Abstract
The ribonuclease Dicer plays a central role in the microRNA pathway by processing microRNA precursors (pre-microRNAs) into microRNAs, a class of 19- to 24-nucleotide non-coding RNAs that regulate expression of ≈60% of the genes in humans. To gain further insights into the function and regulation of Dicer in human cells, we performed a yeast two-hybrid (Y2HB) screen using human Dicer double-stranded RNA-binding domain (dsRBD) as bait. This approach identified tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) as a Dicer-interacting protein candidate. Confocal immunofluorescence microscopy revealed the colocalization of Dicer and TWEAK proteins at the perinuclear region of HeLa cells. The Dicer-TWEAK protein interaction was confirmed by coimmunoprecipitation and found not likely to be mediated by RNA. TWEAK dose-dependently reduced pre-microRNA conversion into mature microRNA in Dicer activity assays using extracts of transfected human HEK 293 cells. TWEAK expression also impaired microRNA-guided RNA silencing of a reporter gene induced by a pre-microRNA. These findings suggest a role for TWEAK—a pro-inflammatory cytokine—in regulating Dicer function and microRNA biogenesis, and its possible involvement in regulating gene expression during inflammatory processes and diseases.
Highlights
MicroRNAs are short, 19- to 24-nucleotide non-coding RNA species that play a vital role in post-transcriptional gene expression, as they modulate ≈60% of the genes in humans [1]
These studies revealed that transactivating response RNA-binding protein (TRBP) and protein kinase R activator (PACT) may act as co-factors of the microRNA processing activity of Dicer [5,6,7], while Dicer interaction with Argonaute 2 (Ago2) may facilitate effector RNA-induced silencing complex (RISC) assembly after microRNA maturation [8]
To gain further insights into the function and regulation of human Dicer, we focused on its C-terminal catalytic RNase III domain, which contains the tandem RNase IIIa/IIIb motifs and double-stranded RNA-binding domain (dsRBD), with the aim of identifying cellular proteins interacting with Dicer, and possibly modulating its enzyme activity
Summary
MicroRNAs are short, 19- to 24-nucleotide (nt) non-coding RNA species that play a vital role in post-transcriptional gene expression, as they modulate ≈60% of the genes in humans [1]. Coimmunoprecipitation approaches have been central to identifying Dicer-interacting proteins, such as transactivating response RNA-binding protein (TRBP) [5,6], protein kinase R activator (PACT) [7], and Argonaute 2 (Ago2) [8], which helped elucidate the role and function of Dicer. These studies revealed that TRBP and PACT may act as co-factors of the microRNA processing activity of Dicer [5,6,7], while Dicer interaction with Ago may facilitate effector RNA-induced silencing complex (RISC) assembly after microRNA maturation [8]. Investigating the functional relationship between Dicer and downstream effector proteins, our group has reported that human fragile X mental retardation protein (FMRP) can act as a microRNA acceptor protein for the ribonuclease Dicer and facilitate the assembly of microRNAs on specific target RNA sequences [9]
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