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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.


Sequence diversity of regular shRNA products. Deep sequencing analysis of the Ago2-bound RNAs for shRT5 (a), mut 6 (b), and mut7 (c) shows a variety of shRNA/AgoshRNA products. The cumulative incidence (y axis, depicted as % of total reads) of distinct 5' ends (black bars) and 3' ends (white bars) is shown along the shRNA sequence (x-axis, in capitals with the promoter and loop area in small letters). The total number of reads is indicated. Marked are the predicted transcription start site (boxed in gray), the predicted Dicer cleavage site (Dicerexp; ◅) and the observed Dicer cleavage site (Dicerobs; ←). The major cleavage products (Ago2, 5p, 3p and an extra fragment*) are indicated per graph.
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fig2: Sequence diversity of regular shRNA products. Deep sequencing analysis of the Ago2-bound RNAs for shRT5 (a), mut 6 (b), and mut7 (c) shows a variety of shRNA/AgoshRNA products. The cumulative incidence (y axis, depicted as % of total reads) of distinct 5' ends (black bars) and 3' ends (white bars) is shown along the shRNA sequence (x-axis, in capitals with the promoter and loop area in small letters). The total number of reads is indicated. Marked are the predicted transcription start site (boxed in gray), the predicted Dicer cleavage site (Dicerexp; ◅) and the observed Dicer cleavage site (Dicerobs; ←). The major cleavage products (Ago2, 5p, 3p and an extra fragment*) are indicated per graph.

Mentions: We extracted Ago2-bound small RNAs to determine the relative amount of Dicer and Ago2 products by deep sequencing. Dominant reads (>50 copies in the library) are listed in Supplementary Table S1. For shRT5 and the two mutants, 96–99% of the plasmid-matching reads aligned to the expressed shRNA (Table 1). This high coverage permitted us to carefully map the content of the Ago2-bound RNAs. A major advantage of the AgoshRNA design over miRNA-like designs is that the Drosha-processing step is not needed to release the hairpin RNA, thus limiting the requirements for processing. Both the 5p and 3p Dicer products of shRT5 are equally present in Ago2 (Figure 2a). This confirms the symmetric nature of shRT5 and explains the silencing activity measured for both strands (Figure 1b; Luc). The reduced activity of the 3p strand compared to the 5p strand may reflect differential silencing activity of the two strands. A clear shift to 5p/AgoshRNA products was apparent for mutants 6 and 7 (Figure 2b,c). More Ago2-cleaved product was observed for mut 7 (9.7% of all reads) and mut 6 (1.3%) over the wild-type (wt) shRT5 (0.1%). This correlates with increased AgoshRNA production by mut 6 and especially mutant 7 on northern blot.28 Surprisingly, the observed Ago2-cleaved product (←) was 2 nt shorter than the expected AgoshRNA product (◄) for wt and the two mutants (Figure 1b). Increased AgoshRNA loading coincided with decreased 3p-strand loading for both mutants but especially mutant 6, although 5p loading was maintained (Figure 2b,c). This 3p loading-deficiency correlates with the reported loss of 3p-mediated Luc silencing activity, although northern blotting did show normal 3p-strand production.28


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)

Sequence diversity of regular shRNA products. Deep sequencing analysis of the Ago2-bound RNAs for shRT5 (a), mut 6 (b), and mut7 (c) shows a variety of shRNA/AgoshRNA products. The cumulative incidence (y axis, depicted as % of total reads) of distinct 5' ends (black bars) and 3' ends (white bars) is shown along the shRNA sequence (x-axis, in capitals with the promoter and loop area in small letters). The total number of reads is indicated. Marked are the predicted transcription start site (boxed in gray), the predicted Dicer cleavage site (Dicerexp; ◅) and the observed Dicer cleavage site (Dicerobs; ←). The major cleavage products (Ago2, 5p, 3p and an extra fragment*) are indicated per graph.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Sequence diversity of regular shRNA products. Deep sequencing analysis of the Ago2-bound RNAs for shRT5 (a), mut 6 (b), and mut7 (c) shows a variety of shRNA/AgoshRNA products. The cumulative incidence (y axis, depicted as % of total reads) of distinct 5' ends (black bars) and 3' ends (white bars) is shown along the shRNA sequence (x-axis, in capitals with the promoter and loop area in small letters). The total number of reads is indicated. Marked are the predicted transcription start site (boxed in gray), the predicted Dicer cleavage site (Dicerexp; ◅) and the observed Dicer cleavage site (Dicerobs; ←). The major cleavage products (Ago2, 5p, 3p and an extra fragment*) are indicated per graph.
Mentions: We extracted Ago2-bound small RNAs to determine the relative amount of Dicer and Ago2 products by deep sequencing. Dominant reads (>50 copies in the library) are listed in Supplementary Table S1. For shRT5 and the two mutants, 96–99% of the plasmid-matching reads aligned to the expressed shRNA (Table 1). This high coverage permitted us to carefully map the content of the Ago2-bound RNAs. A major advantage of the AgoshRNA design over miRNA-like designs is that the Drosha-processing step is not needed to release the hairpin RNA, thus limiting the requirements for processing. Both the 5p and 3p Dicer products of shRT5 are equally present in Ago2 (Figure 2a). This confirms the symmetric nature of shRT5 and explains the silencing activity measured for both strands (Figure 1b; Luc). The reduced activity of the 3p strand compared to the 5p strand may reflect differential silencing activity of the two strands. A clear shift to 5p/AgoshRNA products was apparent for mutants 6 and 7 (Figure 2b,c). More Ago2-cleaved product was observed for mut 7 (9.7% of all reads) and mut 6 (1.3%) over the wild-type (wt) shRT5 (0.1%). This correlates with increased AgoshRNA production by mut 6 and especially mutant 7 on northern blot.28 Surprisingly, the observed Ago2-cleaved product (←) was 2 nt shorter than the expected AgoshRNA product (◄) for wt and the two mutants (Figure 1b). Increased AgoshRNA loading coincided with decreased 3p-strand loading for both mutants but especially mutant 6, although 5p loading was maintained (Figure 2b,c). This 3p loading-deficiency correlates with the reported loss of 3p-mediated Luc silencing activity, although northern blotting did show normal 3p-strand production.28

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.