Mapping effective microRNA pairing beyond the seed using abasic mutations
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1131823 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR29707079.fastq.gz | Fastqsanger.gz | |||
| SRR29707080.fastq.gz | Fastqsanger.gz | |||
| SRR29707081.fastq.gz | Fastqsanger.gz | |||
| SRR29707082.fastq.gz | Fastqsanger.gz | |||
| SRR29707083.fastq.gz | Fastqsanger.gz |
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Project description:MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by base-pairing to complementary sites in mRNAs. The primary element for site recognition is the seed region (nucleotides 2-8 in the miRNA), but for a minority of sites pairing outside the seed increases efficiency, with the supplementary region (nucleotides 13-16) typically having the greatest impact. However, the structural determinants of effective pairing outside the seed are not fully understood. Here, we use abasic modified nucleotides to disrupt pairing to residues 13 and 14 of miR-34a and measure the effect of this modification compared to wild-type miR-34a on the cellular transcriptome and proteome using RNA-seq and mass spectrometry. We find that a subset of sites with predicted supplementary pairing are affected by miRNA transfection, with up to two-fold decreases in site repression at the mRNA level, although the effect at the protein level is less pronounced. Overall, this study demonstrates a novel methodological approach for elucidating the role of specific miRNA residues in target site selection, advancing our understanding of miRNA-mediated gene regulation.
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Project description:MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by base-pairing to complementary sites in mRNAs. The most important element for site recognition is the seed region (nucleotides 2-8 in the miRNA), but for a minority of sites pairing outside the seed increases efficiency, with the supplementary region (nucleotides 13-16) typically having the greatest impact. However, it is not clear how large an effect supplementary pairing has on target site efficiency, or which sites are most affected by it. Here, we use abasic modified nucleotides to disrupt pairing to residues 13 and 14 of miR-34a and measure the effect of this mutation compared to wild-type miR-34a on the cellular transcriptome using RNA-seq. We find that a subset of sites with predicted supplementary pairing are affected 24 hours after miRNA overexpression, with up to two-fold decreases in site repression. We show that miR-34a 3'-pairing is sensitive to GU wobble pairs in a position-specific manner and favors bulges in the miRNA over the target. These results were validated with luciferase reporter assays. Overall, this study demonstrates a novel methodological approach for elucidating the role of specific miRNA residues in target site selection, advancing our understanding of miRNA-mediated gene regulation.
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Project description:Functional microRNA targeting without seed pairing
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Project description:MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to serve as guides, directing binding to partially complementary sites in mRNAs, ultimately causing post-transcriptional repression. Complementarity to the miRNA seed (miRNA nucleotides 2–7) is typically necessary and sufficient for repression. Here, we investigate unusual sites with extensive complementarity to the miRNA 3′ region (nucleotide 9 and onwards) but without complementarity to the seed. The top examples of these 3′-only sites bind as well as top canonical sites and impart similar repression, which can be further boosted by as few as 2–3 additional pairs to the miRNA seed. Despite these similarities, 3′-only sites have slower association and dissociation rates than seed-matched sites. They also impart different conformations to bound AGO–miRNA complexes than do seed-matched sites, and individual miRNAs differ substantially with respect to how well they bind their respective 3′-only sites. Thus, pairing to the seed is not always required for binding and repression, or for a target to gain access to the 3′ region of the guide. For miRNAs that recognize 3′-only sites, those sites are estimated to constitute <1% of the set of endogenous target sites, a proportion resembling that of other rare but functional site types such as 3′-compensatory sites.
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Project description:Mammalian microRNAs (miRNAs) pair to 3'UTRs of mRNAs to direct their posttranscriptional repression. Important for target recognition are approximately 7 nt sites that match the seed region of the miRNA. However, these seed matches are not always sufficient for repression, indicating that other characteristics help specify targeting. By combining computational and experimental approaches, we uncovered five general features of site context that boost site efficacy: AU-rich nucleotide composition near the site, proximity to sites for coexpressed miRNAs (which leads to cooperative action), proximity to residues pairing to miRNA nucleotides 13-16, positioning within the 3'UTR at least 15 nt from the stop codon, and positioning away from the center of long UTRs. A model combining these context determinants quantitatively predicts site performance both for exogenously added miRNAs and for endogenous miRNA-message interactions. Because it predicts site efficacy without recourse to evolutionary conservation, the model also identifies effective nonconserved sites and siRNA off-targets.
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