Yukio Kawahara

TDP-43 promotes microRNA biogenesis as a component of the Drosha and Dicer complexes

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 (pri-miRNAs). Furthermore, cytoplasmic TDP-43, which interacts with the Dicer complex, promotes the processing of some of these pre-miRNAs 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.

Circulating p53-Responsive MicroRNAs Are Predictive Indicators of Heart Failure After Acute Myocardial Infarction

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. Although various microRNAs have been proposed as diagnostic indicators for AMI, no microRNAs have been established as predictors of ischemic HF that develops after AMI. Objective: We attempted to identify circulating microRNAs that can serve as reliable predictors of ischemic HF in post-AMI patients. Methods and Results: Using sera collected a median of 18 days after AMI onset, we screened microRNAs in 21 patients who experienced development of HF within 1 year after AMI and in 65 matched controls without subsequent cardiovascular events after discharge. Among the 377 examined microRNAs, the serum level of only miR-192 was significantly upregulated in AMI patients with development of ischemic HF. Because miR-192 is reported to be p53-responsive, the serum levels of 2 other p53-responsive microRNAs, miR-194 and miR-34a, also were investigated. Interestingly, both microRNAs were coordinately increased with miR-192, particularly in exosomes, suggesting that these microRNAs function as circulating regulators of HF development via the p53 pathway. Furthermore, miR-194 and miR-34a expression levels were significantly correlated with left ventricular end-diastolic dimension 1 year after AMI. Conclusions: In the sera of post-AMI patients who experienced development of de-novo HF within 1 year of AMI onset, the levels of 3 p53-responsive microRNAs had been elevated by the early convalescent stage of AMI. Further investigations are warranted to confirm the usefulness of these circulating microRNAs for predicting the risk of development of ischemic HF after AMI.

Tissue- and Plasma-Specific MicroRNA Signatures for Atherosclerotic Abdominal Aortic Aneurysm

Background Atherosclerotic 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 Results AAA 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). Conclusions The 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.

A subset of circulating microRNAs are predictive for cardiac death after discharge for acute myocardial infarction

To investigate the prognostic impact of circulating microRNAs (miRs) in patients who survived acute myocardial infarction (AMI), we compared the circulating miR signature at the time of survival discharge among samples in the serum bank of the Osaka Acute Coronary Insufficiency Study. Using a high-throughput array consisting of 667 miRs, 11 miRs were found to be differentially expressed in the serum among patients at high-risk for cardiac death. Real-time RT-PCR confirmed that the serum levels of miR-155 and miR-380* were approximately 4- and 3-fold higher, respectively, in patients who experienced cardiac death within 1 year after discharge. Accordingly, a subset of circulating miRs might be predictive for cardiac death in post-AMI patients.

The cleavage pattern of TDP-43 determines its rate of clearance and cytotoxicity

TAR DNA-binding protein of 43 kDa (TDP-43) and its C-terminal fragment of 25 kDa (CTF25) play critical roles in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Although the overexpression of TDP-43 in cultured cells and animals results in the production of CTF25, the cleavage site that generates CTF25 and biological significance of the cleavage remain undetermined. Here we identify Asp174 as a cleavage site for CTF25. TDP-43 is cleaved initially after Asp174, which activates caspase-3/7 to accelerate TDP-43 fragmentation. Consequently, blockage of this cleavage results in a severe delay in TDP-43 clearance and prolonged necrotic cell death. We further show that the endoplasmic reticulum membrane-bound caspase-4 is the enzyme responsible for the cleavage after Asp174 and inhibition of caspase-4 activity slows TDP-43 fragmentation and reduces cell viability. These findings suggest that caspase-4-mediated cleavage after Asp174 is an initiator of TDP-43 clearance, which is required to avoid cell death induced by overexpressed TDP-43.

Human diseases caused by germline and somatic abnormalities in microRNA and microRNA-related genes

The human genome harbors approximately 2000 genes that encode microRNAs (miRNAs), small non‐coding RNAs of approximately 20–22 nt that mediate post‐transcriptional gene silencing. MiRNAs are generated from long transcripts through stepwise processing by the Drosha/DGCR8, Exportin‐5/RanGTP and Dicer/TRBP complexes. Given that the expression of each individual miRNA is tightly regulated, the altered expression of certain miRNAs plays a pivotal role in human diseases. For instance, germline and somatic mutations in the genes encoding the miRNA processing machinery have been reported in different cancers. Furthermore, certain miRNA genes are encoded within regions that are deleted or duplicated in individuals with chromosomal abnormalities, and the fact that the knockout of these miRNAs in animal models results in lethality or the abnormal development of certain tissues indicates that these miRNA genes contribute to the disease phenotypes. It has also been reported that mutations in miRNA genes or in miRNA‐binding sites, which result in the impairment of tight regulation of target mRNA expression, cause human genetic diseases, although these cases are rare. This is in contrast to the aberrant expression of certain miRNAs that results from the impairment of transcriptional or post‐transcriptional regulation, which has been reported frequently in various human diseases. The present review focuses on human diseases caused by mutations in genes encoding miRNAs and the miRNA processing machinery as well as in miRNA‐binding sites. Furthermore, human diseases caused by chromosomal abnormalities that involve the deletion or duplication of regions harboring genes that encode miRNAs or the miRNA processing machinery are also introduced.

Quantification of adenosine-to-inosine editing of microRNAs using a conventional method

In this protocol, I describe a method for measuring the frequency of adenosine-to-inosine RNA editing of primary, precursor and mature forms of specific microRNAs (miRNAs) derived from the same source. The procedure involves reverse transcription (RT)-PCR amplification of regions containing the editing sites followed by subcloning of the PCR products and sequencing. In contrast to deep sequencing, this method does not require any specialized equipment. Pri-miRNAs, which are relatively long primary transcripts, are amplified using a conventional RT-PCR method. Therefore, this method can be adapted for any known RNA-editing sites. In contrast, 3′ polyadenylation followed by 5′ adaptor ligation is indispensable for amplification of pre-miRNAs and mature miRNAs. The complete protocol takes ∼1 week. I also include details of direct sequence analysis of the PCR products derived from pri-miRNAs as an alternative method. Although it is not as precise as the subcloning method, this procedure enables us to study RNA-editing events of many samples.

Cross-Linking and Immunoprecipitation of Nuclear RNA- Binding Proteins

The systematic identification of in vivo targets of nuclear RNA-binding proteins (RBPs) is crucial to elucidate the physiological functions of each RBP. However, it has been difficult to distinguish real targets from nonspecifically bound RNAs and to determine the exact binding sites of each RBP by using a conventional RNA-immunoprecipitation (RIP) method. Photoactivatable Ribonucleoside-Enhanced Cross-linking and Immunoprecipitation (PAR-CLIP) is a recently developed method that relies on RNA-protein cross-linking to reduce the contamination of nonspecifically bound RNAs. Furthermore, in combination with high-throughput sequencing followed by bioinformatic analysis, the exact RBP-binding sites can be identified at a single nucleotide resolution. Here, we describe in detail a PAR-CLIP protocol to prepare cDNA libraries for high-throughput sequencing from RNA fragments that are bound to RBPs not only in the nucleus but also in the cytoplasm.

The RNA-binding protein MARF1 promotes cortical neurogenesis through its RNase activity domain

Cortical neurogenesis is a fundamental process of brain development that is spatiotemporally regulated by both intrinsic and extrinsic cues. Although recent evidence has highlighted the significance of transcription factors in cortical neurogenesis, little is known regarding the role of RNA-binding proteins (RBPs) in the post-transcriptional regulation of cortical neurogenesis. Here, we report that meiosis arrest female 1 (MARF1) is an RBP that is expressed during neuronal differentiation. Cortical neurons expressed the somatic form of MARF1 (sMARF1) but not the oocyte form (oMARF1). sMARF1 was enriched in embryonic brains, and its expression level decreased as brain development progressed. Overexpression of sMARF1 in E12.5 neuronal progenitor cells promoted neuronal differentiation, whereas sMARF1 knockdown decreased neuronal progenitor differentiation in vitro. We also examined the function of sMARF1 in vivo using an in utero electroporation technique. Overexpression of sMARF1 increased neuronal differentiation, whereas knockdown of sMARF1 inhibited differentiation in vivo. Moreover, using an RNase domain deletion mutant of sMARF1, we showed that the RNase domain is required for the effects of sMARF1 on cortical neurogenesis in vitro. Our results further elucidate the mechanisms of post-transcriptional regulation of cortical neurogenesis by RBPs.

Matrin3 binds directly to intronic pyrimidine‐rich sequences and controls alternative splicing

Matrin3 is an RNA‐binding protein that is localized in the nuclear matrix. Although various roles in RNA metabolism have been reported for Matrin3, in vivo target RNAs to which Matrin3 binds directly have not been investigated comprehensively so far. Here, we show that Matrin3 binds predominantly to intronic regions of pre‐mRNAs. Photoactivatable Ribonucleoside‐Enhanced Cross‐linking and Immunoprecipitation (PAR‐CLIP) analysis using human neuronal cells showed that Matrin3 recognized pyrimidine‐rich sequences as binding motifs, including the polypyrimidine tract, a splicing regulatory element. Splicing‐sensitive microarray analysis showed that depletion of Matrin3 preferentially increased the inclusion of cassette exons that were adjacent to introns that contained Matrin3‐binding sites. We further found that although most of the genes targeted by polypyrimidine tract binding protein 1 (PTBP1) were also bound by Matrin3, Matrin3 could control alternative splicing in a PTBP1‐independent manner, at least in part. These findings suggest that Matrin3 is a splicing regulator that targets intronic pyrimidine‐rich sequences.