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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.

Show MeSH
miR-361 can directly bind to pri-miR-484 and prevents its processing by Drosha into pre-miR-484.A. The miR-361 targeting site in pri-miR-484 is shown. B and C. Enforced expression of miR-361 increases the levels of pri-miR-484 and reduces the levels of pre-miR-484. Cardiomyocytes were infected with adenoviral miR-361 or β-gal at indicated time, the expression of pri-miR-484 (B) and pre-miR-484 (C) were analyzed by qRT-PCR. *p<0.05 vs control. D and E. Knockdown of miR-361 induces a reduction in pri-miR-484 levels and an increase in pre-miR-484. Cardiomyocytes were transfected with miR-361 antagomir (anta-361) or the antagomir negative control (anta-NC). 24 h after transfection, the expression of pri-miR-484 (D) and pre-miR-484 (E) were analyzed by qRT-PCR. *p<0.05 vs control. F and G. miR-361 prevents the processing of pri-miR-484 by Drosha into pre-miR-484. Cardiomyocytes were coinfected with the adenoviral Drosha and miR-361, transfected with anta-361 or anta-NC. 48 h after transfection, cells were harvested. pri-miR-484 (F) and pre-miR-484 (G) were analyzed by qRT-PCR. *p<0.05. H. miR-361 can directly bind to pri-miR-484 in vivo. Cardiomyocytes were transfected with biotinylated wild type miR-361 (Bio-wt-361), biotinylated mutant miR-361 (Bio-mut-361) and biotinylated negative control (Bio-NC). 48 h after transfection, cells were harvested for biotin-based pull-down assay. Only “seed” region of wt and mutant miR-361 were shown (upper panel). Pri-miR-484 were analyzed by qRT-PCR (low panel). *p<0.05. I. miR-361 is associated with pri-miR-484 in vivo. pri-miR-484 probe-coated magnetic bead was incubated with cardiomyocyte nulear lysate. 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).
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pgen-1004467-g002: miR-361 can directly bind to pri-miR-484 and prevents its processing by Drosha into pre-miR-484.A. The miR-361 targeting site in pri-miR-484 is shown. B and C. Enforced expression of miR-361 increases the levels of pri-miR-484 and reduces the levels of pre-miR-484. Cardiomyocytes were infected with adenoviral miR-361 or β-gal at indicated time, the expression of pri-miR-484 (B) and pre-miR-484 (C) were analyzed by qRT-PCR. *p<0.05 vs control. D and E. Knockdown of miR-361 induces a reduction in pri-miR-484 levels and an increase in pre-miR-484. Cardiomyocytes were transfected with miR-361 antagomir (anta-361) or the antagomir negative control (anta-NC). 24 h after transfection, the expression of pri-miR-484 (D) and pre-miR-484 (E) were analyzed by qRT-PCR. *p<0.05 vs control. F and G. miR-361 prevents the processing of pri-miR-484 by Drosha into pre-miR-484. Cardiomyocytes were coinfected with the adenoviral Drosha and miR-361, transfected with anta-361 or anta-NC. 48 h after transfection, cells were harvested. pri-miR-484 (F) and pre-miR-484 (G) were analyzed by qRT-PCR. *p<0.05. H. miR-361 can directly bind to pri-miR-484 in vivo. Cardiomyocytes were transfected with biotinylated wild type miR-361 (Bio-wt-361), biotinylated mutant miR-361 (Bio-mut-361) and biotinylated negative control (Bio-NC). 48 h after transfection, cells were harvested for biotin-based pull-down assay. Only “seed” region of wt and mutant miR-361 were shown (upper panel). Pri-miR-484 were analyzed by qRT-PCR (low panel). *p<0.05. I. miR-361 is associated with pri-miR-484 in vivo. pri-miR-484 probe-coated magnetic bead was incubated with cardiomyocyte nulear lysate. 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).

Mentions: To understand the mechanism by which nuclear-located miR-361 regulates the levels of cytoplasmic mature miR-484, we tested whether miR-361 is able to affect the levels of pri-miR-484 located in nucleus. We compared the sequences of miR-361 with that of pri-miR-484 using the bioinformatics program RNAhybrid and noticed that miR-361 is complementary to pri-miR-484 (Figure 2A). Their complementary sequences led us to consider whether miR-361 can directly interact with pri-miR-484 and inhibit its processing into pre-miR-484 in the nucleus. We demonstrated that enforced expression of miR-361 resulted in the strong accumulation of pri-miR-484 (Figure 2B) and the reduction of pre-miR-484 (Figure 2C). Knockdown of miR-361 resulted in the reduction of pri-miR-484 (Figure 2D) and the increase of pre-miR-484 (Figure 2E). Thus, it appears that miR-361 prevents the processing of pri-miR-484 into pre-miR-484 in nucleus.


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)

miR-361 can directly bind to pri-miR-484 and prevents its processing by Drosha into pre-miR-484.A. The miR-361 targeting site in pri-miR-484 is shown. B and C. Enforced expression of miR-361 increases the levels of pri-miR-484 and reduces the levels of pre-miR-484. Cardiomyocytes were infected with adenoviral miR-361 or β-gal at indicated time, the expression of pri-miR-484 (B) and pre-miR-484 (C) were analyzed by qRT-PCR. *p<0.05 vs control. D and E. Knockdown of miR-361 induces a reduction in pri-miR-484 levels and an increase in pre-miR-484. Cardiomyocytes were transfected with miR-361 antagomir (anta-361) or the antagomir negative control (anta-NC). 24 h after transfection, the expression of pri-miR-484 (D) and pre-miR-484 (E) were analyzed by qRT-PCR. *p<0.05 vs control. F and G. miR-361 prevents the processing of pri-miR-484 by Drosha into pre-miR-484. Cardiomyocytes were coinfected with the adenoviral Drosha and miR-361, transfected with anta-361 or anta-NC. 48 h after transfection, cells were harvested. pri-miR-484 (F) and pre-miR-484 (G) were analyzed by qRT-PCR. *p<0.05. H. miR-361 can directly bind to pri-miR-484 in vivo. Cardiomyocytes were transfected with biotinylated wild type miR-361 (Bio-wt-361), biotinylated mutant miR-361 (Bio-mut-361) and biotinylated negative control (Bio-NC). 48 h after transfection, cells were harvested for biotin-based pull-down assay. Only “seed” region of wt and mutant miR-361 were shown (upper panel). Pri-miR-484 were analyzed by qRT-PCR (low panel). *p<0.05. I. miR-361 is associated with pri-miR-484 in vivo. pri-miR-484 probe-coated magnetic bead was incubated with cardiomyocyte nulear lysate. 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).
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pgen-1004467-g002: miR-361 can directly bind to pri-miR-484 and prevents its processing by Drosha into pre-miR-484.A. The miR-361 targeting site in pri-miR-484 is shown. B and C. Enforced expression of miR-361 increases the levels of pri-miR-484 and reduces the levels of pre-miR-484. Cardiomyocytes were infected with adenoviral miR-361 or β-gal at indicated time, the expression of pri-miR-484 (B) and pre-miR-484 (C) were analyzed by qRT-PCR. *p<0.05 vs control. D and E. Knockdown of miR-361 induces a reduction in pri-miR-484 levels and an increase in pre-miR-484. Cardiomyocytes were transfected with miR-361 antagomir (anta-361) or the antagomir negative control (anta-NC). 24 h after transfection, the expression of pri-miR-484 (D) and pre-miR-484 (E) were analyzed by qRT-PCR. *p<0.05 vs control. F and G. miR-361 prevents the processing of pri-miR-484 by Drosha into pre-miR-484. Cardiomyocytes were coinfected with the adenoviral Drosha and miR-361, transfected with anta-361 or anta-NC. 48 h after transfection, cells were harvested. pri-miR-484 (F) and pre-miR-484 (G) were analyzed by qRT-PCR. *p<0.05. H. miR-361 can directly bind to pri-miR-484 in vivo. Cardiomyocytes were transfected with biotinylated wild type miR-361 (Bio-wt-361), biotinylated mutant miR-361 (Bio-mut-361) and biotinylated negative control (Bio-NC). 48 h after transfection, cells were harvested for biotin-based pull-down assay. Only “seed” region of wt and mutant miR-361 were shown (upper panel). Pri-miR-484 were analyzed by qRT-PCR (low panel). *p<0.05. I. miR-361 is associated with pri-miR-484 in vivo. pri-miR-484 probe-coated magnetic bead was incubated with cardiomyocyte nulear lysate. 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).
Mentions: To understand the mechanism by which nuclear-located miR-361 regulates the levels of cytoplasmic mature miR-484, we tested whether miR-361 is able to affect the levels of pri-miR-484 located in nucleus. We compared the sequences of miR-361 with that of pri-miR-484 using the bioinformatics program RNAhybrid and noticed that miR-361 is complementary to pri-miR-484 (Figure 2A). Their complementary sequences led us to consider whether miR-361 can directly interact with pri-miR-484 and inhibit its processing into pre-miR-484 in the nucleus. We demonstrated that enforced expression of miR-361 resulted in the strong accumulation of pri-miR-484 (Figure 2B) and the reduction of pre-miR-484 (Figure 2C). Knockdown of miR-361 resulted in the reduction of pri-miR-484 (Figure 2D) and the increase of pre-miR-484 (Figure 2E). Thus, it appears that miR-361 prevents the processing of pri-miR-484 into pre-miR-484 in nucleus.

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