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MicroRNA (miRNA) are short, regulatory RNA transcribed from introns (non-coding regions of a gene) or intergenic regions (stretches of DNA present between genes). Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself, forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA...
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RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
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Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome
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miRepress: modelling gene expression regulation by microRNA with non-conventional binding sites.

Suman Ghosal, Shekhar Saha1, Shaoli Das

  • 1Department of Biological Chemistry, Indian Association for the Cultivation of Science, Kolkata, West Bengal, 700032, India.

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This study reveals non-conventional microRNA (miRNA) interactions binding to mRNA 3' ends, impacting protein regulation. A predictive model and experiments confirm these alternative miRNA binding sites influence gene expression.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • MicroRNA (miRNA)-mediated gene regulation is crucial in cellular processes.
  • Conventional miRNA targeting relies on seed region pairing with mRNA.
  • Alternative miRNA binding modes, particularly involving the miRNA 3' end, have been suggested but require further investigation.

Purpose of the Study:

  • To investigate non-conventional microRNA (miRNA)-target interactions involving the miRNA 3' end.
  • To develop a predictive model for miRNA-mediated gene regulation based on both conventional and non-conventional binding sites.
  • To experimentally validate the functional impact of predicted non-conventional miRNA-target interactions.

Main Methods:

  • Analysis of AGO PAR-CLIP data to identify miRNA binding sites lacking conventional seed pairing.
  • Correlation analysis of global protein fold changes with the presence of 6-mer and 7-mer target sites involving the miRNA 3' end.
  • Development of a computational model to predict miRNA target regulation based on binding site types.
  • Experimental validation using luciferase assays and immunoblot analysis in MCF-7 cells, modulating miRNA mimics for predicted non-conventional interactions (miR-367-5p/WNT1, miR-181a-5p/MYH10).

Main Results:

  • Identification of mRNA target regions with non-conventional binding sites at the miRNA 3' end, distinct from canonical seed pairing.
  • Significant association found between global protein fold changes and target sites involving the miRNA 3' end (6-mer and 7-mer sites).
  • A predictive model integrating conventional and non-conventional sites showed significant correlation with observed protein expression changes.
  • Experimental validation confirmed that non-conventional interactions can indeed mediate target repression, as demonstrated for WNT1 and MYH10.

Conclusions:

  • Non-conventional microRNA (miRNA) binding sites, particularly those engaging the miRNA 3' end, represent a significant mode of gene regulation.
  • The developed predictive model accurately reflects the contribution of both conventional and non-conventional sites to miRNA-mediated protein regulation.
  • These findings expand the understanding of miRNA targeting mechanisms and offer new avenues for predicting and manipulating gene expression.