Limits...
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.

Show MeSH

Related in: MedlinePlus

miRNA seed regions are defined by structural backbone rigidity. (A) Schematic for splinted ligation. (B) Probing of mir-58 and mir-52 miRNPs by SHAPE. DMSO (control) or NMIA were added to miRNPs. The miRNAs were extracted, ligated to a donor sequence and modifications detected. (C) Quantitation of NMIA reactivity of mir-58 and mir-52 in naked RNA or in miRNPs. For each nucleotide position, the intensity in the NMIA-modified lane relative to the DMSO control was determined. Results from three independent experiments are plotted. (D) Measurement of the miRNP induced reactivity, NMIA chemical reactivity of mir-58 and mir-52 containing miRNPs relative to the chemical reactivity of the corresponding naked miRNA. For each position, the reactivity of the modified miRNP backbone position determined in (C) was divided by reactivity of the naked miRNA at that position (also determined in (C).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3113585&req=5

Figure 2: miRNA seed regions are defined by structural backbone rigidity. (A) Schematic for splinted ligation. (B) Probing of mir-58 and mir-52 miRNPs by SHAPE. DMSO (control) or NMIA were added to miRNPs. The miRNAs were extracted, ligated to a donor sequence and modifications detected. (C) Quantitation of NMIA reactivity of mir-58 and mir-52 in naked RNA or in miRNPs. For each nucleotide position, the intensity in the NMIA-modified lane relative to the DMSO control was determined. Results from three independent experiments are plotted. (D) Measurement of the miRNP induced reactivity, NMIA chemical reactivity of mir-58 and mir-52 containing miRNPs relative to the chemical reactivity of the corresponding naked miRNA. For each position, the reactivity of the modified miRNP backbone position determined in (C) was divided by reactivity of the naked miRNA at that position (also determined in (C).

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)

miRNA seed regions are defined by structural backbone rigidity. (A) Schematic for splinted ligation. (B) Probing of mir-58 and mir-52 miRNPs by SHAPE. DMSO (control) or NMIA were added to miRNPs. The miRNAs were extracted, ligated to a donor sequence and modifications detected. (C) Quantitation of NMIA reactivity of mir-58 and mir-52 in naked RNA or in miRNPs. For each nucleotide position, the intensity in the NMIA-modified lane relative to the DMSO control was determined. Results from three independent experiments are plotted. (D) Measurement of the miRNP induced reactivity, NMIA chemical reactivity of mir-58 and mir-52 containing miRNPs relative to the chemical reactivity of the corresponding naked miRNA. For each position, the reactivity of the modified miRNP backbone position determined in (C) was divided by reactivity of the naked miRNA at that position (also determined in (C).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: miRNA seed regions are defined by structural backbone rigidity. (A) Schematic for splinted ligation. (B) Probing of mir-58 and mir-52 miRNPs by SHAPE. DMSO (control) or NMIA were added to miRNPs. The miRNAs were extracted, ligated to a donor sequence and modifications detected. (C) Quantitation of NMIA reactivity of mir-58 and mir-52 in naked RNA or in miRNPs. For each nucleotide position, the intensity in the NMIA-modified lane relative to the DMSO control was determined. Results from three independent experiments are plotted. (D) Measurement of the miRNP induced reactivity, NMIA chemical reactivity of mir-58 and mir-52 containing miRNPs relative to the chemical reactivity of the corresponding naked miRNA. For each position, the reactivity of the modified miRNP backbone position determined in (C) was divided by reactivity of the naked miRNA at that position (also determined in (C).
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
Related in: MedlinePlus