Young-Kook Kim

Re-evaluation of the roles of DROSHA, Exportin 5, and DICER in microRNA biogenesis

Significance MicroRNAs (miRNAs) are noncoding RNAs with diverse roles in development and pathogenesis. Biogenesis of canonical miRNA requires nuclear processing by DROSHA, nuclear export by Exportin 5, and cytoplasmic processing by DICER. To gain a deeper understanding of the maturation processes, we here ablated the DROSHA, Exportin 5, and DICER genes using the same human cell line. Canonical miRNA production was abolished in DROSHA-deleted cells, revealing an irreplaceable role of DROSHA. Interestingly, however, some canonical miRNAs were still produced without DICER albeit at markedly reduced levels, and many were detected in Exportin 5-deleted cells at only modestly decreased levels. Our study allows us to understand differential contributions of key biogenesis factors, and provides valuable resources for miRNA research. Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by DROSHA, nuclear export of precursor miRNA (pre-miRNA) by Exportin 5 (XPO5), and cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted DROSHA, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in DROSHA-deleted cells, whereas we detected a few DROSHA-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to DROSHA knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3′ end, yielding miRNAs from the 5′ strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of DROSHA and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research.

TALEN-based knockout library for human microRNAs

Various technical tools have been developed to probe the functions of microRNAs (miRNAs), yet their application has been limited by low efficacy and specificity. To overcome the limitations, we used transcription activator–like effector nucleases (TALENs) to knock out human miRNA genes. We designed and produced a library of 540 pairs of TALENs for 274 miRNA loci, focusing on potentially important miRNAs. The knockout procedure takes only 2–4 weeks and can be applied to any cell type. As a case study, we generated knockout cells for two related miRNAs, miR-141 and miR-200c, which belong to the highly conserved miR-200 family. Interestingly, miR-141 and miR-200c, despite their overall similarity, suppress largely nonoverlapping groups of targets, thus suggesting that functional miRNA-target interaction requires strict seed-pairing. Our study illustrates the potency of TALEN technology and provides useful resources for miRNA research.

Extracellular microRNAs as Biomarkers in Human Disease

Dysregulation of microRNA (miRNA) levels is observed in diverse disease states. Early studies showed that by analyzing the expression profile of miRNAs in the tissue sample of a diseased person, it was possible to classify the disease into a specific subtype. To be used for diagnostic purposes more practically, however, a less invasive method than tissue biopsy is required. Surprisingly, it was discovered that a notable amount of extracellular miRNAs circulate throughout the body fluids with high stability. Moreover, the expression profile of miRNAs was shown to differ considerably between healthy and diseased people. In addition, evidence has been accumulating of extracellular miRNAs acting as signaling molecules between distantly located cells. If the expression profile faithfully reflects the disease states, the profiling of extracellular miRNAs will become a useful means of early warning or diagnosis of diverse diseases, replacing more invasive biopsy methods.

Identification of the Role of miR-142-5p in Alzheimer’s Disease by Comparative Bioinformatics and Cellular Analysis

Alzheimer’s disease (AD) is the most common neurodegenerative disease characterized by the formation of amyloid beta (Aβ) or tau protein aggregates, the hallmark of cognitive decline. MicroRNAs (miRNAs) have emerged as critical factors in neurogenesis and synaptic functions in the central nervous system (CNS). Recent studies have reported alterations in miRNA expression in patients with AD. However, miRNAs associated with AD varied with patient groups or experimental models, suggesting the need for a comparative study to identify miRNAs commonly dysregulated in diverse AD models. Here, we investigated the miRNAs that show dysregulated expression in two different human AD groups and mouse and cellular AD models. After selection of commonly dysregulated miRNAs in these groups, we investigated the pathophysiological significance of miR-142-5p in SH-SY5Y neuronal cells. We found that miR-142-5p was increased upon treatment with Aβ peptide 1–42 (Aβ42). Inhibition of miR-142-5p rescued the Aβ42-mediated synaptic dysfunctions, as indicated by the expression of postsynaptic density protein 95 (PSD-95). Among genes with decreased expression in Aβ42-treated SH-SY5Y cells, the predicted miR-142-5p target genes were significantly related with neuronal function and synapse plasticity. These findings suggest that dysregulation in miR-142-5p expression contributes the pathogenesis of AD by triggering synaptic dysfunction associated with Aβ42-mediated pathophysiology.

The microRNA miR‐124 inhibits vascular smooth muscle cell proliferation by targeting S100 calcium‐binding protein A4 (S100A4)

S100 calcium‐binding protein A4 (S100A4) induces proliferation and migration of vascular smooth muscle cells (VSMCs). We aimed to find the microRNA regulating S100A4 expression. S100A4 transcripts are abruptly increased in the acute phase of carotid arterial injury 1 day later (at day 1) but gradually decreases at days 7 and 14. Bioinformatics analysis reveals that miR‐124 targets S100A4. VSMC survival is attenuated by miR‐124 mimic but increased by miR‐124 inhibitor. miR‐124 decreases immediately after carotid arterial injury but dramatically increases at days 7 and 14. miR‐124 inhibitor‐induced cell proliferation is blocked by S100A4 siRNA, whereas miR‐124‐induced cell death is recovered by S100A4. Our findings suggest that miR‐124 is a novel regulator of VSMC proliferation and may play a role in the development of neointimal proliferation.

A positive regulatory role of the watermelon ClWRKY70 gene for disease resistance in transgenic Arabidopsis thaliana

A pathogen-inducible WRKY cDNA was cloned from the leaves of watermelon (Citrullus lanatus) seedlings 24 h after inoculation with Cladosporium cucumerinum. The deduced protein of the gene, designated as ClWRKY70, was classified as a group III WRKY protein based on its single WRKY domain containing a Cys2HisCys zinc-finger motif. Its Arabidopsis thaliana sequence homologue (AtWRKY70) has been described as playing an important role in the plant defense response. ClWRKY70 gene transcripts were highly accumulated in watermelon by salicylic acid treatment, but not by jasmonic acid. By evaluating target gene expression in transgenic Arabidopsis overexpressing the ClWRKY70 gene, it is suggested that the watermelon WRKY gene may play a positive regulatory role in plant resistance against pathogen attack.

Transcript Accumulation from the rpoS Gene Encoding a Stationary-Phase Sigma Factor in Pseudomonas chlororaphis Strain O6 Is Regulated by the Polyphosphate Kinase Gene

Polyphosphate levels are modulated by the actions of polyphosphate kinase, encoded by ppk, and exopolyphosphatase, encoded by ppx. The genes ppk and ppx are adjacent to each other in the genome of the root colonizer, Pseudomonas chlororaphis O6. A ppk-deficient mutant was more sensitive to oxidative stress than the wild-type and the ppx mutant. Transcripts from ppx increased as cultures matured from mid- to late-logarithmic and stationary phases, whereas abundance was greater for ppk in the late-logarithmic phase than in the stationary phase. Transcript accumulation from the rpoS gene, encoding the stationary-phase sigma factor RpoS, was decreased in the mid- and late-logarithmic and stationary phases in the ppk mutant. Thus, ppk regulates rpoS transcript accumulation in P. chlororaphis O6. However, mutations in either the ppk or ppx genes had no effect on induction of systemic resistance in plants colonized by P. chlororaphis O6.

Precise mapping of the transcription start sites of human microRNAs using DROSHA knockout cells

BackgroundThe expression of microRNAs (miRNAs) is primarily regulated during their transcription. However, the transcriptional regulation of miRNA genes has not been studied extensively owing to the lack of sufficient information about the promoters and transcription start sites of most miRNAs.ResultsIn this study, we identified the transcription start sites of human primary miRNAs (pri-miRNAs) using DROSHA knockout cells. DROSHA knockout resulted in increased accumulation of pri-miRNAs and facilitated the precise mapping of their 5′ end nucleotides using the rapid amplification of cDNA ends (RACE) technique. By analyzing the promoter region encompassing the transcription start sites of miRNAs, we found that the unrelated miRNAs in their sequences have many common elements in their promoters for binding the same transcription factors. Moreover, by analyzing intronic miRNAs, we also obtained comprehensive evidence that miRNA-harboring introns are spliced more slowly than other introns.ConclusionsThe precisely mapped transcription start sites of pri-miRNAs, and the list of transcription factors for pri-miRNAs regulation, will be valuable resources for future studies to understand the regulatory network of miRNAs.

Knockout of miR-221 and miR-222 reveals common and specific targets for paralogous miRNAs

ABSTRACT MicroRNAs (miRNAs) regulate the expression of mRNA through sequence-specific binding of the 3′ untranslated region (UTR). The seed sequence of miRNAs is the key determinant for target site recognition. Paralogous miRNAs, which share the same seed sequences but differ in their 3′ regions, are known to regulate largely overlapping groups of mRNAs. However, no study has analyzed functional differences between paralogous miRNAs with proper experimental methods. In this study, we compared the targets of paralogous miRNAs, miR-221 and miR-222. Using a nuclease-mediated genome engineering technique, we established knockout cell lines for these miRNAs, and precisely analyzed differences in target regulation. We found that miR-221 and miR-222 suppress the previously identified targets, CDKN1B and CDKN1C, differentially. Whereas both miRNAs suppressed CDKN1B, only miR-221 suppressed CDKN1C. From transcriptome analyses, we found that several different target mRNAs were regulated by each of miR-221 and miR-222 independently, although a large number of mRNAs responded commonly to miR-221 and miR-222. This is the first study to compare the mRNA regulations by paralogous miRNAs and illustrate that paralogous miRNAs with the same seed sequence also have difference in target regulation.

New Aspects of Vascular Calcification: Histone Deacetylases and Beyond

Vascular calcification is a pathologic phenomenon in which calcium phosphate is ectopically deposited in the arteries. Previously, calcification was considered to be a passive process in response to metabolic diseases, vascular or valvular diseases, or even aging. However, now calcification is recognized as a highly-regulated consequence, like bone formation, and many clinical trials have been carried out to elucidate the correlation between vascular calcification and cardiovascular events and mortality. As a result, vascular calcification has been implicated as an independent risk factor in cardiovascular diseases. Many molecules are now known to be actively associated with this process. Recently, our laboratory found that posttranslational modification of histone deacetylase (HDAC) 1 is actively involved in the development of vascular calcification. In addition, we found that modulation of the activity of HDAC as well as its protein stability by MDM2, an HDAC1-E3 ligase, may be a therapeutic target in vascular calcification. In the present review, we overview the pathomechanism of vascular calcification and the involvement of posttranslational modification of epigenetic regulators.