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Deep Sequence Analysis of AgoshRNA Processing Reveals 3' A Addition and Trimming.

Harwig A, Herrera-Carrillo E, Jongejan A, van Kampen AH, Berkhout B - Mol Ther Nucleic Acids (2015)

Bottom Line: Here we present the results of a deep sequence study on the processing of shRNAs with different stem length and a top G·U wobble base pair (bp).First, we confirmed the gradual shift from Dicer to Ago2 processing upon shortening of the hairpin length.Fourth, the Ago2-processed AgoshRNAs acquire a short 3' tail of 1-3 A-nucleotides (nt) and we present evidence that this product is subsequently trimmed by the poly(A)-specific ribonuclease (PARN).

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

ABSTRACT
The RNA interference (RNAi) pathway, in which microprocessor and Dicer collaborate to process microRNAs (miRNA), was recently expanded by the description of alternative processing routes. In one of these noncanonical pathways, Dicer action is replaced by the Argonaute2 (Ago2) slicer function. It was recently shown that the stem-length of precursor-miRNA or short hairpin RNA (shRNA) molecules is a major determinant for Dicer versus Ago2 processing. Here we present the results of a deep sequence study on the processing of shRNAs with different stem length and a top G·U wobble base pair (bp). This analysis revealed some unexpected properties of these so-called AgoshRNA molecules that are processed by Ago2 instead of Dicer. First, we confirmed the gradual shift from Dicer to Ago2 processing upon shortening of the hairpin length. Second, hairpins with a stem larger than 19 base pair are inefficiently cleaved by Ago2 and we noticed a shift in the cleavage site. Third, the introduction of a top G·U bp in a regular shRNA can promote Ago2-cleavage, which coincides with a loss of Ago2-loading of the Dicer-cleaved 3' strand. Fourth, the Ago2-processed AgoshRNAs acquire a short 3' tail of 1-3 A-nucleotides (nt) and we present evidence that this product is subsequently trimmed by the poly(A)-specific ribonuclease (PARN).

No MeSH data available.


Knockdown of PARN. HEK-293T was transfected with 5 μg of the indicated AgoshRNA constructs and 50 nmol/l siRNA against PARN (PARN) and control (Ctrl). (a) Total RNA was isolated and analyzed by northern blot using an locked nucleic acid probe directed against the 5' side of the hairpin. The AgoshTRIM and Dicer products are indicated. Size markers were included in the far left lanes and the length is indicated. Ethidium bromide staining of small rRNAs and tRNAs are shown as loading controls below the blot. (b) Quantification of AgoshTRIM and Dicer products. The siCtrl-treated samples were set at 100%.
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fig5: Knockdown of PARN. HEK-293T was transfected with 5 μg of the indicated AgoshRNA constructs and 50 nmol/l siRNA against PARN (PARN) and control (Ctrl). (a) Total RNA was isolated and analyzed by northern blot using an locked nucleic acid probe directed against the 5' side of the hairpin. The AgoshTRIM and Dicer products are indicated. Size markers were included in the far left lanes and the length is indicated. Ethidium bromide staining of small rRNAs and tRNAs are shown as loading controls below the blot. (b) Quantification of AgoshTRIM and Dicer products. The siCtrl-treated samples were set at 100%.

Mentions: To further investigate the involvement of PARN in the generation of AgoshTRIM products, we performed a PARN knockdown experiment with 18GC and 19GC. These constructs were chosen because they generate distinct AgoshRNA products.28 The total RNA content of transfected cells was separated on a denaturing gel, blotted and probed with a locked nucleic acid probe directed against the 5' side of the hairpin (Figure 5a). This probe should thus detect Ago2 cleavage products as well as the Dicer-cleaved 5p strand. The knockdown of PARN caused the selective disappearance of the AgoshTRIM product, while leaving the Dicer cleaved product intact (Figure 5b).


Deep Sequence Analysis of AgoshRNA Processing Reveals 3' A Addition and Trimming.

Harwig A, Herrera-Carrillo E, Jongejan A, van Kampen AH, Berkhout B - Mol Ther Nucleic Acids (2015)

Knockdown of PARN. HEK-293T was transfected with 5 μg of the indicated AgoshRNA constructs and 50 nmol/l siRNA against PARN (PARN) and control (Ctrl). (a) Total RNA was isolated and analyzed by northern blot using an locked nucleic acid probe directed against the 5' side of the hairpin. The AgoshTRIM and Dicer products are indicated. Size markers were included in the far left lanes and the length is indicated. Ethidium bromide staining of small rRNAs and tRNAs are shown as loading controls below the blot. (b) Quantification of AgoshTRIM and Dicer products. The siCtrl-treated samples were set at 100%.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561654&req=5

fig5: Knockdown of PARN. HEK-293T was transfected with 5 μg of the indicated AgoshRNA constructs and 50 nmol/l siRNA against PARN (PARN) and control (Ctrl). (a) Total RNA was isolated and analyzed by northern blot using an locked nucleic acid probe directed against the 5' side of the hairpin. The AgoshTRIM and Dicer products are indicated. Size markers were included in the far left lanes and the length is indicated. Ethidium bromide staining of small rRNAs and tRNAs are shown as loading controls below the blot. (b) Quantification of AgoshTRIM and Dicer products. The siCtrl-treated samples were set at 100%.
Mentions: To further investigate the involvement of PARN in the generation of AgoshTRIM products, we performed a PARN knockdown experiment with 18GC and 19GC. These constructs were chosen because they generate distinct AgoshRNA products.28 The total RNA content of transfected cells was separated on a denaturing gel, blotted and probed with a locked nucleic acid probe directed against the 5' side of the hairpin (Figure 5a). This probe should thus detect Ago2 cleavage products as well as the Dicer-cleaved 5p strand. The knockdown of PARN caused the selective disappearance of the AgoshTRIM product, while leaving the Dicer cleaved product intact (Figure 5b).

Bottom Line: Here we present the results of a deep sequence study on the processing of shRNAs with different stem length and a top G·U wobble base pair (bp).First, we confirmed the gradual shift from Dicer to Ago2 processing upon shortening of the hairpin length.Fourth, the Ago2-processed AgoshRNAs acquire a short 3' tail of 1-3 A-nucleotides (nt) and we present evidence that this product is subsequently trimmed by the poly(A)-specific ribonuclease (PARN).

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

ABSTRACT
The RNA interference (RNAi) pathway, in which microprocessor and Dicer collaborate to process microRNAs (miRNA), was recently expanded by the description of alternative processing routes. In one of these noncanonical pathways, Dicer action is replaced by the Argonaute2 (Ago2) slicer function. It was recently shown that the stem-length of precursor-miRNA or short hairpin RNA (shRNA) molecules is a major determinant for Dicer versus Ago2 processing. Here we present the results of a deep sequence study on the processing of shRNAs with different stem length and a top G·U wobble base pair (bp). This analysis revealed some unexpected properties of these so-called AgoshRNA molecules that are processed by Ago2 instead of Dicer. First, we confirmed the gradual shift from Dicer to Ago2 processing upon shortening of the hairpin length. Second, hairpins with a stem larger than 19 base pair are inefficiently cleaved by Ago2 and we noticed a shift in the cleavage site. Third, the introduction of a top G·U bp in a regular shRNA can promote Ago2-cleavage, which coincides with a loss of Ago2-loading of the Dicer-cleaved 3' strand. Fourth, the Ago2-processed AgoshRNAs acquire a short 3' tail of 1-3 A-nucleotides (nt) and we present evidence that this product is subsequently trimmed by the poly(A)-specific ribonuclease (PARN).

No MeSH data available.