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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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Interaction between MDRL and miR-361.A. MDRL RNA contains a site complementary to miR-361. B. Luciferase assay. miR-361 binding site in MDRL RNA wild type form (Luc-MDRL-wt) and the mutated form (Luc-MDRL-mut) are shown in upper panel. HEK293 cells were infected with adenoviral miR-361 or β-gal, then transfected with the luciferase constructs of Luc-MDRL-wt or Luc-MDRL-mut. The luciferase activity was analyzed. *p<0.05. C. Wild type and the mutated form of biotin-labeled miR-361 sequence are shown. D. miR-361 can bind directly to MDRL in vivo. Cardiomyocytes were transfected with biotinylated wild type miR-361 (Bio-wt-361) or biotinylated mutant miR-361 (Bio-mut-361). A biotinylated miRNA that is not complementary to MDRL was used as a negative control (Bio-NC). 48 h after transfection, cells were harvested for biotin-based pull-down assay. MDRL expression levels were analyzed by real time PCR. *p<0.05 vs Bio-NC. E. MDRL can bind to miR-361 in vivo. Cardiomyocyte nuclear lysate was incubated with MDRL probe or random probe-coated magnetic bead. After washing and enrichment of beads/RNA complex, RNA was eluted from the streptavidin beads and was analyzed by northern blot. I, input (10% samples were loaded); P, pellet (100% samples were loaded). F and G. Enforced expression of MDRL induces the decreases of pri-miR-484 expression levels and the increases pre-miR-484 expression levels. Cardiomyocytes were infected with adenoviral MDRL or β-gal, the expression of pri-miR-484 (F) and pre-miR-484 (G) were analyzed by qRT-PCR. *p<0.05 vs control. H. Knockdown of miR-361 attenuated the inhibitory effects of MDRL knockdown on the processing of pri-miR-484 induced by Drosha. Cardiomyocytes were coinfected with the adenoviral Drosha, MDRL-siRNA and MDRL-sc, transfected with anta-361 or anta-NC. 48 h after transfection, cells were harvested. pri-miR-484 expression levels were analyzed by qRT-PCR. *p<0.05.
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pgen-1004467-g005: Interaction between MDRL and miR-361.A. MDRL RNA contains a site complementary to miR-361. B. Luciferase assay. miR-361 binding site in MDRL RNA wild type form (Luc-MDRL-wt) and the mutated form (Luc-MDRL-mut) are shown in upper panel. HEK293 cells were infected with adenoviral miR-361 or β-gal, then transfected with the luciferase constructs of Luc-MDRL-wt or Luc-MDRL-mut. The luciferase activity was analyzed. *p<0.05. C. Wild type and the mutated form of biotin-labeled miR-361 sequence are shown. D. miR-361 can bind directly to MDRL in vivo. Cardiomyocytes were transfected with biotinylated wild type miR-361 (Bio-wt-361) or biotinylated mutant miR-361 (Bio-mut-361). A biotinylated miRNA that is not complementary to MDRL was used as a negative control (Bio-NC). 48 h after transfection, cells were harvested for biotin-based pull-down assay. MDRL expression levels were analyzed by real time PCR. *p<0.05 vs Bio-NC. E. MDRL can bind to miR-361 in vivo. Cardiomyocyte nuclear lysate was incubated with MDRL probe or random probe-coated magnetic bead. After washing and enrichment of beads/RNA complex, RNA was eluted from the streptavidin beads and was analyzed by northern blot. I, input (10% samples were loaded); P, pellet (100% samples were loaded). F and G. Enforced expression of MDRL induces the decreases of pri-miR-484 expression levels and the increases pre-miR-484 expression levels. Cardiomyocytes were infected with adenoviral MDRL or β-gal, the expression of pri-miR-484 (F) and pre-miR-484 (G) were analyzed by qRT-PCR. *p<0.05 vs control. H. Knockdown of miR-361 attenuated the inhibitory effects of MDRL knockdown on the processing of pri-miR-484 induced by Drosha. Cardiomyocytes were coinfected with the adenoviral Drosha, MDRL-siRNA and MDRL-sc, transfected with anta-361 or anta-NC. 48 h after transfection, cells were harvested. pri-miR-484 expression levels were analyzed by qRT-PCR. *p<0.05.

Mentions: To understand the mechanism by which MDRL regulates the levels of miR-361, we tested whether MDRL can interact with miR-361. We compared the sequences of MDRL with that of miR-361 using the bioinformatics program RNAhybrid and noticed that MDRL contains a target site of miR-361 (Figure 5A). The wild type luciferase construct of MDRL (Luc-MDRL-wt) and a mutated form (Luc-MDRL-mut) were produced by inserting the sequence of putative miR-361 binding site into the report constructs (Figure 5B, upper panel). Luciferase assay revealed that miR-361 could suppress the luciferase activity of MDRL, but it had less effect on the mutated form of MDRL compared to the wild type (Figure 5B). Our results further showed that the mutated form of MDRL had no effect on miR-361 activity (Figure S3C) and it also lost the ability to counteract miR-361 (Figure S3D). These results revealed that MDRL may interact with miR-361 by this putative binding site.


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)

Interaction between MDRL and miR-361.A. MDRL RNA contains a site complementary to miR-361. B. Luciferase assay. miR-361 binding site in MDRL RNA wild type form (Luc-MDRL-wt) and the mutated form (Luc-MDRL-mut) are shown in upper panel. HEK293 cells were infected with adenoviral miR-361 or β-gal, then transfected with the luciferase constructs of Luc-MDRL-wt or Luc-MDRL-mut. The luciferase activity was analyzed. *p<0.05. C. Wild type and the mutated form of biotin-labeled miR-361 sequence are shown. D. miR-361 can bind directly to MDRL in vivo. Cardiomyocytes were transfected with biotinylated wild type miR-361 (Bio-wt-361) or biotinylated mutant miR-361 (Bio-mut-361). A biotinylated miRNA that is not complementary to MDRL was used as a negative control (Bio-NC). 48 h after transfection, cells were harvested for biotin-based pull-down assay. MDRL expression levels were analyzed by real time PCR. *p<0.05 vs Bio-NC. E. MDRL can bind to miR-361 in vivo. Cardiomyocyte nuclear lysate was incubated with MDRL probe or random probe-coated magnetic bead. After washing and enrichment of beads/RNA complex, RNA was eluted from the streptavidin beads and was analyzed by northern blot. I, input (10% samples were loaded); P, pellet (100% samples were loaded). F and G. Enforced expression of MDRL induces the decreases of pri-miR-484 expression levels and the increases pre-miR-484 expression levels. Cardiomyocytes were infected with adenoviral MDRL or β-gal, the expression of pri-miR-484 (F) and pre-miR-484 (G) were analyzed by qRT-PCR. *p<0.05 vs control. H. Knockdown of miR-361 attenuated the inhibitory effects of MDRL knockdown on the processing of pri-miR-484 induced by Drosha. Cardiomyocytes were coinfected with the adenoviral Drosha, MDRL-siRNA and MDRL-sc, transfected with anta-361 or anta-NC. 48 h after transfection, cells were harvested. pri-miR-484 expression levels were analyzed by qRT-PCR. *p<0.05.
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pgen-1004467-g005: Interaction between MDRL and miR-361.A. MDRL RNA contains a site complementary to miR-361. B. Luciferase assay. miR-361 binding site in MDRL RNA wild type form (Luc-MDRL-wt) and the mutated form (Luc-MDRL-mut) are shown in upper panel. HEK293 cells were infected with adenoviral miR-361 or β-gal, then transfected with the luciferase constructs of Luc-MDRL-wt or Luc-MDRL-mut. The luciferase activity was analyzed. *p<0.05. C. Wild type and the mutated form of biotin-labeled miR-361 sequence are shown. D. miR-361 can bind directly to MDRL in vivo. Cardiomyocytes were transfected with biotinylated wild type miR-361 (Bio-wt-361) or biotinylated mutant miR-361 (Bio-mut-361). A biotinylated miRNA that is not complementary to MDRL was used as a negative control (Bio-NC). 48 h after transfection, cells were harvested for biotin-based pull-down assay. MDRL expression levels were analyzed by real time PCR. *p<0.05 vs Bio-NC. E. MDRL can bind to miR-361 in vivo. Cardiomyocyte nuclear lysate was incubated with MDRL probe or random probe-coated magnetic bead. After washing and enrichment of beads/RNA complex, RNA was eluted from the streptavidin beads and was analyzed by northern blot. I, input (10% samples were loaded); P, pellet (100% samples were loaded). F and G. Enforced expression of MDRL induces the decreases of pri-miR-484 expression levels and the increases pre-miR-484 expression levels. Cardiomyocytes were infected with adenoviral MDRL or β-gal, the expression of pri-miR-484 (F) and pre-miR-484 (G) were analyzed by qRT-PCR. *p<0.05 vs control. H. Knockdown of miR-361 attenuated the inhibitory effects of MDRL knockdown on the processing of pri-miR-484 induced by Drosha. Cardiomyocytes were coinfected with the adenoviral Drosha, MDRL-siRNA and MDRL-sc, transfected with anta-361 or anta-NC. 48 h after transfection, cells were harvested. pri-miR-484 expression levels were analyzed by qRT-PCR. *p<0.05.
Mentions: To understand the mechanism by which MDRL regulates the levels of miR-361, we tested whether MDRL can interact with miR-361. We compared the sequences of MDRL with that of miR-361 using the bioinformatics program RNAhybrid and noticed that MDRL contains a target site of miR-361 (Figure 5A). The wild type luciferase construct of MDRL (Luc-MDRL-wt) and a mutated form (Luc-MDRL-mut) were produced by inserting the sequence of putative miR-361 binding site into the report constructs (Figure 5B, upper panel). Luciferase assay revealed that miR-361 could suppress the luciferase activity of MDRL, but it had less effect on the mutated form of MDRL compared to the wild type (Figure 5B). Our results further showed that the mutated form of MDRL had no effect on miR-361 activity (Figure S3C) and it also lost the ability to counteract miR-361 (Figure S3D). These results revealed that MDRL may interact with miR-361 by this putative binding site.

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