Kinetic analysis of small silencing RNA production by human and Drosophila dicer enzymes in vitro

Susan E. Liao, Ryuya Fukunaga

Research output: ResearchChapter

Abstract

Dicer enzymes produce small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), which then are loaded into Argonaute proteins and act as sequence-specific guides. A powerful tool to understand the molecular mechanism of small silencing RNA production by Dicers is an in vitro RNA processing assay using recombinant Dicer proteins. Such biochemical analyses have elucidated the substrate specificities and kinetics of Dicers, the mechanism by which the length of small RNAs produced by Dicers is determined, and the effects of Dicer-partner proteins and endogenous small molecules such as ATP and inorganic phosphate on small RNA production by Dicers, among others. Here, we describe methods for in vitro small RNA production assay using recombinant human and Drosophila Dicer proteins.

LanguageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages101-121
Number of pages21
Volume1680
DOIs
StatePublished - 2018

Publication series

NameMethods in Molecular Biology
Volume1680
ISSN (Print)1064-3745

Fingerprint

Ribonuclease III
RNA Interference
Drosophila
RNA
In Vitro Techniques
Argonaute Proteins
Drosophila Proteins
Substrate Specificity
MicroRNAs
Recombinant Proteins
Small Interfering RNA
Adenosine Triphosphate
Phosphates
Proteins
human DICER1 protein

Keywords

  • Dicer
  • Kinetics
  • miRNA
  • RNA silencing
  • siRNA

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Liao, S. E., & Fukunaga, R. (2018). Kinetic analysis of small silencing RNA production by human and Drosophila dicer enzymes in vitro. In Methods in Molecular Biology (Vol. 1680, pp. 101-121). (Methods in Molecular Biology; Vol. 1680). Humana Press Inc.. DOI: 10.1007/978-1-4939-7339-2_7

Kinetic analysis of small silencing RNA production by human and Drosophila dicer enzymes in vitro. / Liao, Susan E.; Fukunaga, Ryuya.

Methods in Molecular Biology. Vol. 1680 Humana Press Inc., 2018. p. 101-121 (Methods in Molecular Biology; Vol. 1680).

Research output: ResearchChapter

Liao, SE & Fukunaga, R 2018, Kinetic analysis of small silencing RNA production by human and Drosophila dicer enzymes in vitro. in Methods in Molecular Biology. vol. 1680, Methods in Molecular Biology, vol. 1680, Humana Press Inc., pp. 101-121. DOI: 10.1007/978-1-4939-7339-2_7
Liao SE, Fukunaga R. Kinetic analysis of small silencing RNA production by human and Drosophila dicer enzymes in vitro. In Methods in Molecular Biology. Vol. 1680. Humana Press Inc.2018. p. 101-121. (Methods in Molecular Biology). Available from, DOI: 10.1007/978-1-4939-7339-2_7
Liao, Susan E. ; Fukunaga, Ryuya. / Kinetic analysis of small silencing RNA production by human and Drosophila dicer enzymes in vitro. Methods in Molecular Biology. Vol. 1680 Humana Press Inc., 2018. pp. 101-121 (Methods in Molecular Biology).
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