Biochemistry of RNA Silencing

Abstract

RNA interference is an inhibitory mechanism able to downregulate the expression of mRNA (messenger ribonucleic acid) in a very specific manner. This posttranscriptional gene regulation pathway relies on the action of a small noncoding RNA (miRNA). MiRNA is an imperfectly matched double‐stranded molecule deriving from large primary transcript (pri‐miRNA). The action of Drosha and DGCR8 proteins initiates the miRNA maturation producing shorter pre‐miRNA molecule. Following Exportin‐5:RanGTP‐driven nuclear export pre‐miRNA is subjected to Dicer processing, resulting in mature miRNA molecule. In addition to this canonical pathway, alternative miRNA biogenesis routes have been described. The understanding of canonical and noncanonical miRNA biogenesis is important to define the roles of miRNAs in cellular homeostasis processes and human diseases as well as to develop potential miRNA‐based treatments.

Key Concepts

  • The canonical maturation of pri‐miRNA involves processing of RNA polymerase II transcripts by Drosha and Dicer proteins.
  • Within the Microprocessor complex, DGCR8 is needed to orientate pri‐miRNA substrate and permit efficient Drosha‐mediated processing.
  • Other proteins such as Rbfox3, GSk3b, BRCA1 and HP1BP3 indirectly regulate the expression levels of pri‐miRNAs.
  • Drosha cleavage product, named pre‐miRNA, is translocated from the nucleus to the cytosol by the formation of a complex with Exportin‐5 and RanGTP.
  • TRBP and PACT are cofactor partners of Dicer protein and help the pre‐miRNA loading and processing.
  • Mature miRNA can also be produced by splicing mechanisms without the involvement of Drosha through mirtrons intermediates.
  • Processing of intronic‐derived miRNAs by Drosha generates simtrons with special characteristics.
  • Duplex RNA sequences adjacent to pre‐miRNA can also be processed to form miRNA‐offset RNAs (moRNAs).
  • In some cases, miRNA maturation is directed by Argonaute2 without the intervention of Dicer.
  • 2 nt 3′‐overhang feature is important for miRNA biogenesis.

Keywords: RNA interference; microRNA; Dicer; siRNA; Drosha; Argonaute; miRNA biogenesis; RISC; Exportin

Figure 1. Schematic structure of the Microprocessor complex [Drosha and DGCR8 (DiGeorge Critical Region 8)] and pri‐miRNA hairpin. Primary sequence motifs (UG; UGU; GHG; CNNC) support the Drosha‐mediated cleavage and influence the processing efficiency [H = A, U or G; N = A, U, C or G].
Figure 2. Exp‐5:RanGTP:pre‐miRNA complex. The pre‐miRNA double‐stranded stem is accommodated into the positively charged tunnel of the Exp‐5 protein, the 2nt 3′‐overhang interacts with the base of the mitt‐like structure. Arg 602 is engaged in π–π stacking interactions with the first base pair of pre‐miRNA.
Figure 3. Schematic representation of Dicer, TRBP and pre‐miRNA substrate. TRBP helps the loading of dsRNA molecules into Dicer protein, the presence of 5′‐phosphate group and 2 nt 3′‐overhang stimulates Dicer conformational transition towards active state, resulting in efficient pre‐miRNA processing.
Figure 4. Overview of noncanonical miRNA‐like structures. (a) miRNA‐offset RNAs (moRNAs) derive from sequences located next to miRNA duplex in pri‐miRNA structures; (b) miRNA‐like agotron: Ago2 associates with free 5′‐end and processes the hairpin molecules on 3p arm; (c) Mirtrons are located within introns and exploit the splicing machinery to generate functional miRNA molecules.
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Further Reading

Alagia A and Eritja R (2016) siRNA and RNAi optimization. Wiley Interdisciplinary Reviews: RNA 7: 316–329.

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Zhu L, Jiang H, Sheong FK, et al. (2016) Understanding the core of RNA interference: the dynamic aspects of Argonaute‐mediated processes. Progress in Biophysics and Molecular Biology. DOI: 10.1016/j.pbiomolbio.2016.09.008.

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Alagia, Adele, and Eritja, Ramon(Jun 2017) Biochemistry of RNA Silencing. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021019]