Kazuko Nishikura

TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing

MicroRNAs (miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the post-transcriptional level. Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex. These pre-miRNAs are cleaved by the RNase III Dicer to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence. Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein), which contains three double-stranded, RNA-binding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer–TRBP with Argonaute 2 (Ago2), the catalytic engine of RISC. The physical association of Dicer–TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA (siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer–TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer–TRBP complex not only in miRNA processing but also as a platform for RISC assembly.

Redirection of Silencing Targets by Adenosine-to-Inosine Editing of miRNAs

Primary transcripts of certain microRNA (miRNA) genes are subject to RNA editing that converts adenosine to inosine. However, the importance of miRNA editing remains largely undetermined. Here we report that tissue-specific adenosine-to-inosine editing of miR-376 cluster transcripts leads to predominant expression of edited miR-376 isoform RNAs. One highly edited site is positioned in the middle of the 5′-proximal half “seed” region critical for the hybridization of miRNAs to targets. We provide evidence that the edited miR-376 RNA silences specifically a different set of genes. Repression of phosphoribosyl pyrophosphate synthetase 1, a target of the edited miR-376 RNA and an enzyme involved in the uric-acid synthesis pathway, contributes to tight and tissue-specific regulation of uric-acid levels, revealing a previously unknown role for RNA editing in miRNA-mediated gene silencing.

Modulation of microRNA processing and expression through RNA editing by ADAR deaminases

Adenosine deaminases acting on RNA (ADARs) are involved in editing of adenosine residues to inosine in double-stranded RNA (dsRNA). Although this editing recodes and alters functions of several mammalian genes, its most common targets are noncoding repeat sequences, indicating the involvement of this editing system in currently unknown functions other than recoding of protein sequences. Here we show that specific adenosine residues of certain microRNA (miRNA) precursors are edited by ADAR1 and ADAR2. Editing of pri–miR-142, the precursor of miRNA-142, expressed in hematopoietic tissues, resulted in suppression of its processing by Drosha. The edited pri–miR-142 was degraded by Tudor-SN, a component of RISC and also a ribonuclease specific to inosine-containing dsRNAs. Consequently, mature miRNA-142 expression levels increased substantially in ADAR1 null or ADAR2 null mice. Our results demonstrate a new function of RNA editing in the control of miRNA biogenesis.

RNA editing of the microRNA-151 precursor blocks cleavage by the Dicer-TRBP complex

MicroRNAs (miRNAs) mediate translational repression or degradation of their target messenger RNAs by RNA interference (RNAi). The primary transcripts of miRNA genes (pri‐miRNAs) are sequentially processed by the nuclear Drosha–DGCR8 complex to approximately 60–70 nucleotide (nt) intermediates (pre‐miRNAs) and then by the cytoplasmic Dicer–TRBP complex to approximately 20–22 nt mature miRNAs. Certain pri‐miRNAs are subject to RNA editing that converts adenosine to inosine (A → I RNA editing); however, the fate of edited pri‐miRNAs is mostly unknown. Here, we provide evidence that RNA editing of pri‐miR‐151 results in complete blockage of its cleavage by Dicer and accumulation of edited pre‐miR‐151 RNAs. Our results indicate that A → I conversion at two specific positions of the pre‐miRNA foldback structure can affect its interaction with the Dicer–TRBP complex, showing a new regulatory role of A → I RNA editing in miRNA biogenesis.

Regulation of innate immune responses by DAI (DLM-1/ZBP1) and other DNA-sensing molecules

DNA, whether it is microbe-derived or host-derived, evokes immune responses when exposed to the cytosol of a cell. We previously reported that DNA-dependent activator of IFN regulatory factors (DAI), also referred to as DLM-1/ZBP1, functions as a DNA sensor that activates the innate immune system. In the present study, we examined the regulation of the complex DNA-sensing system by DAI and other molecules. We first show that DAI directly interacts with DNA in vitro and that it requires three DNA-binding domains for full activation in vivo. We also show that the artificially induced dimerization of DAI results in the DNA-independent activation of type I IFN genes, thereby providing a better understanding for the molecular basis of DAI activation. Furthermore, we provide evidence for the presence of additional DNA sensors, either positively or negatively regulating cytosolic DNA-mediated innate immune responses. These results in toto provide insights into the mechanism of DAI activation and reveal the complex regulatory mechanisms underlying DNA-mediated protective and pathologic immune responses.

Frequency and fate of microRNA editing in human brain

Primary transcripts of certain microRNA (miRNA) genes (pri-miRNAs) are subject to RNA editing that converts adenosine to inosine (A→I RNA editing). However, the frequency of the pri-miRNA editing and the fate of edited pri-miRNAs remain largely to be determined. Examination of already known pri-miRNA editing sites indicated that adenosine residues of the UAG triplet sequence might be edited more frequently. In the present study, therefore, we conducted a large-scale survey of human pri-miRNAs containing the UAG triplet sequence. By direct sequencing of RT–PCR products corresponding to pri-miRNAs, we examined 209 pri-miRNAs and identified 43 UAG and also 43 non-UAG editing sites in 47 pri-miRNAs, which were highly edited in human brain. In vitro miRNA processing assay using recombinant Drosha-DGCR8 and Dicer-TRBP (the human immuno deficiency virus transactivating response RNA-binding protein) complexes revealed that a majority of pri-miRNA editing is likely to interfere with the miRNA processing steps. In addition, four new edited miRNAs with altered seed sequences were identified by targeted cloning and sequencing of the miRNAs that would be processed from edited pri-miRNAs. Our studies predict that ∼16% of human pri-miRNAs are subject to A→I editing and, thus, miRNA editing could have a large impact on the miRNA-mediated gene silencing.

Editor meets silencer: crosstalk between RNA editing and RNA interference.

The most prevalent type of RNA editing is mediated by ADAR (adenosine deaminase acting on RNA) enzymes, which convert adenosines to inosines (a process known as A→I RNA editing) in double-stranded (ds)RNA substrates. A→I RNA editing was long thought to affect only selected transcripts by altering the proteins they encode. However, genome-wide screening has revealed numerous editing sites within inverted Alu repeats in introns and untranslated regions. Also, recent evidence indicates that A→I RNA editing crosstalks with RNA-interference pathways, which, like A→I RNA editing, involve dsRNAs. A→I RNA editing therefore seems to have additional functions, including the regulation of retrotransposons and gene silencing, which adds a new urgency to the challenges of fully understanding ADAR functions.

A-to-I RNA editing and human disease.

The post-transcriptional modification of mammalian transcripts by A-to-I RNA editing has beenrecognized as an important mechanism for the generation of molecular diversity and alsoregulates protein function through recoding of genomic information. As the molecular players ofediting are characterized and an increasing number of genes become identified that are subject toA-to-I modification, the potential impact of editing on the etiology or progression of humandiseases is realized. Here we review the recent knowledge on where disturbances in A-to-I RNAediting have been correlated with human disease phenotypes.

EDITING OF GLUTAMATE RECEPTOR B SUBUNIT ION CHANNEL RNAS BY FOUR ALTERNATIVELY SPLICED DRADA2 DOUBLE-STRANDED RNA ADENOSINE DEAMINASES

Double-stranded (ds) RNA-specific adenosine deaminase converts adenosine residues into inosines in dsRNA and edits transcripts of certain cellular and viral genes such as glutamate receptor (GluR) subunits and hepatitis delta antigen. The first member of this type of deaminase, DRADA1, has been recently cloned based on the amino acid sequence information derived from biochemically purified proteins. Our search for DRADA1-like genes through expressed sequence tag databases led to the cloning of the second member of this class of enzyme, DRADA2, which has a high degree of sequence homology to DRADA1 yet exhibits a distinctive RNA editing site selectivity. There are four differentially spliced isoforms of human DRADA2. These different isoforms of recombinant DRADA2 proteins, including one which is a human homolog of the recently reported rat RED1, were analyzed in vitro for their GluR B subunit (GluR-B) RNA editing site selectivity. As originally reported for rat RED1, the DRADA2a and -2b isoforms edit GluR-B RNA efficiently at the so-called Q/R site, whereas DRADA1 barely edits this site. In contrast, the R/G site of GluR-B RNA was edited efficiently by the DRADA2a and -2b isoforms as well as DRADA1. Isoforms DRADA2c and -2d, which have a distinctive truncated shorter C-terminal structure, displayed weak adenosine-to-inosine conversion activity but no editing activity tested at three known sites of GluR-B RNA. The possible role of these DRADA2c and -2d isoforms in the regulatory mechanism of RNA editing is discussed.

A-to-I editing of coding and non-coding RNAs by ADARs

Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA. This A-to-I editing occurs not only in protein-coding regions of mRNAs, but also frequently in non-coding regions that contain inverted Alu repeats. Editing of coding sequences can result in the expression of functionally altered proteins that are not encoded in the genome, whereas the significance of Alu editing remains largely unknown. Certain microRNA (miRNA) precursors are also edited, leading to reduced expression or altered function of mature miRNAs. Conversely, recent studies indicate that ADAR1 forms a complex with Dicer to promote miRNA processing, revealing a new function of ADAR1 in the regulation of RNA interference.