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microRNAs associated with the different human Argonaute proteins

Dueck, A., Ziegler, C., Eichner, A., Berezikov, E., Meister, G.·2012·Nucleic Acids Research
DOI 10.1093/nar/gks705PMC PMC3479175
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Figure 1. miR-451 associates with Ago2 only. (A) Schematic representation of Ago2-mediated miR-451 processing (upper part) and canonical pre-miRNA processing by Dicer (lower part). (B) Constructs expressing wildtype (wt) pre-miR-451 (upper panel) or miR-451 embedded into a classical Dicer-dependent hairpin (lower panel) were transfected into HEK 293 cells. Ago1 (Lanes 4 and 10), Ago2 (Lanes 5 and 11) and Ago3 (Lanes 6 and 12) were immunoprecipitated using specific monoclonal antibodies. Co-immunoprecipitated miRNAs were analyzed by northern blotting against miR-451. Lanes 3 and 9 show control IPs, Lanes 2 and 8 show input samples and Lane 7 a size marker. (C) RNA was extracted from the indicated mouse tissues and miR-451 was detected by northern blotting. (D) Endogenous mAgo1 (Lanes 4 and 8) or mAgo2 (Lanes 5 and 9) were immunoprecipitated from spleen or liver and co-immunoprecipitated miR-451 (upper panel) or miR-19b (lower panel) was analyzed by northern blotting. Lanes 3 and 7 show control IPs, Lanes 2 and 6 input samples and Lanes 1 and 10 size markers.
Figure 1. miR-451 associates with Ago2 only. (A) Schematic representation of Ago2-mediated miR-451 processing (upper part) and canonical pre-miRNA processing by Dicer (lower part). (B) Constructs expressing wildtype (wt) pre-miR-451 (upper panel) or miR-451 embedded into a classical Dicer-dependent hairpin (lower panel) were transfected into HEK 293 cells. Ago1 (Lanes 4 and 10), Ago2 (Lanes 5 and 11) and Ago3 (Lanes 6 and 12) were immunoprecipitated using specific monoclonal antibodies. Co-immunoprecipitated miRNAs were analyzed by northern blotting against miR-451. Lanes 3 and 9 show control IPs, Lanes 2 and 8 show input samples and Lane 7 a size marker. (C) RNA was extracted from the indicated mouse tissues and miR-451 was detected by northern blotting. (D) Endogenous mAgo1 (Lanes 4 and 8) or mAgo2 (Lanes 5 and 9) were immunoprecipitated from spleen or liver and co-immunoprecipitated miR-451 (upper panel) or miR-19b (lower panel) was analyzed by northern blotting. Lanes 3 and 7 show control IPs, Lanes 2 and 6 input samples and Lanes 1 and 10 size markers.CC BY-NC · PubMed Central
Figure 2. Pre-miR-451-based shRNA expression. (A) An siRNA against GRK4 was either embedded into a pre-miR-451-like hairpin (left part) or a classical Dicer-dependent hairpin (right part). Both constructs were transfected into HEK 293 cells and Ago1 (Lanes 4 and 10), Ago2 (Lanes 5 and 11) or Ago3 (Lanes 6 and 12) was immunoprecipitated from the lysates. GRK4 siRNA incorporation was analyzed by northern blotting. Lanes 3 and 9 show control IPs, Lanes 2 and 8 input samples and Lane 7 a size marker. (B) Both GRK4 shRNAs described in (A) were transfected into HEK 293 cells. Total RNA was extracted at the indicated time points and GRK4 siRNA production was analyzed by northern blotting using a probe complementary to the guide strand of the GRK4 siRNA. (C) Samples described in (B) were analyzed by qPCR for knockdown of GRK4 at the indicated time points. (D) siRNAs against Importin 8 were embedded into a pre-miR-451-like hairpin (two different clones were tested, Lanes 2 and 3) or a classical Dicer-like shRNA (Lane 1). The constructs were transfected into HEK 293 cells and siRNA production was analyzed by northern blotting. (E) Relative expression levels of let-7c were tested by transfecting the genomically encoded (‘genomic’) (Lanes 2 and 6), the classical Dicer-like shRNA (‘classic’) (Lanes 3 and 7) or a pre-451-like hairpin (‘mimic’) (Lanes 4 and 8). As a background control, RNA from untransfected HEK 293 was used (Lanes 1 and 5).
Figure 2. Pre-miR-451-based shRNA expression. (A) An siRNA against GRK4 was either embedded into a pre-miR-451-like hairpin (left part) or a classical Dicer-dependent hairpin (right part). Both constructs were transfected into HEK 293 cells and Ago1 (Lanes 4 and 10), Ago2 (Lanes 5 and 11) or Ago3 (Lanes 6 and 12) was immunoprecipitated from the lysates. GRK4 siRNA incorporation was analyzed by northern blotting. Lanes 3 and 9 show control IPs, Lanes 2 and 8 input samples and Lane 7 a size marker. (B) Both GRK4 shRNAs described in (A) were transfected into HEK 293 cells. Total RNA was extracted at the indicated time points and GRK4 siRNA production was analyzed by northern blotting using a probe complementary to the guide strand of the GRK4 siRNA. (C) Samples described in (B) were analyzed by qPCR for knockdown of GRK4 at the indicated time points. (D) siRNAs against Importin 8 were embedded into a pre-miR-451-like hairpin (two different clones were tested, Lanes 2 and 3) or a classical Dicer-like shRNA (Lane 1). The constructs were transfected into HEK 293 cells and siRNA production was analyzed by northern blotting. (E) Relative expression levels of let-7c were tested by transfecting the genomically encoded (‘genomic’) (Lanes 2 and 6), the classical Dicer-like shRNA (‘classic’) (Lanes 3 and 7) or a pre-451-like hairpin (‘mimic’) (Lanes 4 and 8). As a background control, RNA from untransfected HEK 293 was used (Lanes 1 and 5).CC BY-NC · PubMed Central
Figure 3. Structure and sequence requirements of miR-451 for Ago2-mediated cleavage. (A) A wt miR-451 construct (Lane 2) or a mutant with a changed 5′ end (Lane 3, A to U mutation, mutant #1) were transfected into HEK 293 cells and processing was analyzed by northern blotting. Lane 1 shows a size marker. (B) Nucleotides were inserted into the loop of pre-miR-451 (Lanes 2 to 6) and the constructs were analyzed as described in (A). (C) Mutants of the wt pre-miR-451 hairpin were constructed to test the sequence specificity of Ago2-mediated processing. Constructs were transfected into HEK 293 cells and analyzed on individual northern blots, since each mutation alters the resulting mature sequence. Equal amounts of size marker were loaded to provide comparability. (D) G/U wobble base pairs at the 5′ end (Lane 4) and near the loop (Lane 5) were mutated to G/C Watson–Crick base pairs and the constructs analyzed as in (A).
Figure 3. Structure and sequence requirements of miR-451 for Ago2-mediated cleavage. (A) A wt miR-451 construct (Lane 2) or a mutant with a changed 5′ end (Lane 3, A to U mutation, mutant #1) were transfected into HEK 293 cells and processing was analyzed by northern blotting. Lane 1 shows a size marker. (B) Nucleotides were inserted into the loop of pre-miR-451 (Lanes 2 to 6) and the constructs were analyzed as described in (A). (C) Mutants of the wt pre-miR-451 hairpin were constructed to test the sequence specificity of Ago2-mediated processing. Constructs were transfected into HEK 293 cells and analyzed on individual northern blots, since each mutation alters the resulting mature sequence. Equal amounts of size marker were loaded to provide comparability. (D) G/U wobble base pairs at the 5′ end (Lane 4) and near the loop (Lane 5) were mutated to G/C Watson–Crick base pairs and the constructs analyzed as in (A).CC BY-NC · PubMed Central
Figure 4. Deep-sequencing analysis of small RNAs associated with endogenous Ago proteins. (A) Composition of the small RNA libraries from the input sample and immunoprecipitated Ago1, Ago2, Ago3, Ago4 and control (an unrelated antibody against the complement system protein RmC from rat). (B) Table of the 10 most abundant miRNAs associated with Ago1, Ago2 or Ago3. The colors indicate the same miRNA. (C) Comparison of the sorting profile from the deep-sequencing data (right side) for miR-182 (top panel), miR-92a (middle panel, right) and miR-30a (lower panel) with profiles resulting from northern blotting (left side).
Figure 4. Deep-sequencing analysis of small RNAs associated with endogenous Ago proteins. (A) Composition of the small RNA libraries from the input sample and immunoprecipitated Ago1, Ago2, Ago3, Ago4 and control (an unrelated antibody against the complement system protein RmC from rat). (B) Table of the 10 most abundant miRNAs associated with Ago1, Ago2 or Ago3. The colors indicate the same miRNA. (C) Comparison of the sorting profile from the deep-sequencing data (right side) for miR-182 (top panel), miR-92a (middle panel, right) and miR-30a (lower panel) with profiles resulting from northern blotting (left side).CC BY-NC · PubMed Central
Figure 5. Ago proteins show different global small RNA binding patterns and nontemplated 3′ extension profiles. (A) Length distribution patterns of Ago1 (left), Ago2 (middle) and Ago3 (right). (B) Left side: Ago1, Ago2 and Ago3 associated miRNAs were analyzed for nontemplated 3′ extensions, values were calculated in relation to all miRNA reads of the respective library. Perfect: no nontemplated 3′ extensions; A, U, G, C: amount of single nucleotide 3′ extensions; other: sum of all other 3′ extensions (≥2 nt). Right side: each single nontemplated 3′ extension was set in relation to the total number of nontemplated 3′ extensions in the respective library (Ago1, Ago2 or Ago3).
Figure 5. Ago proteins show different global small RNA binding patterns and nontemplated 3′ extension profiles. (A) Length distribution patterns of Ago1 (left), Ago2 (middle) and Ago3 (right). (B) Left side: Ago1, Ago2 and Ago3 associated miRNAs were analyzed for nontemplated 3′ extensions, values were calculated in relation to all miRNA reads of the respective library. Perfect: no nontemplated 3′ extensions; A, U, G, C: amount of single nucleotide 3′ extensions; other: sum of all other 3′ extensions (≥2 nt). Right side: each single nontemplated 3′ extension was set in relation to the total number of nontemplated 3′ extensions in the respective library (Ago1, Ago2 or Ago3).CC BY-NC · PubMed Central
Figure 6. Ago proteins show distinct length profiles for individual miRNAs. The length pattern of individual miRNAs was extracted from the deep-sequencing data (left side) and compared against the length profiles seen by northern blotting (right side). For this, northern blotting signals for each miRNA band were quantified; the total signal from each lane/each Ago was set to 100%. The length of a miRNA in northern blotting was assessed by comparison with the size marker. miRNA length profiles were separated into: (A) miRNA length profiles showing divergent Ago association and (B) miRNA length profiles showing matching Ago1–3 association.
Figure 6. Ago proteins show distinct length profiles for individual miRNAs. The length pattern of individual miRNAs was extracted from the deep-sequencing data (left side) and compared against the length profiles seen by northern blotting (right side). For this, northern blotting signals for each miRNA band were quantified; the total signal from each lane/each Ago was set to 100%. The length of a miRNA in northern blotting was assessed by comparison with the size marker. miRNA length profiles were separated into: (A) miRNA length profiles showing divergent Ago association and (B) miRNA length profiles showing matching Ago1–3 association.CC BY-NC · PubMed Central

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Metadata

Authors
Dueck, A., Ziegler, C., Eichner, A., Berezikov, E., & Meister, G.
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2012-07-25
Volume
40
Issue
19
Pages
9850-9862
ISSN
  • 0305-1048
  • 1362-4962
DOI
10.1093/nar/gks705
PMC
PMC3479175
Cited by
195
References
3
Language
en
Licence
creativecommons.org/licenses/by-nc/3.0 (vor, 1566 d)

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