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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.


AgoshRNA processing and stem length. (a) Model for AgoshRNA processing. The AgoshRNA is cleaved by Ago2 into an Agosh guide and a small 3' A-tail is added to create AgoshA, which is subsequently trimmed to AgoshTRIM. (b) The 3' ends of the AgoshTRIM population (incidence on y axis, depicted as % of total AgoshTRIM reads) is shown along the AgoshRNA sequence (x-axis). X is C or U, depending on the mutant analyzed. On the left a small cartoon of the top AgoshRNA is shown with the major observed species after trimming (grey triangle). Average and standard variation was calculated for the complete shRNA/AgoshRNAs set. The major 3' end of AgoshTRIM is marked as a gray triangle in the RNA structure cartoon. (c,d) The reads encoded by the GC set (panel c) and GU set (panel d) were used to calculate the incidence of Dicer (black line), AgoshA (striped line), and AgoshTRIM (gray line) products. Plotted is their percentage (y-axis, % of total reads) along the stem length (x-axis).
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fig4: AgoshRNA processing and stem length. (a) Model for AgoshRNA processing. The AgoshRNA is cleaved by Ago2 into an Agosh guide and a small 3' A-tail is added to create AgoshA, which is subsequently trimmed to AgoshTRIM. (b) The 3' ends of the AgoshTRIM population (incidence on y axis, depicted as % of total AgoshTRIM reads) is shown along the AgoshRNA sequence (x-axis). X is C or U, depending on the mutant analyzed. On the left a small cartoon of the top AgoshRNA is shown with the major observed species after trimming (grey triangle). Average and standard variation was calculated for the complete shRNA/AgoshRNAs set. The major 3' end of AgoshTRIM is marked as a gray triangle in the RNA structure cartoon. (c,d) The reads encoded by the GC set (panel c) and GU set (panel d) were used to calculate the incidence of Dicer (black line), AgoshA (striped line), and AgoshTRIM (gray line) products. Plotted is their percentage (y-axis, % of total reads) along the stem length (x-axis).

Mentions: Inspired by recent findings for miR-451 and small nucleolar RNA (snoRNA), we reasoned that 3' adenylation may present a signal for PARN to trim the AgoshA into the unpaired AgoshTRIM molecule (Figure 4a).38,39 Indeed, a distinct RNA population was observed that starts at the TSS and terminates just 3' of the shRNA loop (Figure 4b).


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)

AgoshRNA processing and stem length. (a) Model for AgoshRNA processing. The AgoshRNA is cleaved by Ago2 into an Agosh guide and a small 3' A-tail is added to create AgoshA, which is subsequently trimmed to AgoshTRIM. (b) The 3' ends of the AgoshTRIM population (incidence on y axis, depicted as % of total AgoshTRIM reads) is shown along the AgoshRNA sequence (x-axis). X is C or U, depending on the mutant analyzed. On the left a small cartoon of the top AgoshRNA is shown with the major observed species after trimming (grey triangle). Average and standard variation was calculated for the complete shRNA/AgoshRNAs set. The major 3' end of AgoshTRIM is marked as a gray triangle in the RNA structure cartoon. (c,d) The reads encoded by the GC set (panel c) and GU set (panel d) were used to calculate the incidence of Dicer (black line), AgoshA (striped line), and AgoshTRIM (gray line) products. Plotted is their percentage (y-axis, % of total reads) along the stem length (x-axis).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: AgoshRNA processing and stem length. (a) Model for AgoshRNA processing. The AgoshRNA is cleaved by Ago2 into an Agosh guide and a small 3' A-tail is added to create AgoshA, which is subsequently trimmed to AgoshTRIM. (b) The 3' ends of the AgoshTRIM population (incidence on y axis, depicted as % of total AgoshTRIM reads) is shown along the AgoshRNA sequence (x-axis). X is C or U, depending on the mutant analyzed. On the left a small cartoon of the top AgoshRNA is shown with the major observed species after trimming (grey triangle). Average and standard variation was calculated for the complete shRNA/AgoshRNAs set. The major 3' end of AgoshTRIM is marked as a gray triangle in the RNA structure cartoon. (c,d) The reads encoded by the GC set (panel c) and GU set (panel d) were used to calculate the incidence of Dicer (black line), AgoshA (striped line), and AgoshTRIM (gray line) products. Plotted is their percentage (y-axis, % of total reads) along the stem length (x-axis).
Mentions: Inspired by recent findings for miR-451 and small nucleolar RNA (snoRNA), we reasoned that 3' adenylation may present a signal for PARN to trim the AgoshA into the unpaired AgoshTRIM molecule (Figure 4a).38,39 Indeed, a distinct RNA population was observed that starts at the TSS and terminates just 3' of the shRNA loop (Figure 4b).

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.