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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.


The shRT5 mutants with varying stem length. The 19/5 shRNA was elongated or shortened at the stem base to create a set of length mutants as described. The terminal top bp was mutated from G-C into a less stable G·U wobble bp (lower set, G·U wobble in a black box). Predicted Dicer (◅), predicted Ago2 (◄) and observed Ago2 (←) cleavage sites are indicated.
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fig3: The shRT5 mutants with varying stem length. The 19/5 shRNA was elongated or shortened at the stem base to create a set of length mutants as described. The terminal top bp was mutated from G-C into a less stable G·U wobble bp (lower set, G·U wobble in a black box). Predicted Dicer (◅), predicted Ago2 (◄) and observed Ago2 (←) cleavage sites are indicated.

Mentions: Next, the shift from Dicer to Ago2 cleavage was analyzed for a second set of shRNAs with decreasing stem length and either a G·U or G-C top base pair. The expected cleavage products of Ago2 (◄) and Dicer (◅) are indicated on the hairpin templates in Figure 3. Some unexpected details of AgoshRNA processing were disclosed that will be discussed before we address the general shift from Dicer to Ago2 cleavage. As observed for mut 6 and 7, we witnessed a shift in the actual Ago2 cleavage site, and an intriguing pattern became apparent. For mutants 17/18/19, cleavage occurred exactly at the predicted position between bp 10–11 from the bottom of the stem, but cleavage shifted to bp 11–12 and 12–13 for mutants 20 and 21, respectively. The observed Ago2 cleavage sites are marked in Figure 3 as arrows (←) and were used for quantitation of the AgoshRNA products. Inspection of the reads indicates that the transcripts with a shifted Ago2 cleavage site (mutants 20/21) do start at the same -1 position as mutants 17/18/19.


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)

The shRT5 mutants with varying stem length. The 19/5 shRNA was elongated or shortened at the stem base to create a set of length mutants as described. The terminal top bp was mutated from G-C into a less stable G·U wobble bp (lower set, G·U wobble in a black box). Predicted Dicer (◅), predicted Ago2 (◄) and observed Ago2 (←) cleavage sites are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: The shRT5 mutants with varying stem length. The 19/5 shRNA was elongated or shortened at the stem base to create a set of length mutants as described. The terminal top bp was mutated from G-C into a less stable G·U wobble bp (lower set, G·U wobble in a black box). Predicted Dicer (◅), predicted Ago2 (◄) and observed Ago2 (←) cleavage sites are indicated.
Mentions: Next, the shift from Dicer to Ago2 cleavage was analyzed for a second set of shRNAs with decreasing stem length and either a G·U or G-C top base pair. The expected cleavage products of Ago2 (◄) and Dicer (◅) are indicated on the hairpin templates in Figure 3. Some unexpected details of AgoshRNA processing were disclosed that will be discussed before we address the general shift from Dicer to Ago2 cleavage. As observed for mut 6 and 7, we witnessed a shift in the actual Ago2 cleavage site, and an intriguing pattern became apparent. For mutants 17/18/19, cleavage occurred exactly at the predicted position between bp 10–11 from the bottom of the stem, but cleavage shifted to bp 11–12 and 12–13 for mutants 20 and 21, respectively. The observed Ago2 cleavage sites are marked in Figure 3 as arrows (←) and were used for quantitation of the AgoshRNA products. Inspection of the reads indicates that the transcripts with a shifted Ago2 cleavage site (mutants 20/21) do start at the same -1 position as mutants 17/18/19.

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.