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Related Concept Videos

MicroRNAs01:22

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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...
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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 cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...
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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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A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools
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An optimized microRNA backbone for effective single-copy RNAi.

Christof Fellmann1, Thomas Hoffmann2, Vaishali Sridhar1

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Researchers optimized synthetic short hairpin RNA (shRNA) constructs, called shRNAmirs, for more effective gene silencing. The new miR-E backbone enhances microRNA processing, leading to potent gene knockdown from single genomic copies.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Short hairpin RNA (shRNA) is crucial for gene repression.
  • Synthetic shRNAs (shRNAmirs) integrated into microRNA contexts offer versatile RNA interference (RNAi) tools.
  • Current shRNAmirs often show limited knockdown efficacy due to incomplete understanding of microRNA biogenesis.

Purpose of the Study:

  • To systematically optimize the microRNA-30 backbone for enhanced shRNAmir processing and function.
  • To identify key elements within the microRNA backbone essential for potent gene knockdown.
  • To develop an improved platform for generating effective single-copy shRNA libraries.

Main Methods:

  • Systematic optimization of the microRNA-30 backbone for synthetic shRNAmirs.
  • Identification and implementation of a conserved element 3' of the basal stem.
  • Validation of the optimized "miR-E" backbone for increased mature shRNA levels and knockdown efficacy.

Main Results:

  • A conserved element 3' of the basal stem was identified as critical for shRNAmir processing.
  • The optimized "miR-E" backbone significantly increased mature shRNA levels.
  • The miR-E system demonstrated potent gene knockdown efficacy, even from a single genomic copy.

Conclusions:

  • The developed "miR-E" backbone represents a significant advancement in shRNAmir technology.
  • This optimized platform facilitates the creation of effective single-copy shRNA libraries for genome-wide functional annotation.
  • The findings improve the reliability and efficiency of RNAi-based gene silencing experiments.