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The conformation of microRNA seed regions in native microRNPs is prearranged for presentation to mRNA targets.

Lambert NJ, Gu SG, Zahler AM - Nucleic Acids Res. (2011)

Bottom Line: Probing the Watson-Crick edges of the bases shows that bases 2-4 are largely inaccessible to solvent, while seed region bases 5-8 are readily modified; collectively our probing results suggest a model in which these bases are primed for initiating base pairing with the target mRNA.In addition, an unusual DMS reactivity with U at position 6 is observed.We propose that interaction of miRNAs with argonaute proteins pre-organizes the structure of the seed sequence for specific recognition of target mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of MCD Biology and the Center for Molecular Biology of RNA, University of California, Santa Cruz, CA 95064, USA.

ABSTRACT
MicroRNAs control gene expression by post-transcriptional down-regulation of their target mRNAs. Complementarity between the seed region (nucleotides 2-8) of a microRNA and the 3'-UTR of its target mRNA is the key determinant in recognition. However, the structural basis of the ability of the seed region to dominate target recognition in eukaryotic argonaute complexes has not been directly demonstrated. To better understand this problem, we performed chemical probing of microRNAs held in native argonaute-containing complexes isolated from Caenorhabditis elegans. Direct probing of the RNA backbone in isolated native microRNP complexes shows that the conformation of the seed region is uniquely constrained, while the rest of the microRNA structure is conformationally flexible. Probing the Watson-Crick edges of the bases shows that bases 2-4 are largely inaccessible to solvent, while seed region bases 5-8 are readily modified; collectively our probing results suggest a model in which these bases are primed for initiating base pairing with the target mRNA. In addition, an unusual DMS reactivity with U at position 6 is observed. We propose that interaction of miRNAs with argonaute proteins pre-organizes the structure of the seed sequence for specific recognition of target mRNAs.

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Seed region nucleotides exhibit unusual chemical reactivity to DMS and show protections from chemical modification (A–C). DMS or kethoxal were used to probe accessibility of bases of enriched C. elegans miRNPs, or miRNAs extracted from the enriched miRNPs. For mir-58 in (B), modifications were detected by splinted ligation. For the others, a 32P labeled oligonucleotide complementary to the terminal 10 nt of each indicated miRNA was used as primer. ‘+’ and ‘++’ indicate DMS incubation times of 10 and 20 min. Note the DMS reactive uracils at position 6 of mir-58 and (C) mir-80. (D) RNP-induced reactivity is plotted for three adjacent seed region bases. Naked miRNA and miRNP DMS reactivity was quantified. Reactivity of the modified position in miRNPs relative to the DMS reactivity in naked miRNAs is the RNP-induced reactivity.
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Figure 3: Seed region nucleotides exhibit unusual chemical reactivity to DMS and show protections from chemical modification (A–C). DMS or kethoxal were used to probe accessibility of bases of enriched C. elegans miRNPs, or miRNAs extracted from the enriched miRNPs. For mir-58 in (B), modifications were detected by splinted ligation. For the others, a 32P labeled oligonucleotide complementary to the terminal 10 nt of each indicated miRNA was used as primer. ‘+’ and ‘++’ indicate DMS incubation times of 10 and 20 min. Note the DMS reactive uracils at position 6 of mir-58 and (C) mir-80. (D) RNP-induced reactivity is plotted for three adjacent seed region bases. Naked miRNA and miRNP DMS reactivity was quantified. Reactivity of the modified position in miRNPs relative to the DMS reactivity in naked miRNAs is the RNP-induced reactivity.

Mentions: SHAPE chemical probing data were quantified using semi-automated footprinting analysis (SAFA) (38). The quantitations for each nucleotide position for either miRNP or miRNA complexes are simply normalized to the background in the DMSO (no NMIA) control (Figure 2C) or are displayed as enhancement of chemical reactivity in miRNPs (reactivity in lanes ± NMIA) over that of miRNAs (reactivity in lanes ± NMIA) (Figure 2D). For quantitation of DMS reactivity in Figure 3, ImageJ software (39) was used to determine the area under the peak for each band in the unmodified control and DMS-modifed lanes for the different bands in .tiff images.


The conformation of microRNA seed regions in native microRNPs is prearranged for presentation to mRNA targets.

Lambert NJ, Gu SG, Zahler AM - Nucleic Acids Res. (2011)

Seed region nucleotides exhibit unusual chemical reactivity to DMS and show protections from chemical modification (A–C). DMS or kethoxal were used to probe accessibility of bases of enriched C. elegans miRNPs, or miRNAs extracted from the enriched miRNPs. For mir-58 in (B), modifications were detected by splinted ligation. For the others, a 32P labeled oligonucleotide complementary to the terminal 10 nt of each indicated miRNA was used as primer. ‘+’ and ‘++’ indicate DMS incubation times of 10 and 20 min. Note the DMS reactive uracils at position 6 of mir-58 and (C) mir-80. (D) RNP-induced reactivity is plotted for three adjacent seed region bases. Naked miRNA and miRNP DMS reactivity was quantified. Reactivity of the modified position in miRNPs relative to the DMS reactivity in naked miRNAs is the RNP-induced reactivity.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113585&req=5

Figure 3: Seed region nucleotides exhibit unusual chemical reactivity to DMS and show protections from chemical modification (A–C). DMS or kethoxal were used to probe accessibility of bases of enriched C. elegans miRNPs, or miRNAs extracted from the enriched miRNPs. For mir-58 in (B), modifications were detected by splinted ligation. For the others, a 32P labeled oligonucleotide complementary to the terminal 10 nt of each indicated miRNA was used as primer. ‘+’ and ‘++’ indicate DMS incubation times of 10 and 20 min. Note the DMS reactive uracils at position 6 of mir-58 and (C) mir-80. (D) RNP-induced reactivity is plotted for three adjacent seed region bases. Naked miRNA and miRNP DMS reactivity was quantified. Reactivity of the modified position in miRNPs relative to the DMS reactivity in naked miRNAs is the RNP-induced reactivity.
Mentions: SHAPE chemical probing data were quantified using semi-automated footprinting analysis (SAFA) (38). The quantitations for each nucleotide position for either miRNP or miRNA complexes are simply normalized to the background in the DMSO (no NMIA) control (Figure 2C) or are displayed as enhancement of chemical reactivity in miRNPs (reactivity in lanes ± NMIA) over that of miRNAs (reactivity in lanes ± NMIA) (Figure 2D). For quantitation of DMS reactivity in Figure 3, ImageJ software (39) was used to determine the area under the peak for each band in the unmodified control and DMS-modifed lanes for the different bands in .tiff images.

Bottom Line: Probing the Watson-Crick edges of the bases shows that bases 2-4 are largely inaccessible to solvent, while seed region bases 5-8 are readily modified; collectively our probing results suggest a model in which these bases are primed for initiating base pairing with the target mRNA.In addition, an unusual DMS reactivity with U at position 6 is observed.We propose that interaction of miRNAs with argonaute proteins pre-organizes the structure of the seed sequence for specific recognition of target mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of MCD Biology and the Center for Molecular Biology of RNA, University of California, Santa Cruz, CA 95064, USA.

ABSTRACT
MicroRNAs control gene expression by post-transcriptional down-regulation of their target mRNAs. Complementarity between the seed region (nucleotides 2-8) of a microRNA and the 3'-UTR of its target mRNA is the key determinant in recognition. However, the structural basis of the ability of the seed region to dominate target recognition in eukaryotic argonaute complexes has not been directly demonstrated. To better understand this problem, we performed chemical probing of microRNAs held in native argonaute-containing complexes isolated from Caenorhabditis elegans. Direct probing of the RNA backbone in isolated native microRNP complexes shows that the conformation of the seed region is uniquely constrained, while the rest of the microRNA structure is conformationally flexible. Probing the Watson-Crick edges of the bases shows that bases 2-4 are largely inaccessible to solvent, while seed region bases 5-8 are readily modified; collectively our probing results suggest a model in which these bases are primed for initiating base pairing with the target mRNA. In addition, an unusual DMS reactivity with U at position 6 is observed. We propose that interaction of miRNAs with argonaute proteins pre-organizes the structure of the seed sequence for specific recognition of target mRNAs.

Show MeSH