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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 regulates Dicer expression through the DICER1 3′UTR. (A) Schematic of the parent reporter plasmid pEGFP and the pEGFP-DICER1(3′) reporter plasmid bearing the DICER1 3′UTR. (B) HeLa cells were transfected with a reporter construct, together with either Ctrl siRNA or AUF1 siRNA; 48 h after transfection, the levels of EGFP, AUF1 and β-actin were assessed by western blot analysis (left) and the levels of EGFP and EGFP-DICER(3′) mRNAs (normalized to GAPDH mRNA levels and represented as % of EGFP mRNA levels in Ctrl siRNA-trasfected cells) were assessed by RT-qPCR analysis (right). (C) Fluorescence microscopy to visualize GFP in cells transfected as in (B). The data (means and ±S.D.) are representative of three independent experiments.
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gks930-F3: AUF1 regulates Dicer expression through the DICER1 3′UTR. (A) Schematic of the parent reporter plasmid pEGFP and the pEGFP-DICER1(3′) reporter plasmid bearing the DICER1 3′UTR. (B) HeLa cells were transfected with a reporter construct, together with either Ctrl siRNA or AUF1 siRNA; 48 h after transfection, the levels of EGFP, AUF1 and β-actin were assessed by western blot analysis (left) and the levels of EGFP and EGFP-DICER(3′) mRNAs (normalized to GAPDH mRNA levels and represented as % of EGFP mRNA levels in Ctrl siRNA-trasfected cells) were assessed by RT-qPCR analysis (right). (C) Fluorescence microscopy to visualize GFP in cells transfected as in (B). The data (means and ±S.D.) are representative of three independent experiments.

Mentions: To investigate if AUF1 regulates DICER1 mRNA stability through the region where it binds more extensively (DICER1 3′UTR), we prepared the heterologous reporter construct pEGFP-DICER1(3′) (Figure 3A). Expression of the heterologous reporter protein EGFP from EGFP mRNA (encoded by the CR of the parent control vector, pEGFP) was compared to EGFP expressed from CR of the chimeric EGFP-DICER1(3′) mRNA. By western blot analysis, EGFP protein levels expressed from pEGFP were unaltered by AUF1 silencing; in contrast, the levels of EGFP protein expressed from pEGFP-DICER1(3′) were substantially higher after silencing AUF1 (Figure 3B left). These changes in EGFP protein abundance were mirrored by changes in the expression of encoding transcripts [EGFP and EGFP-DICER(3′) mRNAs, Figure 3B right] as well as by the green fluorescence of the transfected cultures (Figure 3C). These findings indicate that there are negative regulatory elements within the 3′UTR of DICER1 mRNA and that AUF1 elicits at least part of its repressive actions through this region.Figure 3.


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 regulates Dicer expression through the DICER1 3′UTR. (A) Schematic of the parent reporter plasmid pEGFP and the pEGFP-DICER1(3′) reporter plasmid bearing the DICER1 3′UTR. (B) HeLa cells were transfected with a reporter construct, together with either Ctrl siRNA or AUF1 siRNA; 48 h after transfection, the levels of EGFP, AUF1 and β-actin were assessed by western blot analysis (left) and the levels of EGFP and EGFP-DICER(3′) mRNAs (normalized to GAPDH mRNA levels and represented as % of EGFP mRNA levels in Ctrl siRNA-trasfected cells) were assessed by RT-qPCR analysis (right). (C) Fluorescence microscopy to visualize GFP in cells transfected as in (B). The data (means and ±S.D.) 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-F3: AUF1 regulates Dicer expression through the DICER1 3′UTR. (A) Schematic of the parent reporter plasmid pEGFP and the pEGFP-DICER1(3′) reporter plasmid bearing the DICER1 3′UTR. (B) HeLa cells were transfected with a reporter construct, together with either Ctrl siRNA or AUF1 siRNA; 48 h after transfection, the levels of EGFP, AUF1 and β-actin were assessed by western blot analysis (left) and the levels of EGFP and EGFP-DICER(3′) mRNAs (normalized to GAPDH mRNA levels and represented as % of EGFP mRNA levels in Ctrl siRNA-trasfected cells) were assessed by RT-qPCR analysis (right). (C) Fluorescence microscopy to visualize GFP in cells transfected as in (B). The data (means and ±S.D.) are representative of three independent experiments.
Mentions: To investigate if AUF1 regulates DICER1 mRNA stability through the region where it binds more extensively (DICER1 3′UTR), we prepared the heterologous reporter construct pEGFP-DICER1(3′) (Figure 3A). Expression of the heterologous reporter protein EGFP from EGFP mRNA (encoded by the CR of the parent control vector, pEGFP) was compared to EGFP expressed from CR of the chimeric EGFP-DICER1(3′) mRNA. By western blot analysis, EGFP protein levels expressed from pEGFP were unaltered by AUF1 silencing; in contrast, the levels of EGFP protein expressed from pEGFP-DICER1(3′) were substantially higher after silencing AUF1 (Figure 3B left). These changes in EGFP protein abundance were mirrored by changes in the expression of encoding transcripts [EGFP and EGFP-DICER(3′) mRNAs, Figure 3B right] as well as by the green fluorescence of the transfected cultures (Figure 3C). These findings indicate that there are negative regulatory elements within the 3′UTR of DICER1 mRNA and that AUF1 elicits at least part of its repressive actions through this region.Figure 3.

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