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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.


Canonical and noncanonical miRNA/shRNA processing. (a) Left panel: Dicer-dependent (canonical) processing is shown for miRNA and shRNA. Dicer cleavage (◅) yields 5' (5p) and 3' (3p) strands, the guide is loaded in RNA-induced silencing complex-Ago2 (black line with seed), the passenger is degraded (grey strand). Right panel: The Dicer-independent (non-canonical) miR-451 and AgoshRNA are processed by Ago2 (◄) to yield a single guide that is partially basepaired. Subsequent trimming of the 3' end opens miR-451. For AgoshRNAs, no such trimming has been described. (b) The wild-type shRT5 and two G-U mutants (mutations boxed in black) were studied by deep sequencing. Indicated are the predicted cleavage sites for Dicer and Ago2, but also the observed Ago2 cleavage site (←). Some key activities as measured previously are summarized below: Dicer and Ago2 products as scored on northern blot (see Figure 7 in reference 28) and their activity on Luc-reporter assays (see Figure 6 Luc 5p: antisense; Luc 3p: sense in reference 28).
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fig1: Canonical and noncanonical miRNA/shRNA processing. (a) Left panel: Dicer-dependent (canonical) processing is shown for miRNA and shRNA. Dicer cleavage (◅) yields 5' (5p) and 3' (3p) strands, the guide is loaded in RNA-induced silencing complex-Ago2 (black line with seed), the passenger is degraded (grey strand). Right panel: The Dicer-independent (non-canonical) miR-451 and AgoshRNA are processed by Ago2 (◄) to yield a single guide that is partially basepaired. Subsequent trimming of the 3' end opens miR-451. For AgoshRNAs, no such trimming has been described. (b) The wild-type shRT5 and two G-U mutants (mutations boxed in black) were studied by deep sequencing. Indicated are the predicted cleavage sites for Dicer and Ago2, but also the observed Ago2 cleavage site (←). Some key activities as measured previously are summarized below: Dicer and Ago2 products as scored on northern blot (see Figure 7 in reference 28) and their activity on Luc-reporter assays (see Figure 6 Luc 5p: antisense; Luc 3p: sense in reference 28).

Mentions: The RNA interference (RNAi) mechanism uses small RNA molecules to control cellular gene expression.1,2 These small molecules are processed from short RNA hairpin precursors of which the ends are defined by microprocessor cleavage (miRNAs)3,4 or transcription initiation and termination (shRNAs).5 In the canonical pathway, Dicer will cleave the top of the RNA hairpin to create a small RNA duplex with a 5' (5p) and 3' (3p) strand (Figure 1a; left panel).6,7 Many of the Dicer-cleaved miRNA duplexes are subject to 3' end modification through addition of a few tailing nucleotides, specifically U or A.8,9,10 The addition of 3' U is thought to mark the RNA for degradation11,12,13 or processing.14 Addition of 3'A is believed to promote stability,15 but other reports failed to reproduce this effect.8 The processed RNA duplex is loaded into the Argonaute (Ago)-containing RNA-induced silencing complex (RISC).16,17,18 The PIWI domain of the Ago2 protein has slicing activity that cleaves the passenger strand, thus activating the guide strand.19,20,21 The selection of guide/passenger is determined by the thermodynamic stability of the duplex ends.22,23 The strand of which the 5' end is located at the least stable duplex end is more likely to be selected as guide. Guide-loaded RISC will target partially complementary messenger RNAs (mRNAs) for translational suppression or destruction.18,24


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)

Canonical and noncanonical miRNA/shRNA processing. (a) Left panel: Dicer-dependent (canonical) processing is shown for miRNA and shRNA. Dicer cleavage (◅) yields 5' (5p) and 3' (3p) strands, the guide is loaded in RNA-induced silencing complex-Ago2 (black line with seed), the passenger is degraded (grey strand). Right panel: The Dicer-independent (non-canonical) miR-451 and AgoshRNA are processed by Ago2 (◄) to yield a single guide that is partially basepaired. Subsequent trimming of the 3' end opens miR-451. For AgoshRNAs, no such trimming has been described. (b) The wild-type shRT5 and two G-U mutants (mutations boxed in black) were studied by deep sequencing. Indicated are the predicted cleavage sites for Dicer and Ago2, but also the observed Ago2 cleavage site (←). Some key activities as measured previously are summarized below: Dicer and Ago2 products as scored on northern blot (see Figure 7 in reference 28) and their activity on Luc-reporter assays (see Figure 6 Luc 5p: antisense; Luc 3p: sense in reference 28).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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fig1: Canonical and noncanonical miRNA/shRNA processing. (a) Left panel: Dicer-dependent (canonical) processing is shown for miRNA and shRNA. Dicer cleavage (◅) yields 5' (5p) and 3' (3p) strands, the guide is loaded in RNA-induced silencing complex-Ago2 (black line with seed), the passenger is degraded (grey strand). Right panel: The Dicer-independent (non-canonical) miR-451 and AgoshRNA are processed by Ago2 (◄) to yield a single guide that is partially basepaired. Subsequent trimming of the 3' end opens miR-451. For AgoshRNAs, no such trimming has been described. (b) The wild-type shRT5 and two G-U mutants (mutations boxed in black) were studied by deep sequencing. Indicated are the predicted cleavage sites for Dicer and Ago2, but also the observed Ago2 cleavage site (←). Some key activities as measured previously are summarized below: Dicer and Ago2 products as scored on northern blot (see Figure 7 in reference 28) and their activity on Luc-reporter assays (see Figure 6 Luc 5p: antisense; Luc 3p: sense in reference 28).
Mentions: The RNA interference (RNAi) mechanism uses small RNA molecules to control cellular gene expression.1,2 These small molecules are processed from short RNA hairpin precursors of which the ends are defined by microprocessor cleavage (miRNAs)3,4 or transcription initiation and termination (shRNAs).5 In the canonical pathway, Dicer will cleave the top of the RNA hairpin to create a small RNA duplex with a 5' (5p) and 3' (3p) strand (Figure 1a; left panel).6,7 Many of the Dicer-cleaved miRNA duplexes are subject to 3' end modification through addition of a few tailing nucleotides, specifically U or A.8,9,10 The addition of 3' U is thought to mark the RNA for degradation11,12,13 or processing.14 Addition of 3'A is believed to promote stability,15 but other reports failed to reproduce this effect.8 The processed RNA duplex is loaded into the Argonaute (Ago)-containing RNA-induced silencing complex (RISC).16,17,18 The PIWI domain of the Ago2 protein has slicing activity that cleaves the passenger strand, thus activating the guide strand.19,20,21 The selection of guide/passenger is determined by the thermodynamic stability of the duplex ends.22,23 The strand of which the 5' end is located at the least stable duplex end is more likely to be selected as guide. Guide-loaded RISC will target partially complementary messenger RNAs (mRNAs) for translational suppression or destruction.18,24

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.