A comprehensive analysis of precursor microRNA cleavage by human Dicer

Abstract

Dicer cleaves double-stranded RNAs (dsRNAs) or precursor microRNAs (pre-miRNAs) to yield~22-nt RNA duplexes. The premiRNA structure requirement for human Dicer activity is incompletely understood. By large-scale in vitro dicing assays and mutagenesis studies, we showed that human Dicer cleaves most, although not all, of the 161 tested human pre-miRNAs efficiently. The stable association of RNAs with Dicer, as examined by gel shift assays, appears important but is not sufficient for cleavage. Human Dicer tolerates remarkable structural variation in its pre-miRNA substrates, although the dsRNA feature in the stem region and the 2-nt 39-overhang structure in a pre-miRNA contribute to its binding and cleavage by Dicer, and a large terminal loop further enhances pre-miRNA cleavage. Dicer binding protects the terminal loop from digestion by S1 nuclease, suggesting that Dicer interacts directly with the terminal loop region.

miRNA sensitivity to Drosha levels correlates with pre-miRNA secondary structure

“…Dicer cleavage is further regulated by Lin28, which binds to pre-miRNAs from the let-7 family (Nam et al 2011). Dicer acts by binding the 3 ′ -overhang and cleaves the pri-miRNA ∼22 nt from the Drosha cutting site to remove the terminal loop, resulting in an imperfect ∼22 nt called miRNA:miRNA * (Hutvagner et al 2001;Bernstein and Allis 2005;Feng et al 2012). The miRNA enters the RNA-induced silencing complex (RISC), and the miRNA * is degraded, in a process where strand selection is variable (Bartel 2004;Du and Zamore 2005;Cullen 2006;Kim and Nam 2006).…”

miRNA biogenesis: Biological impact in the development of cancer

“…19 Once in the cytoplasm, Dicer, another RNase III, digests the pre-miRNA into a 22nt mature duplex miRNA (miRNA:miRNA*, where miRNA* is called as the passenger strand). 20,21 During this process, Dicer is associated with other proteins like TAR RNA binding protein (TRBP) and kinase R-activating protein (PACT) to increase its stability and its processing activity. 22,23 Dicer is an essential protein of miRNA maturation and its down-regulation decreases the mature miRNA levels.…”

A Variety of Dicer Substrates in Human and C. elegans

“…Also sRNAs from transcript starts and ends have been described (Seila et al, 2008;Valen et al, 2011;Zamudio et al, 2014). Recent findings highlight that primary sequence, RNA structure, size, and position of stem and loop, the accessibility of 3 0 /5 0 ends, and cofactors are all relevant for Dicer substrate recognition and/or cleavage (Feng et al, 2012;Fukunaga et al, 2012;Gu et al, 2012;Lau et al, 2012;Park et al, 2011;Tian et al, 2014). Furthermore, in vitro studies (Feng et al, 2012) and electron microscopy (Taylor et al, 2013) show that affinity and cleavage efficiency can be uncoupled properties of the bound substrate.…”

“…Recent findings highlight that primary sequence, RNA structure, size, and position of stem and loop, the accessibility of 3 0 /5 0 ends, and cofactors are all relevant for Dicer substrate recognition and/or cleavage (Feng et al, 2012;Fukunaga et al, 2012;Gu et al, 2012;Lau et al, 2012;Park et al, 2011;Tian et al, 2014). Furthermore, in vitro studies (Feng et al, 2012) and electron microscopy (Taylor et al, 2013) show that affinity and cleavage efficiency can be uncoupled properties of the bound substrate. In C. elegans, Dicer binds the lncRNA rncs-1 in vitro but does not process it to sRNA, leading to sequestration of Dicer and inhibition of its function (Hellwig and Bass, 2008).…”