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RNA-binding protein AUF1 represses Dicer expression.

Abdelmohsen K, Tominaga-Yamanaka K, Srikantan S, Yoon JH, Kang MJ, Gorospe M - Nucleic Acids Res. (2012)

Bottom Line: Despite its critical function, the mechanisms that regulate Dicer expression are not well understood.Here we report that the RNA-binding protein (RBP) AUF1 (AU-binding factor 1) associates with the endogenous DICER1 mRNA and can interact with several segments of DICER1 mRNA within the coding region (CR) and the 3'-untranslated region (UTR).In summary, AUF1 suppresses miRNA production by reducing Dicer production.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology and Immunology, National Institute on Aging-Intramural Research Program, NIH, 251 Bayview Blvd., Baltimore, MD 21224, USA. abdelmohsenk@mail.nih.gov

ABSTRACT
MicroRNA (miRNA) biogenesis is tightly regulated by numerous proteins. Among them, Dicer is required for the processing of the precursor (pre-)miRNAs into the mature miRNA. Despite its critical function, the mechanisms that regulate Dicer expression are not well understood. Here we report that the RNA-binding protein (RBP) AUF1 (AU-binding factor 1) associates with the endogenous DICER1 mRNA and can interact with several segments of DICER1 mRNA within the coding region (CR) and the 3'-untranslated region (UTR). Through these interactions, AUF1 lowered DICER1 mRNA stability, since silencing AUF1 lengthened DICER1 mRNA half-life and increased Dicer expression, while overexpressing AUF1 lowered DICER1 mRNA and Dicer protein levels. Given that Dicer is necessary for the synthesis of mature miRNAs, the lowering of Dicer levels by AUF1 diminished the levels of miRNAs tested, but not the levels of the corresponding pre-miRNAs. In summary, AUF1 suppresses miRNA production by reducing Dicer production.

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AUF1 overexpression decreases miRNA levels. (A) Forty-eight hours after transfection with pcDNA, AUF1 overexpression plasmids (described in Figure 2A), and Ctrl or Dicer siRNAs (as in Figure 6A), the levels of several miRNAs were measured using RT-qPCR and normalized to U6. Data in (A) are the means ± S.D. from three independent experiments; *P < 0.05; **P < 0.01. (B) Forty-eight hours after transfection of the plasmids and siRNAs shown, the levels of miR-24, let-7 and loading control U6 were assessed by northern blot analysis. Data in (B) are representative of three independent experiments.
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gks930-F7: AUF1 overexpression decreases miRNA levels. (A) Forty-eight hours after transfection with pcDNA, AUF1 overexpression plasmids (described in Figure 2A), and Ctrl or Dicer siRNAs (as in Figure 6A), the levels of several miRNAs were measured using RT-qPCR and normalized to U6. Data in (A) are the means ± S.D. from three independent experiments; *P < 0.05; **P < 0.01. (B) Forty-eight hours after transfection of the plasmids and siRNAs shown, the levels of miR-24, let-7 and loading control U6 were assessed by northern blot analysis. Data in (B) are representative of three independent experiments.

Mentions: Since Dicer promotes miRNA maturation, we investigated the influence of AUF1-modulated Dicer on the abundance of miRNAs. First, under conditions of reduced Dicer levels achieved by overexpressing AUF1 (Figure 2B) or by siRNA-mediated silencing of Dicer (Figure 6A), we monitored the abundance of several miRNAs. In AUF1-overexpressing HeLa cells (which expressed lower Dicer levels) as well as in cells in which Dicer was silenced, all of the miRNAs tested showed lesser abundance compared with those measured in the control population (Figure 7A). In addition, for abundant endogenous miRNAs (miR-24 and let-7), these differences were confirmed by northern blot analysis. As shown in Figure 7B, miRNA levels were higher when AUF1 was silenced and were lower when AUF1 was overexpressed and when Dicer was silenced. We attempted to study other miRNAs, but they were below the detection level of the assay.Figure 7.


RNA-binding protein AUF1 represses Dicer expression.

Abdelmohsen K, Tominaga-Yamanaka K, Srikantan S, Yoon JH, Kang MJ, Gorospe M - Nucleic Acids Res. (2012)

AUF1 overexpression decreases miRNA levels. (A) Forty-eight hours after transfection with pcDNA, AUF1 overexpression plasmids (described in Figure 2A), and Ctrl or Dicer siRNAs (as in Figure 6A), the levels of several miRNAs were measured using RT-qPCR and normalized to U6. Data in (A) are the means ± S.D. from three independent experiments; *P < 0.05; **P < 0.01. (B) Forty-eight hours after transfection of the plasmids and siRNAs shown, the levels of miR-24, let-7 and loading control U6 were assessed by northern blot analysis. Data in (B) are representative of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526313&req=5

gks930-F7: AUF1 overexpression decreases miRNA levels. (A) Forty-eight hours after transfection with pcDNA, AUF1 overexpression plasmids (described in Figure 2A), and Ctrl or Dicer siRNAs (as in Figure 6A), the levels of several miRNAs were measured using RT-qPCR and normalized to U6. Data in (A) are the means ± S.D. from three independent experiments; *P < 0.05; **P < 0.01. (B) Forty-eight hours after transfection of the plasmids and siRNAs shown, the levels of miR-24, let-7 and loading control U6 were assessed by northern blot analysis. Data in (B) are representative of three independent experiments.
Mentions: Since Dicer promotes miRNA maturation, we investigated the influence of AUF1-modulated Dicer on the abundance of miRNAs. First, under conditions of reduced Dicer levels achieved by overexpressing AUF1 (Figure 2B) or by siRNA-mediated silencing of Dicer (Figure 6A), we monitored the abundance of several miRNAs. In AUF1-overexpressing HeLa cells (which expressed lower Dicer levels) as well as in cells in which Dicer was silenced, all of the miRNAs tested showed lesser abundance compared with those measured in the control population (Figure 7A). In addition, for abundant endogenous miRNAs (miR-24 and let-7), these differences were confirmed by northern blot analysis. As shown in Figure 7B, miRNA levels were higher when AUF1 was silenced and were lower when AUF1 was overexpressed and when Dicer was silenced. We attempted to study other miRNAs, but they were below the detection level of the assay.Figure 7.

Bottom Line: Despite its critical function, the mechanisms that regulate Dicer expression are not well understood.Here we report that the RNA-binding protein (RBP) AUF1 (AU-binding factor 1) associates with the endogenous DICER1 mRNA and can interact with several segments of DICER1 mRNA within the coding region (CR) and the 3'-untranslated region (UTR).In summary, AUF1 suppresses miRNA production by reducing Dicer production.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology and Immunology, National Institute on Aging-Intramural Research Program, NIH, 251 Bayview Blvd., Baltimore, MD 21224, USA. abdelmohsenk@mail.nih.gov

ABSTRACT
MicroRNA (miRNA) biogenesis is tightly regulated by numerous proteins. Among them, Dicer is required for the processing of the precursor (pre-)miRNAs into the mature miRNA. Despite its critical function, the mechanisms that regulate Dicer expression are not well understood. Here we report that the RNA-binding protein (RBP) AUF1 (AU-binding factor 1) associates with the endogenous DICER1 mRNA and can interact with several segments of DICER1 mRNA within the coding region (CR) and the 3'-untranslated region (UTR). Through these interactions, AUF1 lowered DICER1 mRNA stability, since silencing AUF1 lengthened DICER1 mRNA half-life and increased Dicer expression, while overexpressing AUF1 lowered DICER1 mRNA and Dicer protein levels. Given that Dicer is necessary for the synthesis of mature miRNAs, the lowering of Dicer levels by AUF1 diminished the levels of miRNAs tested, but not the levels of the corresponding pre-miRNAs. In summary, AUF1 suppresses miRNA production by reducing Dicer production.

Show MeSH