Limits...
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.


Models to explain aberrant cleavage of AgoshRNAs with an extended stem. (a) A regular AgoshRNA with a stem of 17–19 bp will dock into Ago2 with the 3' and 5' ends near the PAZ and MID domains, respectively. PIWI then cleaves the stem between bp 10–11. With increasing stem length (20–21 bp), AgoshRNA processing is profoundly reduced (right-hand column) and a remarkable shift in cleavage site occurs. (b) A model is proposed in which loading of the extended hairpin triggers rearrangement of Ago2 domains, causing the observed loss of cleavage efficiency and shift in cleavage site. Cleavage of the AgoshRNA yields the AgoshA, which is subsequently trimmed by PARN into the AgoshTRIM molecule. After trimming, the single-stranded 3' end of AgoshTRIM can dock into the PAZ-domain to establish a stable Ago2-RNA complex.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4561654&req=5

fig6: Models to explain aberrant cleavage of AgoshRNAs with an extended stem. (a) A regular AgoshRNA with a stem of 17–19 bp will dock into Ago2 with the 3' and 5' ends near the PAZ and MID domains, respectively. PIWI then cleaves the stem between bp 10–11. With increasing stem length (20–21 bp), AgoshRNA processing is profoundly reduced (right-hand column) and a remarkable shift in cleavage site occurs. (b) A model is proposed in which loading of the extended hairpin triggers rearrangement of Ago2 domains, causing the observed loss of cleavage efficiency and shift in cleavage site. Cleavage of the AgoshRNA yields the AgoshA, which is subsequently trimmed by PARN into the AgoshTRIM molecule. After trimming, the single-stranded 3' end of AgoshTRIM can dock into the PAZ-domain to establish a stable Ago2-RNA complex.

Mentions: Cleavage at the predicted site confirmed Ago2 cleavage for the shorter hairpins, but we noticed an upward shift in cleavage site for the less efficient AgoshRNAs larger than 19 bp for both mutant sets (Figures 1b and 3). An intriguing pattern became apparent: cleavage of mutants 17/18/19 occurs exactly at the predicted position in the 3' arm between bp 10 and 11 from the bottom of the stem, but was shifted between bp 11–12 and 12–13 for mutants 20 and 21, respectively (Figure 6a). Mutants 19/20/21 support the new concept of “cleavage at 9 bp from the loop” instead of the well-established “10 bp from the bottom” rule. In search for a mechanistic explanation, it is important to realize that the extended hairpins are processed less efficiently by Ago2 (Figure 4c,d). We present a mechanistic model in which the 5' end of the AgoshRNA docks in the MID domain and the loop is near the PAZ domain (Figure 6b). We propose a structural realignment in the Ago2-containing complex because the extended hairpins sterically clash with the PAZ and/or MID domains that are in close contact with the hairpin. This structural Ago2 rearrangement may explain the loss of slicer activity. A similar steric problem was suggested to occur for Ago2-loaded hairpins with a large loop.29 Structural rearrangement pushes the domains of Ago2 out of the optimal position, which can explain the cleavage to shift 1 or 2 bp further up in the stem, albeit at a greatly reduced efficiency. This mechanistic model also explains the unexpected PIWI cleavage sites observed for the initial mutant set with extended stems of 21 bp (Figure 2). Thus, the sequence of an AgoshRNA does not influence the actual cleavage site, which seems dictated primarily by the duplex length (and secondarily by the loop size, see Liu et al.29). Cleavage and subsequent trimming by PARN may thus be essential to create a free 3' end that can dock into the PAZ domain to reach a stable configuration.40,41,42 Single-stranded AgoshTRIM products will be accommodated in this stable Ago2 complex, thus blocking further PARN trimming and explaining the discrete trimming pattern (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)

Models to explain aberrant cleavage of AgoshRNAs with an extended stem. (a) A regular AgoshRNA with a stem of 17–19 bp will dock into Ago2 with the 3' and 5' ends near the PAZ and MID domains, respectively. PIWI then cleaves the stem between bp 10–11. With increasing stem length (20–21 bp), AgoshRNA processing is profoundly reduced (right-hand column) and a remarkable shift in cleavage site occurs. (b) A model is proposed in which loading of the extended hairpin triggers rearrangement of Ago2 domains, causing the observed loss of cleavage efficiency and shift in cleavage site. Cleavage of the AgoshRNA yields the AgoshA, which is subsequently trimmed by PARN into the AgoshTRIM molecule. After trimming, the single-stranded 3' end of AgoshTRIM can dock into the PAZ-domain to establish a stable Ago2-RNA complex.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Models to explain aberrant cleavage of AgoshRNAs with an extended stem. (a) A regular AgoshRNA with a stem of 17–19 bp will dock into Ago2 with the 3' and 5' ends near the PAZ and MID domains, respectively. PIWI then cleaves the stem between bp 10–11. With increasing stem length (20–21 bp), AgoshRNA processing is profoundly reduced (right-hand column) and a remarkable shift in cleavage site occurs. (b) A model is proposed in which loading of the extended hairpin triggers rearrangement of Ago2 domains, causing the observed loss of cleavage efficiency and shift in cleavage site. Cleavage of the AgoshRNA yields the AgoshA, which is subsequently trimmed by PARN into the AgoshTRIM molecule. After trimming, the single-stranded 3' end of AgoshTRIM can dock into the PAZ-domain to establish a stable Ago2-RNA complex.
Mentions: Cleavage at the predicted site confirmed Ago2 cleavage for the shorter hairpins, but we noticed an upward shift in cleavage site for the less efficient AgoshRNAs larger than 19 bp for both mutant sets (Figures 1b and 3). An intriguing pattern became apparent: cleavage of mutants 17/18/19 occurs exactly at the predicted position in the 3' arm between bp 10 and 11 from the bottom of the stem, but was shifted between bp 11–12 and 12–13 for mutants 20 and 21, respectively (Figure 6a). Mutants 19/20/21 support the new concept of “cleavage at 9 bp from the loop” instead of the well-established “10 bp from the bottom” rule. In search for a mechanistic explanation, it is important to realize that the extended hairpins are processed less efficiently by Ago2 (Figure 4c,d). We present a mechanistic model in which the 5' end of the AgoshRNA docks in the MID domain and the loop is near the PAZ domain (Figure 6b). We propose a structural realignment in the Ago2-containing complex because the extended hairpins sterically clash with the PAZ and/or MID domains that are in close contact with the hairpin. This structural Ago2 rearrangement may explain the loss of slicer activity. A similar steric problem was suggested to occur for Ago2-loaded hairpins with a large loop.29 Structural rearrangement pushes the domains of Ago2 out of the optimal position, which can explain the cleavage to shift 1 or 2 bp further up in the stem, albeit at a greatly reduced efficiency. This mechanistic model also explains the unexpected PIWI cleavage sites observed for the initial mutant set with extended stems of 21 bp (Figure 2). Thus, the sequence of an AgoshRNA does not influence the actual cleavage site, which seems dictated primarily by the duplex length (and secondarily by the loop size, see Liu et al.29). Cleavage and subsequent trimming by PARN may thus be essential to create a free 3' end that can dock into the PAZ domain to reach a stable configuration.40,41,42 Single-stranded AgoshTRIM products will be accommodated in this stable Ago2 complex, thus blocking further PARN trimming and explaining the discrete trimming pattern (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.