our present study reveals a novel regulating model of mitochondrial fission program which is composed of MDRL, miR-361 and miR-484. Our work not only expands the function of the lncRNA pathway in gene regulation but also establishes a new mechanism for controlling miRNA expression.

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MDRL can regulate miR-361 expression and activity.A. LncRNAs expression levels upon treatment with A/R. Cardiomyocytes were untreated (control) or treated with A/R. LncRNAs expressed in heart in lncRNA array from Affymetrix company (http://www.noncode.org) were analyzed by qRT-PCR. *p<0.05 vs control. B. Knockdown of MDRL induces the decrease of MDRL expression levels. Cardiomyocytes were infected with adenoviral MDRL siRNA (MDRL-siRNA) and its scramble form (MDRL-sc). 24 h after infection MDRL levels were analyzed by real time PCR. *p<0.05 vs control. C. Knockdown of MDRL upregulates miR-361 expression levels. Cardiomyocytes were infected with adenoviral MDRL siRNA (MDRL-siRNA) and its scramble form (MDRL-sc). 24 h after infection miR-361 levels were analyzed by northern blot. D. Knockdown of MDRL induces miR-361 activity. Cardiomyocytes were infected with adenoviral MDRL-siRNA and its scramble, then transfected with miR-361 sensor. Luciferase activity was analyzed. *p<0.05 vs miR-361 sensor alone. E. Enforced expression of MDRL reduces the expression levels of miR-361. Cardiomyocytes were infected with adenoviral MDRL or β-gal. 24 h after infection MDRL levels were analyzed by real time PCR (low panel), and miR-361 levels were analyzed by northern blot (upper panel). F. MDRL reduces miR-361 activity. Cardiomyocytes were infected with adenoviral MDRL or β-gal, then transfected with miR-361 sensor. Luciferase activity was analyzed. *p<0.05 vs miR-361 sensor alone. G. MDRL acts as a sponge for miR-361 activity. Cardiomyocytes were infected with adenoviral miR-361, MDRL or β-gal, then transfected with miR-361 sensor. Luciferase activity was analyzed. *p<0.05.
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pgen-1004467-g004: MDRL can regulate miR-361 expression and activity.A. LncRNAs expression levels upon treatment with A/R. Cardiomyocytes were untreated (control) or treated with A/R. LncRNAs expressed in heart in lncRNA array from Affymetrix company (http://www.noncode.org) were analyzed by qRT-PCR. *p<0.05 vs control. B. Knockdown of MDRL induces the decrease of MDRL expression levels. Cardiomyocytes were infected with adenoviral MDRL siRNA (MDRL-siRNA) and its scramble form (MDRL-sc). 24 h after infection MDRL levels were analyzed by real time PCR. *p<0.05 vs control. C. Knockdown of MDRL upregulates miR-361 expression levels. Cardiomyocytes were infected with adenoviral MDRL siRNA (MDRL-siRNA) and its scramble form (MDRL-sc). 24 h after infection miR-361 levels were analyzed by northern blot. D. Knockdown of MDRL induces miR-361 activity. Cardiomyocytes were infected with adenoviral MDRL-siRNA and its scramble, then transfected with miR-361 sensor. Luciferase activity was analyzed. *p<0.05 vs miR-361 sensor alone. E. Enforced expression of MDRL reduces the expression levels of miR-361. Cardiomyocytes were infected with adenoviral MDRL or β-gal. 24 h after infection MDRL levels were analyzed by real time PCR (low panel), and miR-361 levels were analyzed by northern blot (upper panel). F. MDRL reduces miR-361 activity. Cardiomyocytes were infected with adenoviral MDRL or β-gal, then transfected with miR-361 sensor. Luciferase activity was analyzed. *p<0.05 vs miR-361 sensor alone. G. MDRL acts as a sponge for miR-361 activity. Cardiomyocytes were infected with adenoviral miR-361, MDRL or β-gal, then transfected with miR-361 sensor. Luciferase activity was analyzed. *p<0.05.

Mentions: Recent studies have suggested that lncRNAs may act as endogenous sponge RNA to interact with miRNAs and influence the expression of miRNA [9], [25]–[27]. To explore the underlying mechanism responsible for miR-361 upregulation in response to A/R treatment, we tested whether lncRNA could regulate miR-361 expression. We carried out qRT-PCR to detect lncRNAs levels in response to A/R treatment. LncRNAs were chosen from the lncRNA array published online by Fantom company. Among 100 lncRNAs, AK009271 which we named mitochondrial dynamic related lncRNA (MDRL), was substantially reduced (Figure 4A). The MDRL is 1039 nt in length and the subcellular location showed that MDRL was expressed both in nucleus and cytoplasm (Figure S3B). Further, our results showed that miR-361 levels were elevated in the cells upon knockdown of endogenous MDRL (Figures 4B and 4C). To know whether MDRL can affect miR-361 activity, we constructed miR-361 sensor (with a perfect miR-361 binding site). The lucifease activity of miR-361 sensor was decreased in cells treated with MDRL siRNA (Figure 4D), suggesting the induction of miR-361 activity. Enforced expression of MDRL induced a reduction in miR-361 expression (Figure 4E) and activity (Figure 4F). To further test whether MDRL may act as a miR-361 sponge, we transfected the miR-361 sensor luciferase reporter, along with adenoviral miR-361, MDRL or β-gal. The luciferase activity showed that MDRL counteracted the effect of miR-361 (Figure 4G), suggesting that MDRL is a functional sponge for miR-361. Taken together, these data suggest that MDRL is able to regulate miR-361 levels and activity.


MDRL lncRNA regulates the processing of miR-484 primary transcript by targeting miR-361.

Wang K, Sun T, Li N, Wang Y, Wang JX, Zhou LY, Long B, Liu CY, Liu F, Li PF - PLoS Genet. (2014)

MDRL can regulate miR-361 expression and activity.A. LncRNAs expression levels upon treatment with A/R. Cardiomyocytes were untreated (control) or treated with A/R. LncRNAs expressed in heart in lncRNA array from Affymetrix company (http://www.noncode.org) were analyzed by qRT-PCR. *p<0.05 vs control. B. Knockdown of MDRL induces the decrease of MDRL expression levels. Cardiomyocytes were infected with adenoviral MDRL siRNA (MDRL-siRNA) and its scramble form (MDRL-sc). 24 h after infection MDRL levels were analyzed by real time PCR. *p<0.05 vs control. C. Knockdown of MDRL upregulates miR-361 expression levels. Cardiomyocytes were infected with adenoviral MDRL siRNA (MDRL-siRNA) and its scramble form (MDRL-sc). 24 h after infection miR-361 levels were analyzed by northern blot. D. Knockdown of MDRL induces miR-361 activity. Cardiomyocytes were infected with adenoviral MDRL-siRNA and its scramble, then transfected with miR-361 sensor. Luciferase activity was analyzed. *p<0.05 vs miR-361 sensor alone. E. Enforced expression of MDRL reduces the expression levels of miR-361. Cardiomyocytes were infected with adenoviral MDRL or β-gal. 24 h after infection MDRL levels were analyzed by real time PCR (low panel), and miR-361 levels were analyzed by northern blot (upper panel). F. MDRL reduces miR-361 activity. Cardiomyocytes were infected with adenoviral MDRL or β-gal, then transfected with miR-361 sensor. Luciferase activity was analyzed. *p<0.05 vs miR-361 sensor alone. G. MDRL acts as a sponge for miR-361 activity. Cardiomyocytes were infected with adenoviral miR-361, MDRL or β-gal, then transfected with miR-361 sensor. Luciferase activity was analyzed. *p<0.05.
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pgen-1004467-g004: MDRL can regulate miR-361 expression and activity.A. LncRNAs expression levels upon treatment with A/R. Cardiomyocytes were untreated (control) or treated with A/R. LncRNAs expressed in heart in lncRNA array from Affymetrix company (http://www.noncode.org) were analyzed by qRT-PCR. *p<0.05 vs control. B. Knockdown of MDRL induces the decrease of MDRL expression levels. Cardiomyocytes were infected with adenoviral MDRL siRNA (MDRL-siRNA) and its scramble form (MDRL-sc). 24 h after infection MDRL levels were analyzed by real time PCR. *p<0.05 vs control. C. Knockdown of MDRL upregulates miR-361 expression levels. Cardiomyocytes were infected with adenoviral MDRL siRNA (MDRL-siRNA) and its scramble form (MDRL-sc). 24 h after infection miR-361 levels were analyzed by northern blot. D. Knockdown of MDRL induces miR-361 activity. Cardiomyocytes were infected with adenoviral MDRL-siRNA and its scramble, then transfected with miR-361 sensor. Luciferase activity was analyzed. *p<0.05 vs miR-361 sensor alone. E. Enforced expression of MDRL reduces the expression levels of miR-361. Cardiomyocytes were infected with adenoviral MDRL or β-gal. 24 h after infection MDRL levels were analyzed by real time PCR (low panel), and miR-361 levels were analyzed by northern blot (upper panel). F. MDRL reduces miR-361 activity. Cardiomyocytes were infected with adenoviral MDRL or β-gal, then transfected with miR-361 sensor. Luciferase activity was analyzed. *p<0.05 vs miR-361 sensor alone. G. MDRL acts as a sponge for miR-361 activity. Cardiomyocytes were infected with adenoviral miR-361, MDRL or β-gal, then transfected with miR-361 sensor. Luciferase activity was analyzed. *p<0.05.
Mentions: Recent studies have suggested that lncRNAs may act as endogenous sponge RNA to interact with miRNAs and influence the expression of miRNA [9], [25]–[27]. To explore the underlying mechanism responsible for miR-361 upregulation in response to A/R treatment, we tested whether lncRNA could regulate miR-361 expression. We carried out qRT-PCR to detect lncRNAs levels in response to A/R treatment. LncRNAs were chosen from the lncRNA array published online by Fantom company. Among 100 lncRNAs, AK009271 which we named mitochondrial dynamic related lncRNA (MDRL), was substantially reduced (Figure 4A). The MDRL is 1039 nt in length and the subcellular location showed that MDRL was expressed both in nucleus and cytoplasm (Figure S3B). Further, our results showed that miR-361 levels were elevated in the cells upon knockdown of endogenous MDRL (Figures 4B and 4C). To know whether MDRL can affect miR-361 activity, we constructed miR-361 sensor (with a perfect miR-361 binding site). The lucifease activity of miR-361 sensor was decreased in cells treated with MDRL siRNA (Figure 4D), suggesting the induction of miR-361 activity. Enforced expression of MDRL induced a reduction in miR-361 expression (Figure 4E) and activity (Figure 4F). To further test whether MDRL may act as a miR-361 sponge, we transfected the miR-361 sensor luciferase reporter, along with adenoviral miR-361, MDRL or β-gal. The luciferase activity showed that MDRL counteracted the effect of miR-361 (Figure 4G), suggesting that MDRL is a functional sponge for miR-361. Taken together, these data suggest that MDRL is able to regulate miR-361 levels and activity.

Bottom Line: The results showed that miR-361 that predominantly located in nucleus can directly bind to primary transcript of miR-484 (pri-miR-484) and prevent its processing by Drosha into pre-miR-484. miR-361 is able to regulate mitochondrial fission and apoptosis by regulating miR-484 levels.Our present study reveals a novel regulating model of mitochondrial fission program which is composed of MDRL, miR-361 and miR-484.Our work not only expands the function of the lncRNA pathway in gene regulation but also establishes a new mechanism for controlling miRNA expression.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiovascular Research, State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Long noncoding RNAs (lncRNAs) are emerging as new players in gene regulation, but whether lncRNAs operate in the processing of miRNA primary transcript is unclear. Also, whether lncRNAs are involved in the regulation of the mitochondrial network remains to be elucidated. Here, we report that a long noncoding RNA, named mitochondrial dynamic related lncRNA (MDRL), affects the processing of miR-484 primary transcript in nucleus and regulates the mitochondrial network by targeting miR-361 and miR-484. The results showed that miR-361 that predominantly located in nucleus can directly bind to primary transcript of miR-484 (pri-miR-484) and prevent its processing by Drosha into pre-miR-484. miR-361 is able to regulate mitochondrial fission and apoptosis by regulating miR-484 levels. In exploring the underlying molecular mechanism by which miR-361 is regulated, we identified MDRL and demonstrated that it could directly bind to miR-361 and downregulate its expression levels, which promotes the processing of pri-miR-484. MDRL inhibits mitochondrial fission and apoptosis by downregulating miR-361, which in turn relieves inhibition of miR-484 processing by miR-361. Our present study reveals a novel regulating model of mitochondrial fission program which is composed of MDRL, miR-361 and miR-484. Our work not only expands the function of the lncRNA pathway in gene regulation but also establishes a new mechanism for controlling miRNA expression.

Show MeSH
Related in: MedlinePlus