Gene fusions and their products (RNA and protein) were once thought to be unique features to cancer. However, chimeric RNAs can also be found in normal cells. Here, we performed, curated and analyzed nearly 300 RNA-Seq libraries covering 30 different non-neoplastic human tissues and cells as well as 15 mouse tissues. A large number of fusion transcripts were found. Most fusions were detected only once, while 291 were seen in more than one sample. We focused on the recurrent fusions and performed RNA and protein level validations on a subset. We characterized these fusions based on various features of the fusions, and their parental genes. They tend to be expressed at higher levels relative to their parental genes than the non-recurrent ones. Over half of the recurrent fusions involve neighboring genes transcribing in the same direction. A few sequence motifs were found enriched close to the fusion junction sites. We performed functional analyses on a few widely expressed fusions, and found that silencing them resulted in dramatic reduction in normal cell growth and/or motility. Most chimeras use canonical splicing sites, thus are likely products of ‘intergenic splicing’. We also explored the implications of these non-pathological fusions in cancer and in evolution.

The SILENT INFORMATION REGULATOR2 (SIR2) family proteins are NAD 1 -dependent histone deacetylases. Sir2 is involved in chromatin silencing at the mating-type loci, rDNA, and telomeres in yeast and is associated with lifespan extension in yeast, worms, and flies, but also in a broader range of additional functions. In this work, we investigated the role of OsSRT1, one of the two SIR2-related genes found in rice (Oryza sativa). We show that OsSRT1 is a widely expressed nuclear protein with higher levels in rapidly dividing tissues. OsSRT1 RNA interference induced an increase of histone H3K9 (lysine-9 of H3) acetylation and a decrease of H3K9 dimethylation, leading to H 2 O 2 production, DNA fragmentation, cell death, and lesions mimicking plant hypersensitive responses during incompatible interactions with pathogens, whereas overexpression of OsSRT1 enhanced tolerance to oxidative stress. Transcript microarray analysis revealed that the transcription of many transposons and retrotransposons in addition to genes related to hypersensitive response and/or programmed cell death was activated. Chromatin immunoprecipitation assays showed that OsSRT1 down-regulation induced histone H3K9 acetylation on the transposable elements and some of the hypersensitive response-related genes, suggesting that these genes may be among the primary targets of deacetylation regulated by OsSRT1. Our data together suggest that the rice SIR2-like gene is required for safeguard against genome instability and cell damage to ensure plant cell growth, but likely implicates different molecular mechanisms than yeast and animal homologs.

Genes or their encoded products are not expected to mingle with each other unless in some disease situations. In cancer, a frequent mechanism that can produce gene fusions is chromosomal rearrangement. However, recent discoveries of RNA trans-splicing and cis-splicing between adjacent genes (cis-SAGe) support for other mechanisms in generating fusion RNAs. In our transcriptome analyses of 28 prostate normal and cancer samples, 30% fusion RNAs on average are the transcripts that contain exons belonging to same-strand neighboring genes. These fusion RNAs may be the products of cis-SAGe, which was previously thought to be rare. To validate this finding and to better understand the phenomenon, we used LNCaP, a prostate cell line as a model, and identified 16 additional cis-SAGe events by silencing transcription factor CTCF and paired-end RNA sequencing. About half of the fusions are expressed at a significant level compared to their parental genes. Silencing one of the in-frame fusions resulted in reduced cell motility. Most out-of-frame fusions are likely to function as non-coding RNAs. The majority of the 16 fusions are also detected in other prostate cell lines, as well as in the 14 clinical prostate normal and cancer pairs. By studying the features associated with these fusions, we developed a set of rules: 1) the parental genes are same-strand-neighboring genes; 2) the distance between the genes is within 30kb; 3) the 5′ genes are actively transcribing; and 4) the chimeras tend to have the second-to-last exon in the 5′ genes joined to the second exon in the 3′ genes. We then randomly selected 20 neighboring genes in the genome, and detected four fusion events using these rules in prostate cancer and non-cancerous cells. These results suggest that splicing between neighboring gene transcripts is a rather frequent phenomenon, and it is not a feature unique to cancer cells.

RNA-Seq made possible the global identification of fusion transcripts, i.e. “chimeric RNAs”. Even though various software packages have been developed to serve this purpose, they behave differently in different datasets provided by different developers. It is important for both users, and developers to have an unbiased assessment of the performance of existing fusion detection tools. Toward this goal, we compared the performance of 12 well-known fusion detection software packages. We evaluated the sensitivity, false discovery rate, computing time, and memory usage of these tools in four different datasets (positive, negative, mixed, and test). We conclude that some tools are better than others in terms of sensitivity, positive prediction value, time consumption and memory usage. We also observed small overlaps of the fusions detected by different tools in the real dataset (test dataset). This could be due to false discoveries by various tools, but could also be due to the reason that none of the tools are inclusive. We have found that the performance of the tools depends on the quality, read length, and number of reads of the RNA-Seq data. We recommend that users choose the proper tools for their purpose based on the properties of their RNA-Seq data.

Circular RNAs (circRNAs) are a type of newly identified non-coding RNAs through high-throughput deep sequencing, which play important roles in miRNA function and transcriptional controlling in human, animals, and plants. To date, there is no report in wheat seedlings regarding the circRNAs identification and roles in the dehydration stress response. In present study, the total RNA was extracted from leaves of wheat seedlings under dehydration-stressed and well-watered conditions, respectively. Then, the circRNAs enriched library based deep sequencing was performed and the circRNAs were identified using bioinformatics tools. Around 88 circRNAs candidates were isolated in wheat seedlings leaves while 62 were differentially expressed in dehydration-stressed seedlings compared to well-watered control. Among the dehydration responsive circRNAs, six were found to act as 26 corresponding miRNAs sponges in wheat. Sixteen circRNAs including the 6 miRNAs sponges and other 10 randomly selected ones were further validated to be circular by real-time PCR assay, and 14 displayed consistent regulation patterns with the transcriptome sequencing results. After Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the targeted mRNAs functions, the circRNAs were predicted to be involved in dehydration responsive process, such as photosynthesis, porphyrin, and chlorophyll metabolism, oxidative phosphorylation, amino acid biosynthesis, and metabolism, as well as plant hormone signal transduction, involving auxin, brassinosteroid, and salicylic acid. Herein, we revealed a possible connection between the regulations of circRNAs with the expressions of functional genes in wheat leaves associated with dehydration resistance.

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Gene fusions and their chimeric products are common features of neoplasia. Given that many cancers arise by the dysregulated recapitulation of processes in normal development, we hypothesized that comparable chimeric gene products may exist in normal cells. Here, we show that a chimeric RNA, PAX3-FOXO1 , identical to that found in alveolar rhabdomyosarcoma, is transiently present in cells undergoing differentiation from pluripotent cells into skeletal muscle. Unlike cells of rhabdomyosarcoma, these cells do not seem to harbor the t(2;13) chromosomal translocation. Importantly, both PAX3-FOXO1 RNA and protein could be detected in the samples of normal fetal muscle. Overexpression of the chimera led to continuous expression of MYOD and MYOG-two myogenic markers that are overexpressed in rhabdomyosarcoma cells. Our results are consistent with a developmental role of a specifi c chimeric RNA generated in normal cells without the corresponding chromosomal rearrangement at the DNA level seen in neoplastic cells presumably of the same lineage. SIGNIFICANCE:A chimeric fusion RNA, PAX3-FOXO1 , associated with alveolar rhabdomyosarcoma, is also present in normal non-cancer cells and tissues. Its transient expression nature and the absence of t(2;13) chromosomal translocation are consistent with a posttranscriptional mechanism. When constantly expressed, PAX3-FOXO1 interfered with the muscle differentiation process, which presumably contributes to tumorigenesis.

BackgroundLiver tumor initiating cells (TICs) have self-renewal and differentiation properties, accounting for tumor initiation, metastasis and drug resistance. Long noncoding RNAs are involved in many physiological and pathological processes, including tumorigenesis. DNA copy number alterations (CNA) participate in tumor formation and progression, while the CNA of lncRNAs and their roles are largely unknown.MethodsLncRNA CNA was determined by microarray analyses, realtime PCR and DNA FISH. Liver TICs were enriched by surface marker CD133 and oncosphere formation. TIC self-renewal was analyzed by oncosphere formation, tumor initiation and propagation. CRISPRi and ASO were used for lncRNA loss of function. RNA pulldown, western blot and double FISH were used to identify the interaction between lncRNA and CTNNBIP1.ResultsUsing transcriptome microarray analysis, we identified a frequently amplified long noncoding RNA in liver cancer termed linc00210, which was highly expressed in liver cancer and liver TICs. Linc00210 copy number gain is associated with its high expression in liver cancer and liver TICs. Linc00210 promoted self-renewal and tumor initiating capacity of liver TICs through Wnt/β-catenin signaling. Linc00210 interacted with CTNNBIP1 and blocked its inhibitory role in Wnt/β-catenin activation. Linc00210 silencing cells showed enhanced interaction of β-catenin and CTNNBIP1, and impaired interaction of β-catenin and TCF/LEF components. We also confirmed linc00210 copy number gain using primary hepatocellular carcinoma (HCC) samples, and found the correlation between linc00210 CNA and Wnt/β-catenin activation. Of interest, linc00210, CTNNBIP1 and Wnt/β-catenin signaling targeting can efficiently inhibit tumor growth and progression, and liver TIC propagation.ConclusionWith copy-number gain in liver TICs, linc00210 is highly expressed along with liver tumorigenesis. Linc00210 drives the self-renewal and propagation of liver TICs through activating Wnt/β-catenin signaling. Linc00210 interacts with CTNNBIP1 and blocks the combination between CTNNBIP1 and β-catenin, driving the activation of Wnt/β-catenin signaling. Linc00210-CTNNBIP1-Wnt/β-catenin axis can be targeted for liver TIC elimination.