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Related Concept Videos

MicroRNAs01:22

MicroRNAs

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...
MicroRNAs01:22

MicroRNAs

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 ends...
MicroRNAs01:22

MicroRNAs

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 ends...
siRNA - Small Interfering RNAs02:30

siRNA - Small Interfering RNAs

Small interfering RNAs, or siRNAs, are short regulatory RNA molecules that can silence genes post-transcriptionally, as well as the transcriptional level in some cases. siRNAs are important for protecting cells against viral infections and silencing transposable genetic elements.
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the ATP-dependent...
RNA Interference01:23

RNA Interference

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.
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
Experimental RNAi02:15

Experimental RNAi

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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mirMachine: A One-Stop Shop for Plant miRNA Annotation
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miRNA and shRNA expression vectors based on mRNA and miRNA processing.

Ping Wu1, Melissa A Wilmarth, Feng Zhang

  • 1Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, TX, USA.

Methods in Molecular Biology (Clifton, N.J.)
|September 26, 2012
PubMed
Summary

New RNAi vectors, pSM155 and pSM30, enhance gene silencing by integrating artificial miRNA expression into synthetic introns. These tools simplify cloning and improve marker gene expression for research and therapeutics.

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Area of Science:

  • Molecular Biology
  • Gene Regulation
  • Biotechnology

Background:

  • RNA interference (RNAi) is a key mechanism for gene silencing in research and therapy.
  • RNAi relies on small interfering RNAs derived from microRNAs (miRNAs) or small hairpin RNAs (shRNAs).
  • Developing efficient RNAi vectors is crucial for advancing these applications.

Purpose of the Study:

  • To introduce novel RNAi vectors (pSM155, pSM30) for improved miRNA and shRNA expression.
  • To integrate artificial miRNA expression cassettes within synthetic introns to leverage miRNA processing and RNA splicing.
  • To offer a simplified cloning method and enhanced marker gene expression.

Main Methods:

  • Design and construction of pSM155 and pSM30 RNAi vectors.
  • Incorporation of miRNA-based expression cassettes into synthetic introns.
  • Evaluation of enhanced green fluorescent protein (EGFP) expression from the same transcript.
  • Development of protocols for cloning and efficiency assessment of artificial and natural miRNAs/shRNAs.

Main Results:

  • The new vectors significantly improved the expression of an EGFP marker gene.
  • Integration within introns enhanced miRNA processing and RNA splicing efficiency.
  • A simplified cloning procedure was established for the vectors.
  • Demonstrated effectiveness in downregulating target gene expression.

Conclusions:

  • The pSM155 and pSM30 vectors represent a significant advancement in RNAi technology.
  • These vectors offer enhanced gene silencing capabilities through optimized miRNA expression.
  • They provide valuable tools for both basic research and therapeutic development in gene silencing.