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microRNA-9 targets the long non-coding RNA MALAT1 for degradation in the nucleus.

Leucci E, Patella F, Waage J, Holmstrøm K, Lindow M, Porse B, Kauppinen S, Lund AH - Sci Rep (2013)

Bottom Line: microRNAs regulate the expression of over 60% of protein coding genes by targeting their mRNAs to AGO2-containing complexes in the cytoplasm and promoting their translational inhibition and/or degradation.There is little evidence so far for microRNA-mediated regulation of other classes of non-coding RNAs.Our findings reveal a novel direct regulatory link between two important classes of non-coding RNAs, miRs and lncRNAs, and advance our understanding of microRNA functions.

View Article: PubMed Central - PubMed

Affiliation: 1] Biotech Research and Innovation Centre and Centre for Epigenetics, University of Copenhagen, Denmark [2].

ABSTRACT
microRNAs regulate the expression of over 60% of protein coding genes by targeting their mRNAs to AGO2-containing complexes in the cytoplasm and promoting their translational inhibition and/or degradation. There is little evidence so far for microRNA-mediated regulation of other classes of non-coding RNAs. Here we report that microRNA-9 (miR-9) regulates the expression of the Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT-1), one of the most abundant and conserved long non-coding RNAs. Intriguingly, we find that miR-9 targets AGO2-mediated regulation of MALAT1 in the nucleus. Our findings reveal a novel direct regulatory link between two important classes of non-coding RNAs, miRs and lncRNAs, and advance our understanding of microRNA functions.

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miR-9 targets MALAT1 to AGO2 containing complexes in the nuclei (a) RNA fluorescent in situ hybridization for miR-9 in different cell lines. Magnification 63X. DAPI is in blue, miR-9 in green. (b) Expression of miR-9 and MALAT1 in nuclear and cytoplasmic fractions used as inputs for AGO2 IP was measured by RT-qPCR. (c) Nuclear and total fractions were isolated from U87MG cells transfected with a scramble control or an antimiR-9, and immunoprecipitated using an AGO2 antibody. The western blot shows the purity of the extracts and the efficiency of AGO2 immunoprecipitation. The lower and the upper panel derive from the same gel, only the membrane was split in two to perform the incubation with the antibody. A full picture of the gel can be found in the supplementary informations. (d) The amount of miR-9 and MALAT1 was measured in inputs RNA used for the RIP by qPCR. d) The amount of miR-9 and MALAT1 bound to AGO2 was measured by qPCR in nuclear and cytoplasmic fractions in presence of a scramble control or an antimiR-9. P-value (p<0.0001) was calculated by two-ways ANOVA.
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f4: miR-9 targets MALAT1 to AGO2 containing complexes in the nuclei (a) RNA fluorescent in situ hybridization for miR-9 in different cell lines. Magnification 63X. DAPI is in blue, miR-9 in green. (b) Expression of miR-9 and MALAT1 in nuclear and cytoplasmic fractions used as inputs for AGO2 IP was measured by RT-qPCR. (c) Nuclear and total fractions were isolated from U87MG cells transfected with a scramble control or an antimiR-9, and immunoprecipitated using an AGO2 antibody. The western blot shows the purity of the extracts and the efficiency of AGO2 immunoprecipitation. The lower and the upper panel derive from the same gel, only the membrane was split in two to perform the incubation with the antibody. A full picture of the gel can be found in the supplementary informations. (d) The amount of miR-9 and MALAT1 was measured in inputs RNA used for the RIP by qPCR. d) The amount of miR-9 and MALAT1 bound to AGO2 was measured by qPCR in nuclear and cytoplasmic fractions in presence of a scramble control or an antimiR-9. P-value (p<0.0001) was calculated by two-ways ANOVA.

Mentions: MicroRNAs are known to exert their functions mainly, if not exclusively, in the cytoplasm18. However, MALAT1 is a nuclear long ncRNA that localizes to nuclear speckles in association with splicing regulators19 raising the possibility that miR-9 targets MALAT1 in the nucleus. Using in situ hybridization and confocal microscopy, we detected miR-9 both in the cytoplasm and the nucleus of L428 Hodgkin lymphoma cells, HEK293 and U87MG glioblastoma cells (Fig. 4). Since previous studies have already demonstrated the presence of AGO2 in the nucleus202122, we next investigated whether miR-9 regulates MALAT1 in the nucleus in an AGO2-dependent manner. To this end, we transfected U87MG cells and L428 with the antimiR-9 oligonucleotide or a scrambled control (Fig. 4b), isolated the nuclei and immunoprecipitated AGO2 and the associated RNAs. AGO2 was efficiently immunoprecipitated from both total cell extracts and nuclear extracts (Fig. 4c–d). Notably, whereas MALAT1 was readily detected in AGO2 immunoprecipitates from scrambled control treated cells, its levels were significantly reduced in AGO2 complexes purified from cells treated with antimiR-9, similar results were obtained in L428 (supplementary figure 4a–b). These results are consistent with the notion that MALAT1 is targeted by miR-9 in the nucleus in an AGO2-dependent manner (Fig. 2c).


microRNA-9 targets the long non-coding RNA MALAT1 for degradation in the nucleus.

Leucci E, Patella F, Waage J, Holmstrøm K, Lindow M, Porse B, Kauppinen S, Lund AH - Sci Rep (2013)

miR-9 targets MALAT1 to AGO2 containing complexes in the nuclei (a) RNA fluorescent in situ hybridization for miR-9 in different cell lines. Magnification 63X. DAPI is in blue, miR-9 in green. (b) Expression of miR-9 and MALAT1 in nuclear and cytoplasmic fractions used as inputs for AGO2 IP was measured by RT-qPCR. (c) Nuclear and total fractions were isolated from U87MG cells transfected with a scramble control or an antimiR-9, and immunoprecipitated using an AGO2 antibody. The western blot shows the purity of the extracts and the efficiency of AGO2 immunoprecipitation. The lower and the upper panel derive from the same gel, only the membrane was split in two to perform the incubation with the antibody. A full picture of the gel can be found in the supplementary informations. (d) The amount of miR-9 and MALAT1 was measured in inputs RNA used for the RIP by qPCR. d) The amount of miR-9 and MALAT1 bound to AGO2 was measured by qPCR in nuclear and cytoplasmic fractions in presence of a scramble control or an antimiR-9. P-value (p<0.0001) was calculated by two-ways ANOVA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3756333&req=5

f4: miR-9 targets MALAT1 to AGO2 containing complexes in the nuclei (a) RNA fluorescent in situ hybridization for miR-9 in different cell lines. Magnification 63X. DAPI is in blue, miR-9 in green. (b) Expression of miR-9 and MALAT1 in nuclear and cytoplasmic fractions used as inputs for AGO2 IP was measured by RT-qPCR. (c) Nuclear and total fractions were isolated from U87MG cells transfected with a scramble control or an antimiR-9, and immunoprecipitated using an AGO2 antibody. The western blot shows the purity of the extracts and the efficiency of AGO2 immunoprecipitation. The lower and the upper panel derive from the same gel, only the membrane was split in two to perform the incubation with the antibody. A full picture of the gel can be found in the supplementary informations. (d) The amount of miR-9 and MALAT1 was measured in inputs RNA used for the RIP by qPCR. d) The amount of miR-9 and MALAT1 bound to AGO2 was measured by qPCR in nuclear and cytoplasmic fractions in presence of a scramble control or an antimiR-9. P-value (p<0.0001) was calculated by two-ways ANOVA.
Mentions: MicroRNAs are known to exert their functions mainly, if not exclusively, in the cytoplasm18. However, MALAT1 is a nuclear long ncRNA that localizes to nuclear speckles in association with splicing regulators19 raising the possibility that miR-9 targets MALAT1 in the nucleus. Using in situ hybridization and confocal microscopy, we detected miR-9 both in the cytoplasm and the nucleus of L428 Hodgkin lymphoma cells, HEK293 and U87MG glioblastoma cells (Fig. 4). Since previous studies have already demonstrated the presence of AGO2 in the nucleus202122, we next investigated whether miR-9 regulates MALAT1 in the nucleus in an AGO2-dependent manner. To this end, we transfected U87MG cells and L428 with the antimiR-9 oligonucleotide or a scrambled control (Fig. 4b), isolated the nuclei and immunoprecipitated AGO2 and the associated RNAs. AGO2 was efficiently immunoprecipitated from both total cell extracts and nuclear extracts (Fig. 4c–d). Notably, whereas MALAT1 was readily detected in AGO2 immunoprecipitates from scrambled control treated cells, its levels were significantly reduced in AGO2 complexes purified from cells treated with antimiR-9, similar results were obtained in L428 (supplementary figure 4a–b). These results are consistent with the notion that MALAT1 is targeted by miR-9 in the nucleus in an AGO2-dependent manner (Fig. 2c).

Bottom Line: microRNAs regulate the expression of over 60% of protein coding genes by targeting their mRNAs to AGO2-containing complexes in the cytoplasm and promoting their translational inhibition and/or degradation.There is little evidence so far for microRNA-mediated regulation of other classes of non-coding RNAs.Our findings reveal a novel direct regulatory link between two important classes of non-coding RNAs, miRs and lncRNAs, and advance our understanding of microRNA functions.

View Article: PubMed Central - PubMed

Affiliation: 1] Biotech Research and Innovation Centre and Centre for Epigenetics, University of Copenhagen, Denmark [2].

ABSTRACT
microRNAs regulate the expression of over 60% of protein coding genes by targeting their mRNAs to AGO2-containing complexes in the cytoplasm and promoting their translational inhibition and/or degradation. There is little evidence so far for microRNA-mediated regulation of other classes of non-coding RNAs. Here we report that microRNA-9 (miR-9) regulates the expression of the Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT-1), one of the most abundant and conserved long non-coding RNAs. Intriguingly, we find that miR-9 targets AGO2-mediated regulation of MALAT1 in the nucleus. Our findings reveal a novel direct regulatory link between two important classes of non-coding RNAs, miRs and lncRNAs, and advance our understanding of microRNA functions.

Show MeSH
Related in: MedlinePlus