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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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Related in: MedlinePlus

Native C. elegans microRNPs contain ALG-1/ALG-2 and bind to complementary targets. (A) A biotinylated control DNA sequence, or a mixture of biotinylated DNA oligos perfectly complementary to four abundant miRNAs (mir-58, mir-52, mir-66, mir-71) were bound to streptavidin beads and incubated with enriched miRNPs from N2 worms. Northern blots probed with mir-58, mir-52, mir-66, mir-71 and negative control lin-4 show the enrichment. Silver stain of SDS–PAGE shows co-purified proteins. ALG-1 and ALG-2 were identified by mass spectrometry. (B) An in vitro transcribed control RNA and an imperfectly complementary synthetic RNA target for mir-58 were covalently immobilized to beads. RNA-bound beads and a beads-only control were incubated with enriched miRNP fractions from an ALG-1::GFP expressing strain, WM84. The enrichment of mir-58 was detected by northern blot; ALG-1::GFP and TSN-1 were detected by immunoblots.
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Figure 1: Native C. elegans microRNPs contain ALG-1/ALG-2 and bind to complementary targets. (A) A biotinylated control DNA sequence, or a mixture of biotinylated DNA oligos perfectly complementary to four abundant miRNAs (mir-58, mir-52, mir-66, mir-71) were bound to streptavidin beads and incubated with enriched miRNPs from N2 worms. Northern blots probed with mir-58, mir-52, mir-66, mir-71 and negative control lin-4 show the enrichment. Silver stain of SDS–PAGE shows co-purified proteins. ALG-1 and ALG-2 were identified by mass spectrometry. (B) An in vitro transcribed control RNA and an imperfectly complementary synthetic RNA target for mir-58 were covalently immobilized to beads. RNA-bound beads and a beads-only control were incubated with enriched miRNP fractions from an ALG-1::GFP expressing strain, WM84. The enrichment of mir-58 was detected by northern blot; ALG-1::GFP and TSN-1 were detected by immunoblots.

Mentions: To determine the structural basis of the seed region's importance, we isolated native C. elegans microRNP complexes for structural probing experiments. We previously reported the purification of native miRNPs from C. elegans using a combination of size exclusion, anion (monoQ) and cation (monoS) exchange chromatographic steps; >95% of the RNAs purified from this biochemically enriched miRNP fraction consisted of known miRNAs (33). No target mRNAs were detected in these high-throughput sequencing experiments, as all of the RNAs in the enriched miRNP fraction were 21–25 nt in length. To further characterize the protein content of these purified miRNPs, we prepared a mixture of biotinylated DNA oligonucleotides complementary to the four most abundant C. elegans miRNAs (mir-58, mir-52, mir-62 and mir-71), which together account for 43% of the pool of C. elegans miRNAs (33). The oligonucleotides were immobilized on streptavidin beads and used to specifically enrich their miRNP protein partners. Figure 1A shows specific enrichment of the targeted miRNAs on the immobilized oligonucleotides. Specific proteins that co-purified with these abundant miRNAs were analyzed by mass spectrometry. The argonautes ALG-1 and ALG-2, homologs of the human argonaute AGO2 that is found associated with miRNAs (40), were the only proteins from the miRNP-enriched fraction that specifically co-purified with the four abundant miRNAs (Figure 1A).Figure 1.


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)

Native C. elegans microRNPs contain ALG-1/ALG-2 and bind to complementary targets. (A) A biotinylated control DNA sequence, or a mixture of biotinylated DNA oligos perfectly complementary to four abundant miRNAs (mir-58, mir-52, mir-66, mir-71) were bound to streptavidin beads and incubated with enriched miRNPs from N2 worms. Northern blots probed with mir-58, mir-52, mir-66, mir-71 and negative control lin-4 show the enrichment. Silver stain of SDS–PAGE shows co-purified proteins. ALG-1 and ALG-2 were identified by mass spectrometry. (B) An in vitro transcribed control RNA and an imperfectly complementary synthetic RNA target for mir-58 were covalently immobilized to beads. RNA-bound beads and a beads-only control were incubated with enriched miRNP fractions from an ALG-1::GFP expressing strain, WM84. The enrichment of mir-58 was detected by northern blot; ALG-1::GFP and TSN-1 were detected by immunoblots.
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Figure 1: Native C. elegans microRNPs contain ALG-1/ALG-2 and bind to complementary targets. (A) A biotinylated control DNA sequence, or a mixture of biotinylated DNA oligos perfectly complementary to four abundant miRNAs (mir-58, mir-52, mir-66, mir-71) were bound to streptavidin beads and incubated with enriched miRNPs from N2 worms. Northern blots probed with mir-58, mir-52, mir-66, mir-71 and negative control lin-4 show the enrichment. Silver stain of SDS–PAGE shows co-purified proteins. ALG-1 and ALG-2 were identified by mass spectrometry. (B) An in vitro transcribed control RNA and an imperfectly complementary synthetic RNA target for mir-58 were covalently immobilized to beads. RNA-bound beads and a beads-only control were incubated with enriched miRNP fractions from an ALG-1::GFP expressing strain, WM84. The enrichment of mir-58 was detected by northern blot; ALG-1::GFP and TSN-1 were detected by immunoblots.
Mentions: To determine the structural basis of the seed region's importance, we isolated native C. elegans microRNP complexes for structural probing experiments. We previously reported the purification of native miRNPs from C. elegans using a combination of size exclusion, anion (monoQ) and cation (monoS) exchange chromatographic steps; >95% of the RNAs purified from this biochemically enriched miRNP fraction consisted of known miRNAs (33). No target mRNAs were detected in these high-throughput sequencing experiments, as all of the RNAs in the enriched miRNP fraction were 21–25 nt in length. To further characterize the protein content of these purified miRNPs, we prepared a mixture of biotinylated DNA oligonucleotides complementary to the four most abundant C. elegans miRNAs (mir-58, mir-52, mir-62 and mir-71), which together account for 43% of the pool of C. elegans miRNAs (33). The oligonucleotides were immobilized on streptavidin beads and used to specifically enrich their miRNP protein partners. Figure 1A shows specific enrichment of the targeted miRNAs on the immobilized oligonucleotides. Specific proteins that co-purified with these abundant miRNAs were analyzed by mass spectrometry. The argonautes ALG-1 and ALG-2, homologs of the human argonaute AGO2 that is found associated with miRNAs (40), were the only proteins from the miRNP-enriched fraction that specifically co-purified with the four abundant miRNAs (Figure 1A).Figure 1.

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