Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals.

Mammalian genomes produce huge numbers of noncoding RNAs (ncRNAs). However, the functions of most ncRNAs are unclear, and novel techniques that can distinguish functional ncRNAs are needed. Studies of mRNAs have revealed that the half-life of each mRNA is closely related to its physiological function, raising the possibility that the RNA stability of an ncRNA reflects its function. In this study, we first determined the half-lives of 11,052 mRNAs and 1418 ncRNAs in HeLa Tet-off (TO) cells by developing a novel genome-wide method, which we named 5'-bromo-uridine immunoprecipitation chase-deep sequencing analysis (BRIC-seq). This method involved pulse-labeling endogenous RNAs with 5'-bromo-uridine and measuring the ongoing decrease in RNA levels over time using multifaceted deep sequencing. By analyzing the relationship between RNA half-lives and functional categories, we found that RNAs with a long half-life (t(1/2) ≥ 4 h) contained a significant proportion of ncRNAs, as well as mRNAs involved in housekeeping functions, whereas RNAs with a short half-life (t(1/2) < 4 h) included known regulatory ncRNAs and regulatory mRNAs. The stabilities of a significant set of short-lived ncRNAs are regulated by external stimuli, such as retinoic acid treatment. In particular, we identified and characterized several novel long ncRNAs involved in cell proliferation from the group of short-lived ncRNAs. We designated this novel class of ncRNAs with a short half-life as Short-Lived noncoding Transcripts (SLiTs). We propose that the strategy of monitoring RNA half-life will provide a powerful tool for investigating hitherto functionally uncharacterized regulatory RNAs.

Identification of cis- and trans-acting factors involved in the localization of MALAT-1 noncoding RNA to nuclear speckles.

MALAT-1 noncoding RNA is localized to nuclear speckles despite its mRNA-like characteristics. Here, we report the identification of several key factors that promote the localization of MALAT-1 to nuclear speckles and also provide evidence that MALAT-1 is involved in the regulation of gene expression. Heterokaryon assays revealed that MALAT-1 does not shuttle between the nucleus and cytoplasm. RNAi-mediated repression of the nuclear speckle proteins, RNPS1, SRm160, or IBP160, which are well-known mRNA processing factors, resulted in the diffusion of MALAT-1 to the nucleoplasm. We demonstrated that MALAT-1 contains two distinct elements directing transcripts to nuclear speckles, which were also capable of binding to RNPS1 in vitro. Depletion of MALAT-1 represses the expression of several genes. Taken together, our results suggest that RNPS1, SRm160, and IBP160 contribute to the localization of MALAT-1 to nuclear speckles, where MALAT-1 could be involved in regulating gene expression.

SALL3 expression balance underlies lineage biases in human induced pluripotent stem cell differentiation.

Clinical applications of human induced pluripotent stem cells (hiPSCs) are expected, but hiPSC lines vary in their differentiation propensity. For efficient selection of hiPSC lines suitable for differentiation into desired cell lineages, here we identify SALL3 as a marker to predict differentiation propensity. SALL3 expression in hiPSCs correlates positively with ectoderm differentiation capacity and negatively with mesoderm/endoderm differentiation capacity. Without affecting self-renewal of hiPSCs, SALL3 knockdown inhibits ectoderm differentiation and conversely enhances mesodermal/endodermal differentiation. Similarly, loss- and gain-of-function studies reveal that SALL3 inversely regulates the differentiation of hiPSCs into cardiomyocytes and neural cells. Mechanistically, SALL3 modulates DNMT3B function and DNA methyltransferase activity, and influences gene body methylation of Wnt signaling-related genes in hiPSCs. These findings suggest that SALL3 switches the differentiation propensity of hiPSCs toward distinct cell lineages by changing the epigenetic profile and serves as a marker for evaluating the hiPSC differentiation propensity.

Tumorigenicity-associated characteristics of human iPS cell lines.

Human induced pluripotent stem cells (hiPSCs) represent promising raw materials of human cell-based therapeutic products (hCTPs). As undifferentiated hiPSCs exhibit intrinsic tumorigenicity properties that enable them to form teratomas, hCTPs containing residual undifferentiated hiPSCs may cause tumor formation following transplantation. We first established quantitative and sensitive tumorigenicity testing of hiPSCs dissociated into single cells using NOD/Shi-scid IL2Rγnull (NOG) mice by inhibiting apoptosis of hiPSCs with a Rho kinase inhibitor. To examine different features in tumorigenicity of various hiPSCs, 10 commonly available hiPSC lines were subjected to in vivo tumorigenicity testing. Transplanted hiPSC lines showed remarkable variation in tumor incidence, formation latency, and volumes. Most of the tumors formed were classified as immature teratomas. However, no signs of malignancies, such as carcinoma and sarcoma, were recognized in the tumors. Characteristics associated tumorigenicity of hiPSCs were investigated with microarray analysis, karyotype analysis, and whole exome sequencing. Gene expression profiling and pathway analysis supported different features of hiPSC lines in tumorigenicity. hiPSC lines showed chromosomal abnormalities in some lines and 61-77 variants of cancer-related genes carrying effective nonsynonymous mutations, which were confirmed in the COSMIC databases. In this study, the chromosomal abnormalities and cancer-related gene mutations observed in hiPSC lines did not lead to the malignancy of tumors derived from hiPSCs. Our results suggest that the potential tumorigenicity risk of hCTPs containing residual undifferentiated hiPSCs is dependent on not only amounts of undifferentiated hiPSCs but also features of the cell lines used as raw materials, a finding that should be considered from the perspective of quality of hCTPs used.

Identification of Minimal p53 Promoter Region Regulated by MALAT1 in Human Lung Adenocarcinoma Cells.

The MALAT1 long noncoding RNA is strongly linked to cancer progression. Here we report a MALAT1 function in repressing the promoter of p53 (TP53) tumor suppressor gene. p21 and FAS, well-known p53 targets, were upregulated by MALAT1 knockdown in A549 human lung adenocarcinoma cells. We found that these upregulations were mediated by transcriptional activation of p53 through MALAT1 depletion. In addition, we identified a minimal MALAT1-responsive region in the P1 promoter of p53 gene. Flow cytometry analysis revealed that MALAT1-depleted cells exhibited G1 cell cycle arrest. These results suggest that MALAT1 affects the expression of p53 target genes through repressing p53 promoter activity, leading to influence the cell cycle progression.

Highly sensitive droplet digital PCR method for detection of residual undifferentiated cells in cardiomyocytes derived from human pluripotent stem cells.

Human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are leading candidate cells as raw materials for cell therapy products, because of their capacity for pluripotent differentiation and unlimited self-renewal. hPSC-derived products have already entered the scope of clinical application. However, the assessment and control of their tumorigenicity remains to be a critical challenge. Sensitive detection of the pluripotent cellular impurities is necessary for the safety and quality control of the hPSC-derived products. In the present study, we established a sensitive assay for detection of the residual undifferentiated hiPSCs in cardiomyocytes, using droplet digital PCR (ddPCR). The ddPCR method with a probe and primers for LIN28 significantly detected as low as 0.001% undifferentiated hiPSCs in primary cardiomyocytes, which is equivalent to the ratio of a single hiPSC to 1 × 105 cardiomyocytes. The ddPCR also showed that LIN28 expression is extremely low in human tissues including liver, heart, pancreas, kidney, spinal cord, corneal epithelium and lung. These results suggest that the ddPCR method targeting LIN28 transcripts is highly sensitive and useful for the quality assessment of various cell therapy products derived from hPSCs.

A novel in vitro method for detecting undifferentiated human pluripotent stem cells as impurities in cell therapy products using a highly efficient culture system.

Innovative applications of cell therapy products (CTPs) derived from human pluripotent stem cells (hPSCs) in regenerative medicine are currently being developed. The presence of residual undifferentiated hPSCs in CTPs is a quality concern associated with tumorigencity. However, no simple in vitro method for direct detection of undifferentiated hPSCs that contaminate CTPs has been developed. Here, we show a novel approach for direct and sensitive detection of a trace amount of undifferentiated human induced pluripotent stem cells (hiPSCs) using a highly efficient amplification method in combination with laminin-521 and Essential 8 medium. Essential 8 medium better facilitated the growth of hiPSCs dissociated into single cells on laminin-521 than in mTeSR1 medium. hiPSCs cultured on laminin-521 in Essential 8 medium were maintained in an undifferentiated state and they maintained the ability to differentiate into various cell types. Essential 8 medium allowed robust hiPSC proliferation plated on laminin-521 at low cell density, whereas mTeSR1 did not enhance the cell growth. The highly efficient culture system using laminin-521 and Essential 8 medium detected hiPSCs spiked into primary human mesenchymal stem cells (hMSCs) or human neurons at the ratio of 0.001%-0.01% as formed colonies. Moreover, this assay method was demonstrated to detect residual undifferentiated hiPSCs in cell preparations during the process of hMSC differentiation from hiPSCs. These results indicate that our highly efficient amplification system using a combination of laminin-521 and Essential 8 medium is able to detect a trace amount of undifferentiated hPSCs contained as impurities in CTPs and would contribute to quality assessment of hPSC-derived CTPs during the manufacturing process.

Long non-coding RNAs in cancer progression.

Recent large-scale transcriptome analyses have revealed that transcription is spread throughout the mammalian genomes, yielding large numbers of transcripts, including long non-coding RNAs (lncRNAs) with little or no protein-coding capacity. Dozens of lncRNAs have been identified as biologically significant. In many cases, lncRNAs act as key molecules in the regulation of processes such as chromatin remodeling, transcription, and post-transcriptional processing. Several lncRNAs (e.g., MALAT1, HOTAIR, and ANRIL) are associated with human diseases, including cancer. Those lncRNAs associated with cancer are often aberrantly expressed. Although the underlying molecular mechanisms by which lncRNAs regulate cancer development are unclear, recent studies have revealed that such aberrant expression of lncRNAs affects the progression of cancers. In this review, we highlight recent findings regarding the roles of lncRNAs in cancer biology.

Identification and characterization of novel genotoxic stress-inducible nuclear long noncoding RNAs in mammalian cells.

Whole transcriptome analyses have revealed a large number of novel transcripts including long and short noncoding RNAs (ncRNAs). Currently, there is great interest in characterizing the functions of the different classes of ncRNAs and their relevance to cellular processes. In particular, nuclear long ncRNAs may be involved in controlling various aspects of biological regulation, such as stress responses. By a combination of bioinformatic and experimental approaches, we identified 25 novel nuclear long ncRNAs from 6,088,565 full-length human cDNA sequences. Some nuclear long ncRNAs were conserved among vertebrates, whereas others were found only among primates. Expression profiling of the nuclear long ncRNAs in human tissues revealed that most were expressed ubiquitously. A subset of the identified nuclear long ncRNAs was induced by the genotoxic agents mitomycin C or doxorubicin, in HeLa Tet-off cells. There were no commonly altered nuclear long ncRNAs between mitomycin C- and doxorubicin-treated cells. These results suggest that distinct sets of nuclear long ncRNAs play roles in cellular defense mechanisms against specific genotoxic agents, and that particular long ncRNAs have the potential to be surrogate indicators of a specific cell stress.

MALAT-1 enhances cell motility of lung adenocarcinoma cells by influencing the expression of motility-related genes.

MALAT-1, a long non-coding RNA, is associated with metastasis, but its role in the metastatic process remains unknown. Here, we show that short-interfering RNA-mediated MALAT-1 silencing impaired in vitro cell motility of lung cancer cells and influenced the expression of numerous genes. In these genes, knockdown of any one of CTHRC1, CCT4, HMMR, or ROD1 clearly inhibited cell migration. In MALAT-1 knockdown cells, pre-mRNA levels were decreased in some but not all genes. Thus, our findings suggest that MALAT-1 is a novel class of non-coding RNA that promotes cell motility through transcriptional and post-transcriptional regulation of motility related gene expression.