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 miRNAmediated gene silencing.Many developmental and cellular processes are regulated by microRNA (miRNA)-mediated RNA interference (RNAi) (1-4). After incorporation into the RNA-induced silencing complex, miRNAs guide the RNAi machinery to their target genes by forming RNA duplexes, resulting in sequence-specific mRNA degradation or translational repression (1,2,4). The generation of mature miRNAs requires the processing of primary transcripts (pri-miRNAs) (5), and A → I RNA editing occurs to certain pri-miRNAs (6-8).Human chromosome 14 and syntenic regions of the distal end of mouse chromosome 12 harbor the miR-376 cluster of miRNA genes (9). The six human miR-376 RNAs (miR-376a2, -376b, -368, -B1, and -B2) (Fig. 1A) and three mouse miR-376a-c RNAs ( fig. S1A) have highly similar sequences ( fig. S2). Expression of miR-376 RNAs is detected in the placenta, developing embryos, and adult tissues (9,10).All of the miR-376 RNA cluster members are transcribed into a long primary transcript encompassing the entire region and (except human miR-B1) undergo extensive and * To whom correspondence should be addressed. ykawahara@wistar.org (Y.K.); kazuko@wistar.org (K.N.). † These authors contributed equally to this work. simultaneous A → I editing at one or both of two specific sites (+4 and +44) in select human and mouse tissues and specific subregions of the brain ( Fig. 2 and table S1) (11). The +4 site of some pri-miR-376 cluster genes (e.g., human -376b and -368) is genomically encoded as G and thus not subject to A → I editing (Fig. 1A). Certain miR-376 members, such as primiR-376a2, -376b, and -368, are nearly 100% edited at the +44 site in the human cortex and medulla (Figs. 1B and 2 and table S1), whereas no editing was detected in other tissues (e.g., the +4 site of human pri-miR-376a1 in liver and the +44 site of mouse pri-miR-376a in all tissues). In select members of the cluster, substantial editing (∼20 to 55%) occurs at the −1 site, and infrequent editing occurs at several additional sites (table S1). In contrast, no editing was detected in human pri-miR-654 and mouse pri-miR-300. Although these two pri-miRNAs are located within the miR-376 cluster, t...

Although aberrant microRNA (miRNA) expression is linked to human diseases including cancer, the mechanisms that regulate the expression of each individual miRNA remain largely unknown. TAR DNA-binding protein-43 (TDP-43) is homologous to the heterogeneous nuclear ribonucleoproteins (hnRNPs), which are involved in RNA processing, and its abnormal cellular distribution is a key feature of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), two neurodegenerative diseases. Here, we show that TDP-43 facilitates the production of a subset of precursor miRNAs (pre-miRNAs) by both interacting with the nuclear Drosha complex and binding directly to the relevant primary miRNAs (primiRNAs). Furthermore, cytoplasmic TDP-43, which interacts with the Dicer complex, promotes the processing of some of these premiRNAs via binding to their terminal loops. Finally, we show that involvement of TDP-43 in miRNA biogenesis is indispensable for neuronal outgrowth. These results support a previously uncharacterized role for TDP-43 in posttranscriptional regulation of miRNA expression in both the nucleus and the cytoplasm.M icroRNAs (miRNAs), small noncoding RNAs of ∼20-22 nt, have emerged as novel regulatory factors of gene expression (1). The expression of each individual miRNA is tightly regulated in a development-and cell-specific manner through transcriptional or posttranscriptional control. As a result, miRNAs can act as regulatory switches for development, organogenesis, and cellular differentiation and control distinct functions that are required for the maintenance of different cell subtypes. Moreover, the altered expression of certain miRNAs is involved in the pathogenesis of developmental abnormalities and human diseases such as cancer and Parkinson's disease (2, 3). As a consequence, understanding the mechanisms that regulate the expression of each individual miRNA is essential to elucidate the molecular pathogenesis of human diseases.MiRNAs are generated from long primary transcripts, termed pri-miRNAs, which consist of a short dsRNA region and a loop. The nuclear Drosha complex cleaves pri-miRNAs to release intermediate precursors that are termed pre-miRNAs. Pre-miRNAs are then transported by Exportin-5 into the cytoplasm where they are cleaved further by the Dicer complex to generate mature miRNAs. Finally, mature miRNAs are incorporated into the RNA-induced silencing complex (RISC), after which they can hybridize to the 3′-untranslated region (UTR) of their target mRNAs to repress translation or degrade these mRNAs (4). DGCR8 is a partner protein that is indispensable for the processing of pri-miRNAs by Drosha (5). In addition, recent work has identified multiple proteins that modulate the processing of specific miRNAs by interacting with the Drosha complex or by binding directly to pri-miRNAs or both (6, 7). Compared with the number of regulatory proteins that are involved in Drosha cleavage, only a few proteins have been identified that regulate the processing of pre-miRNAs by Dicer (6, 7)....

The aetiology of sporadic amyotrophic lateral sclerosis (ALS), a fatal paralytic disease, is largely unknown. Here we show that there is a defect in the editing of the messenger RNA encoding the GluR2 subunit of glutamate AMPA receptors in the spinal motor neurons of individuals affected by ALS. This failure to swap an arginine for a glutamine residue at a crucial site in the subunit, which occurs normally in the affected brain areas of patients with other neurodegenerative diseases, will interfere with the correct functioning of the glutamate receptors and may be a contributory cause of neuronal death in ALS patients.

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.

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 (premiRNAs) 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 primiR-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.

A lthough recent advances in the management of acute myocardial infarction (AMI), including primary percutaneous coronary intervention strategies and evidence-based therapies, have resulted in a substantial decline in mortality, the number of post-AMI patients who survive AMI but experience development of ischemic heart failure (HF) is increasing worldwide.1 Therefore, the identification of biomarkers that can predict risk of HF development in post-AMI patients is needed for optimizing management and treatment strategies. To date, several types of biomarkers, such as N-terminal probrain natriuretic peptide and cardiac troponin T, have been shown to predict cardiovascular events after AMI; however, it remains inconclusive whether these biomarkers can predict future HF in post-AMI patients. Rationale: Despite a recent decline of in-hospital mortality attributable to acute myocardial infarction (AMI), the incidence of ischemic heart failure (HF) in post-AMI patients is increasing. Correspondence to Yukio Kawahara, Laboratory of RNA Function, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan (e-mail ykawahara@rna.med.osaka-u.ac.jp); or Issei Komuro, Department of Cardiovascular Medicine, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan (e-mail komuro-tky@umin.ac.jp). Matsumoto et al Circulating MicroRNAs Predict Heart Failure 323membrane-bound vesicles termed exosomes, which circulate stably in the bloodstream. [3][4][5] Although the physiological significance of circulating microRNAs is not fully understood, they have attracted attention as potential diagnostic and prognostic biomarkers for various diseases, particularly cancer. 3,4 With respect to cardiovascular disease, several cardiac microRNAs, including miR-1, miR-133a, and miR-208a, have been detected in the serum in the acute phase of AMI and thus represent potentially useful diagnostic markers for AMI. 5,6 However, these microRNAs are most likely released from necrotic heart tissue into the blood directly and are not encapsulated within exosomes and, therefore, have short half-lives. For this reason, these cardiac microRNAs are unlikely to be predictive of future HF development in post-AMI patients. 6 The aim of the present study was to identify circulating microRNAs that can serve as predictors of HF development in patients who survive the acute stage of AMI. MethodsWe retrospectively analyzed the records of patients registered in the Osaka Acute Coronary Insufficiency Study, which has been described elsewhere. 7 The study protocol was approved by the ethics committee of each participating hospital, and written informed consent was provided by each patient at the time of registration. On the basis of the results of an initial screening (Online Table I), we performed a second screening to examine the microRNA profiles of an increased number of matched patients (HF group, n=21; control group, n=65; Online Table II ResultsTo identify microRNAs with altered expression ...

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

BackgroundAtherosclerotic abdominal aortic aneurysm (AAA) is a progressive, gradual aortic rupture that results in death in the absence of surgical intervention. Key factors that regulate initiation and progression of AAA are unknown, making targeted interventions difficult. MicroRNAs play a fundamental role in atherosclerosis, and atherosclerotic coronary artery disease is characterized by tissue- and plasma-specific microRNA signatures. However, little is known about microRNAs involved in AAA pathology. This study examined tissue and plasma microRNAs specifically associated with AAA.Methods and ResultsAAA and normal wall tissues were sampled from patients undergoing AAA repair (n=13; mean age, 68±6 years) and aortic valve replacement surgery (n=7; mean age, 66±4 years), respectively. MicroRNA expression was assessed by high-throughput microRNA arrays and validated by real-time polymerase chain reaction for individual microRNAs that showed significant expression differences in the initial screening. MicroRNAs related to fibrosis (miR-29b), inflammation (miR-124a, miR-146a, miR-155, and miR-223), and endothelium (miR-126, let-7 family members, and miR-21) were significantly upregulated in AAA tissue. Significant negative correlations were seen in expression levels of monocyte chemoattractant protein-1 and miR-124a, -146a, and -223; tumor necrosis factor-α and miR-126 and -223; and transforming growth factor-β and miR-146a. Expression of microRNAs, such as miR-29b, miR-124a, miR-155, and miR-223, that were upregulated in AAA tissue was significantly reduced in plasma of patients with AAA (n=23; mean age, 72±9 years) compared to healthy controls (n=12; mean age, 51±11 years) and patients with coronary artery disease (n=17; mean age, 71±9 years).ConclusionsThe expression of some microRNAs was specifically upregulated in AAA tissue, warranting further studies on the microRNA function in AAA pathogenesis and on the possibility of using a microRNA biomarker for AAA diagnosis.