Nodamura virus B2 amino terminal domain sensitivity to small interfering RNA

Nodamura virus (NoV) B2, a suppressor of RNA interference, binds double stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs) corresponding to Dicer substrates and products. Here, we report that the amino terminal domain of NoV B2 (NoV B2 79) specifically binds siRNAs but not dsRNAs. NoV B2 79 oligomerizes on binding to 27 nucleotide siRNA. Mutation of the residues phenylalanine49 and alanine60 to cysteine and methionine, respectively enhances the RNA binding affinity of NoV B2 79. Circular dichroism spectra demonstrated that the wild type and mutant NoV B2 79 have similar secondary structure conformations.     

Dicer 的结构功能及其与肿瘤的关系

Dicer蛋白是RNaseⅢ家族中重要的一员,对miRNA或siRNA的产生起着至关重要的作用。Dicer蛋白通常由1个DEXH盒子或H盒子、1个DUF283结构域、1个PAZ结构域、2个RNaseⅢ结构域(RNaseⅢa和RNaseⅢb)和1个dsRNA结合结构域组成。Dicer蛋白的分子结构决定了其在miRNAs合成中发挥着重要作用。Dicer及生成的miRNA与肿瘤又有着密切关系。本文主要针对Dicer及其与肿瘤的关系作简要综述。     

Structure of precursor microRNA’s terminal loop regulates human Dicer’s dicing activity by switching DExH/D domain

Almost all pre-miRNAs in eukaryotic cytoplasm are recognized and processed into double-stranded microRNAs by the endonuclease Dicer protein comprising of multiple domains. As a key player in the small RNA induced gene silencing pathway, the major domains of Dicer are conserved among different species with the exception of the N-terminal components. Human Dicer’s N-terminal domain has been shown to play an autoinhibitory function of the protein’s dicing activity. Such an auto-inhibition can be released when the human Dicer protein dimerizes with its partner protein, such as TRBP, PACT through the N-terminal DExH/D (ATPase-helicase) domain. The typical feature of a pre-miRNA contains a terminal loop and a stem duplex, which bind to human Dicer’s DExH/D (ATPase-helicase) domain and PAZ domain respectively during the dicing reaction. Here, we show that pre-miRNA’s terminal loop can regulate human Dicer’s enzymatic activity by interacting with the DExH/D (ATPase-helicase) domain. We found that various editing products of pre-miR-151 by the ADAR1P110 protein, an A-to-I editing enzyme that modifies pre-miRNAs sequence, have different terminal loop structures and different activity regulatory effects on human Dicer. Single particle electron microscopy reconstruction revealed that pre-miRNAs with different terminal loop structures induce human Dicer’s DExH/D (ATPase-helicase) domain into different conformational states, in correlation with their activity regulatory effects.     

Functional parameters of Dicer-independent microRNA biogenesis

Until recently, a Dicer-class RNase III enzyme was believed to be essential for microRNA (miRNA) biogenesis in all animals. The conserved vertebrate locus mir-451 defies this expectation and instead matures by direct cleavage of its pre-miRNA hairpin via the Slicer activity of Argonaute2 (Ago2). In this study, we used structure-function analysis to define the functional parameters of Ago2-mediated miRNA biogenesis. These include (1) the requirement for base-pairing at most, but not all, positions within the pre-mir-451 stem; (2) surprisingly little influence of the 5'-nucleotide on Ago sorting; (3) substantial influence of Ago protein stoichiometry on mir-451 maturation; (4) strong influence of G:C content in the distal stem on 3' resection of cleaved mir-451 substrates; and (5) the influence of hairpin length on substrate utilization by Ago2 and Dicer. Unexpectedly, we find that certain hairpin lengths confer competence to mature via both Dicer-mediated and Ago2-mediated pathways, and we show, in fact, that a conventional shRNA can traverse the Dicer-independent pathway. Altogether, these data inform the design of effective Dicer-independent substrates for gene silencing and reveal novel aspects of substrate handling by Ago proteins.     

In vivo structure-function analysis of human Dicer reveals directional processing of precursor miRNAs

Dicer is an RNase III family endoribonuclease and haploinsufficient tumor suppressor that processes mature miRNAs from the 5' (5p) or 3' (3p) arm of hairpin precursors. In murine Dicer knockout fibroblasts, we expressed human Dicer with point mutations in the RNase III, helicase, and PAZ domains and characterized miRNA expression by Northern blot and massively parallel sequencing of small RNAs. We report that inactivation of the RNase IIIA domain results in complete loss of 3p-derived mature miRNAs, but only partial reduction in 5p-derived mature miRNAs. Conversely, inactivation of the RNase IIIB domain by mutation of D1709, a residue mutated in a subset of nonepithelial ovarian cancers, results in complete loss of 5p-derived mature miRNAs, including the tumor-suppressive let-7 family, but only partial reduction in 3p-derived mature miRNAs. Mutation of the PAZ domain results in global reduction of miRNA processing, while mutation of the Walker A motif in the helicase domain of Dicer does not alter miRNA processing. These results provide insight into the biochemical activity of human Dicer in vivo and, furthermore, suggest that mutation of the clinically relevant residue D1709 within the RNase IIIB results in a uniquely miRNA-haploinsufficient state in which the let-7 family of tumor suppressor miRNAs is lost while a complement of 3p-derived miRNAs remains expressed.     

Biochemical mechanisms of the RNA-induced silencing complex

In less than 10 years since its inception, RNA interference （RNAi） has had extraordinary impact on biomedical science. RNAi has been demonstrated to influence numerous biological and disease pathways. Development and adoption of RNAi technologies have been prolific ranging from basic loss-of-function tools, genome-wide screening libraries to pharmaceutical target validation and therapeutic development. However, understanding of the molecular mechanisms of RNAi is far from complete. The purpose of this brief review is to highlight key achievements in elucidating the bio- chemical mechanisms of the RNA-induced silencing complex and to outline major challenges for the field.