A high-resolution map of the human small non-coding transcriptome.

Small noncoding RNAs are regulatory molecules that play important roles in several aspects of cellular biology. They vary from 18 to 30 nucleotides in length and often act through the inactivation of complementary sequences. A variety of small RNA classes have been identified to date and the list is constantly growing, owing to the advent of new sequencing technologies. Among these molecules, microRNAs are the most extensively studied. They are known to play important roles in human diseases, including cancer. More recently, genome-wide analyses have showed that changes in the expression levels of other small noncoding RNAs are also associated with cancer, with correlations between RNA abundance and disease status. Oral squamous cell carcinoma, associated with chronic tobacco and alcohol consumption, is among the leading cancers in the world. The presence of cervical lymph node metastases is currently its strongest prognostic factor. The possibility to evaluate its metastatic potential is relevant to the clinical and molecular oncologist due to frequent asymptomatic development of such cancer in its early stages. In this study small RNA libraries from 30 oral squamous cell carcinoma samples were sequenced for the identification and quantification of known small RNAs. Samples were divided in two groups: those presenting lymph node metastasis at the time of diagnosis and those that did not present this characteristic. Additionally, plasma of 30 patients was accessed for the presence of microRNAs identified as differentially expressed between metastatic and non-metastatic tumor samples as a means to identify circulating molecules that could be considered potential biomarkers for lymph node metastasis. Global expression patterns of small RNA molecules were not associated with cervical metastases. MiR-21, miR-203 and miR-205 were highly expressed throughout samples, in agreement with their role in epithelial cell biology, but disagreeing with studies correlating these molecules with cancer invasion. Nineteen microRNAs, but no other small RNA class, varied consistently between metastatic and non-metastatic cancer samples, some of which were also detected at corresponding levels in plasma. MiR-31 and miR-130b, known to inhibit several steps in the metastatic process, were over-expressed in non-metastatic samples and the expression of miR-130b was confirmed in plasma of patients presenting non-metastatic oral squamous cell carcinoma. MiR-181 and miR-296 were associated with metastasis but were not detected in plasma. In conclusion, we identified microRNAs linked to the metastatic status in a group of patients diagnosed with oral squamous cell carcinoma, both in tissue and plasma samples. We also demonstrate that other small RNA classes are expressed and should, therefore, be involved and contribute to the metastatic phenotype of this disease. Citation Format: Patricia Severino, Liliane Santana Oliveira, Flavia Maziero Andreghetto, Natalia Torres, Otavio Curioni, Patricia Maluf Cury, Tatiana Natasha Toporcov, Alexandre Rossi Paschoal, Alan Mitchell Durham. MicroRNAs and other small RNA molecules expressed in metastatic and non-metastatic oral squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3983. doi:10.1158/1538-7445.AM2015-3983

Integrated small RNA, mRNA, and degradome sequencing reveals the important role of miRNAs in the interactions between parasitic plant Cuscuta australis and its host Trifolium repens

BackgroundMicroRNAs (miRNAs) are short regulatory RNAs derived from longer precursor RNAs. miRNA biogenesis has been studied in animals and plants, recently elucidating more complex aspects, such as non-conserved, species-specific, and heterogeneous miRNA precursor populations. Small RNA sequencing data can help in computationally identifying genomic loci of miRNA precursors. The challenge is to predict a valid miRNA precursor from inhomogeneous read coverage from a complex RNA library: while the mature miRNA typically produces many sequence reads, the remaining part of the precursor is covered very sparsely. As recent results suggest, alternative miRNA biogenesis pathways may lead to a more diverse miRNA precursor population than previously assumed. In plants, the latter manifests itself in e.g. complex secondary structures and expression from multiple loci within precursors. Current miRNA identification algorithms often depend on already existing gene annotation, and/or make use of specific miRNA precursor features such as precursor lengths, secondary structures etc. Consequently and in view of the emerging new understanding of a more complex miRNA biogenesis in plants, current tools may fail to characterise organism-specific and heterogeneous miRNA populations.ResultsmiRA is a new tool to identify miRNA precursors in plants, allowing for heterogeneous and complex precursor populations. miRA requires small RNA sequencing data and a corresponding reference genome, and evaluates precursor secondary structures and precursor processing accuracy; key parameters can be adapted based on the specific organism under investigation. We show that miRA outperforms the currently best plant miRNA prediction tools both in sensitivity and specificity, for data involving Arabidopsis thaliana and the Volvocine algae Chlamydomonas reinhardtii; the latter organism has been shown to exhibit a heterogeneous and complex precursor population with little cross-species miRNA sequence conservation, and therefore constitutes an ideal model organism. Furthermore we identify novel miRNAs in the Chlamydomonas-related organism Volvox carteri.ConclusionsWe propose miRA, a new plant miRNA identification tool that is well adapted to complex precursor populations. miRA is particularly suited for organisms with no existing miRNA annotation, or without a known related organism with well characterized miRNAs. Moreover, miRA has proven its ability to identify species-specific miRNAs. miRA is flexible in its parameter settings, and produces user-friendly output files in various formats (pdf, csv, genome-browser-suitable annotation files, etc.). It is freely available at https://github.com/mhuttner/miRA.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0798-3) contains supplementary material, which is available to authorized users.

While the risks of maternal alcohol abuse during pregnancy are well-established, several preclinical studies suggest that chronic preconception alcohol consumption by either parent may also have significance consequences for offspring health and development. Notably, since isogenic male mice used in these studies are not involved in gestation or rearing of offspring, the cross-generational effects of paternal alcohol exposure suggest a germline-based epigenetic mechanism. Many recent studies have demonstrated that the effects of paternal environmental exposures such as stress or malnutrition can be transmitted to the next generation via alterations to small noncoding RNAs in sperm. Therefore, we used high throughput sequencing to examine the effect of preconception ethanol on small noncoding RNAs in sperm. We found that chronic intermittent ethanol exposure altered several small noncoding RNAs from three of the major small RNA classes in sperm, tRNA-derived small RNA (tDR), mitochondrial small RNA, and microRNA. Six of the ethanol-responsive small noncoding RNAs were evaluated with RT-qPCR on a separate cohort of mice and five of the six were confirmed to be altered by chronic ethanol exposure, supporting the validity of the sequencing results. In addition to altered sperm RNA abundance, chronic ethanol exposure affected post-transcriptional modifications to sperm small noncoding RNAs, increasing two nucleoside modifications previously identified in mitochondrial tRNA. Furthermore, we found that chronic ethanol reduced epididymal expression of a tRNA methyltransferase, Nsun2, known to directly regulate tDR biogenesis. Finally, ethanol-responsive sperm tDR are similarly altered in extracellular vesicles of the epididymis (i.e., epididymosomes), supporting the hypothesis that alterations to sperm tDR emerge in the epididymis and that epididymosomes are the primary source of small noncoding RNAs in sperm. These results add chronic ethanol to the growing list of paternal exposures that can affect small noncoding RNA abundance and nucleoside modifications in sperm. As small noncoding RNAs in sperm have been shown to causally induce heritable phenotypes in offspring, additional research is warranted to understand the potential effects of ethanol-responsive sperm small noncoding RNAs on offspring health and development.

The small noncoding RNAs (sncRNAs) are considered as post-transcriptional key regulators of male germ cell development. In addition to microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), other sncRNAs generated from small nucleolar RNAs (snoRNAs), tRNAs, or rRNAs processing may also play important regulatory roles in spermatogenesis. By next-generation sequencing (NGS), we characterized the sncRNA populations detected at three milestone stages in male germ differentiation: primordial germ cells (PGCs), pubertal spermatogonia cells, and mature spermatozoa. To assess their potential transmission through the spermatozoa during fertilization, the sncRNAs of mouse oocytes and zygotes were also analyzed. Both, microRNAs and snoRNA-derived small RNAs are abundantly expressed in PGCs but transiently replaced by piRNAs in spermatozoa and endo-siRNAs in oocytes and zygotes. Exhaustive analysis of miRNA sequence variants also shows an increment of noncanonical microRNA forms along male germ cell differentiation. RNAs-derived from tRNAs and rRNAs interacting with PIWI proteins are not generated by the ping-pong pathway and could be a source of primary piRNAs. Moreover, our results strongly suggest that the small RNAs-derived from tRNAs and rRNAs are interacting with PIWI proteins, and specifically with MILI. Finally, computational analysis revealed their potential involvement in post-transcriptional regulation of mRNA transcripts suggesting functional convergence among different small RNA classes in germ cells and zygotes.

ABSTRACTSmall RNAs play a critical role in host-pathogen interaction. Indeed, small RNA-mediated silencing or RNA interference (RNAi) is one of the earliest forms of antiviral immunity. Although it represents the main defense system against viruses in many organisms, the antiviral role of RNAi has not been clearly proven in higher vertebrates. However, it is well established that their response to viral infection relies on the recognition of viral RNAs by host pattern recognition receptors (PRRs) to trigger activation of the interferon pathway. In the present work, we report the existence of a novel small noncoding RNA population produced in mammalian cells upon RNA virus infection. Using Sindbis virus (SINV) as a prototypic arbovirus model, we profiled the small RNA population of infected cells in both human and African green monkey cell lines. Here, we provide evidence for the presence of discrete small RNAs of viral origin that are not associated with the RNA-induced silencing complex (RISC), that are highly expressed and detected by Northern blot analysis, and that accumulate as 21- to 28-nucleotide (nt) species during infection. We report that the cellular antiviral endoribonuclease RNase L cleaves the viral genome, producing in turn the small RNAs. Surprisingly, we uncovered the presence of a modification on the 3′-end nucleotide of SINV-derived viral small RNAs (SvsRNAs) that might be at the origin of their stability. Altogether, our findings show that stable modified small viral RNAs could represent a novel way to modulate host-virus interaction upon SINV infection.

Small non-coding RNAs such as miRNAs, piRNAs and endo-siRNAs fine-tune gene expression through post-transcriptional regulation, modulating important processes in development, differentiation, homeostasis and regeneration. Using deep sequencing, we have profiled small non-coding RNAs in Hydra magnipapillata and investigated changes in small RNA expression pattern during head regeneration. Our results reveal a unique repertoire of small RNAs in hydra. We have identified 126 miRNA loci; 123 of these miRNAs are unique to hydra. Less than 50% are conserved across two different strains of Hydra vulgaris tested in this study, indicating a highly diverse nature of hydra miRNAs in contrast to bilaterian miRNAs. We also identified siRNAs derived from precursors with perfect stem–loop structure and that arise from inverted repeats. piRNAs were the most abundant small RNAs in hydra, mapping to transposable elements, the annotated transcriptome and unique non-coding regions on the genome. piRNAs that map to transposable elements and the annotated transcriptome display a ping–pong signature. Further, we have identified several miRNAs and piRNAs whose expression is regulated during hydra head regeneration. Our study defines different classes of small RNAs in this cnidarian model system, which may play a role in orchestrating gene expression essential for hydra regeneration.

Chronic lymphocytic leukemia (CLL) is the most common human leukemia, and transgenic mouse studies indicate that activation of the T-cell leukemia/lymphoma 1 (TCL1) oncogene is a contributing event in the pathogenesis of the aggressive form of this disease. While studying the regulation of TCL1 expression, we identified the microRNA cluster miR-4521/3676 and discovered that these two microRNAs are associated with tRNA sequences and that this region can produce two small RNAs, members of a recently identified class of small noncoding RNAs, tRNA-derived small RNAs (tsRNAs). We further proved that miR-3676 and miR-4521 are tsRNAs using Northern blot analysis. We found that, like ts-3676, ts-4521 is down-regulated and mutated in CLL. Analysis of lung cancer samples revealed that both ts-3676 and ts-4521 are down-regulated and mutated in patient tumor samples. Because tsRNAs are similar in nature to piRNAs [P-element-induced wimpy testis (Piwi)-interacting small RNAs], we investigated whether ts-3676 and ts-4521 can interact with Piwi proteins and found these two tsRNAs in complexes containing Piwi-like protein 2 (PIWIL2). To determine whether other tsRNAs are involved in cancer, we generated a custom microarray chip containing 120 tsRNAs 16 bp or more in size. Microarray hybridization experiments revealed tsRNA signatures in CLL and lung cancer, indicating that, like microRNAs, tsRNAs may have an oncogenic and/or tumor-suppressor function in hematopoietic malignancies and solid tumors. Thus, our results show that tsRNAs are dysregulated in human cancer.

Small RNA sequencing by Illumina's Next Generation technology has revolutionized the transcriptome analysis by facilitating massive parallel sequencing of RNA molecules at low cost. Illumina's Next Generation RNA sequencing is ideal for profiling small RNA (microRNAs, snoRNAs, and piRNAs) libraries in the identification of novel biomarkers for better clinical diagnosis. This method offers significant advantages when compared to microarray analysis with the ability to identify novel transcripts, higher sensitivity, specificity, and detection of rare and low-abundance transcripts. Small RNAs, including microRNAs and snoRNAs, belong to the class of small non-coding RNAs with 50-200 nucleotides in length and are involved in post-transcriptional regulation of gene expression. Executing Illumina's Next Generation Sequencing technology, we have recently deciphered microRNAs and snoRNAs expressed in cerebral cavernous malformations (CCMs). Small RNA library preparation is a prerequisite step prior to RNA sequencing for the identification of microRNAs and snoRNAs. Here, we describe stepwise small RNA library preparation starting from total RNA isolated from CCMs patient until library validation using the Illumina® TruSeq® Small RNA Sample preparation kit. We believe this method will shed light into the functional identification of other novel small non-coding RNAs in CCMs that awaits discovery.

Background: Cancer is a major cause of deaths worldwide, despite improved healthcare and technologies. Recent advancements in genome sequencing have led to the rapid study of the whole transcriptome and small RNAs with their biological functions. Non-coding RNAs (ncRNAs) play an important role in biological processes that greatly impact biomarker development for diagnosis, prognosis, and therapy. Non-coding RNAs such as microRNAs (miRNA), Piwi-interacting-RNAs (piRNAs), small nuclear/nucleolar RNAs (sn/snoRNAs) have recently been studied to understand their biology and pathology. Results: In the present study, we used eight matched colorectal patient tissue samples (benign, tumor, and metastasis) small RNA sequencing data remapped for various small RNA annotation. We identified aberrant expression of 13 miRs in tumor and metastasis specimens [tumor vs benign group (19 miRs) and metastasis vs benign group (38 miRs)] of which five were upregulated, and eight were downregulated, during disease progression. We also investigated pathway analysis on abbarent expression of miRNAs, which showed majority of miRs involved in the colon other types of cancers. Further analysis of piRNAs revealed that six piRNAs in tumor vs benign and 24 in metastasis vs benign samples (commonly in both groups, only two piRNAs). Additionally, we examined other types of small RNAs [sn/snoRNAs, mt_rRNA, misc_RNA, nonsense mediated decay (NMD) and rRNA], we identified 15 in tumor vs benign and 104 metastasis vs benign and only four in commonly expressed. Conclusion: In summary, our results identified multiple sncRNAs during colorectal cancer progression which needs to be further validated and can be used for prognosis, diagnosis, and therapeutic potentials. Citation Format: Srinivas V. Koduru, Amit K. Tiwari, Sprague W. Hazard, Milind K. Mahajan, Dino J. Ravnic. Analysis of small RNA-seq data for differential expression of small noncoding RNAs in human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4447. doi:10.1158/1538-7445.AM2017-4447

MicroRNAs (miRNAs) are ∼22-nt long, non-coding RNAs that regulate gene silencing. It is known that many human miRNAs are deregulated in numerous types of tumors. Here we report the sequencing of small RNAs (17–25 nt) from 23 breast, bladder, colon and lung tumor samples using high throughput sequencing. We identified 49 novel miRNA and miR-sized small RNAs. We further validated the expression of 10 novel small RNAs in 31 different types of blood, normal and tumor tissue samples using two independent platforms, namely microarray and RT–PCR. Some of the novel sequences show a large difference in expression between tumor and tumor-adjacent tissues, between different tumor stages, or between different tumor types. We also report the identification of novel small RNA classes in human: highly expressed small RNA derived from Y-RNA and endogenous siRNA. Finally, we identified dozens of new miRNA sequence variants that demonstrate the existence of miRNA-related SNP or post-transcriptional modifications. Our work extends the current knowledge of the tumor small RNA transcriptome and provides novel candidates for molecular biomarkers and drug targets.

In this study, we reported two featured series of rRNA-derived RNA fragments (rRFs) from the small RNA sequencing (sRNA-seq) data of Amblyomma testudinarium using the Illunima platform. Two series of rRFs (rRF5 and rRF3) were precisely aligned to the 5' and 3' ends of the 5.8S and 28S rRNA gene. The rRF5 and rRF3 series were significantly more highly expressed than the rRFs located in the body of the rRNA genes. These series contained perfectly aligned reads, the lengths of which varied progressively with 1-bp differences. The rRF5 and rRF3 series in the same expression pattern exist ubiquitously from ticks to human. The cellular experiments showed the RNAi knockdown of one 20-nt rRF3 induced the cell apoptosis and inhibited the cell proliferation. In addition, the RNAi knockdown resulted in a significant decrease of H1299 cells in the G2 phase of the cell cycle. These results indicated the rRF5 and rRF3 series were not random intermediates or products during rRNA degradation, but could constitute a new class of small RNAs that deserves further investigation.

BackgroundNon-coding small RNAs, ranging from 20 to 30 nucleotides in length, mediate the regulation of gene expression and play important roles in many biological processes. One class of small RNAs, microRNAs (miRNAs), are highly conserved across taxa and mediate the regulation of the chromatin state and the post-transcriptional regulation of messenger RNA (mRNA). Another class of small RNAs is the Piwi-interacting RNAs, which play important roles in the silencing of transposons and other functional genes. Although the biological functions of the different small RNAs have been elucidated in several laboratory animals, little is known regarding naturally occurring variation in small RNA transcriptomes among closely related species.ResultsWe employed next-generation sequencing technology to compare the expression profiles of brain small RNAs between sympatric species of the Japanese threespine stickleback (Gasterosteus aculeatus). We identified several small RNAs that were differentially expressed between sympatric Pacific Ocean and Japan Sea sticklebacks. Potential targets of several small RNAs were identified as repetitive sequences. Female-biased miRNA expression from the old X chromosome was also observed, and it was attributed to the degeneration of the Y chromosome.ConclusionsOur results suggest that expression patterns of small RNA can differ between incipient species and may be a potential mechanism underlying differential mRNA expression and transposon activity.

Background: Prostate cancer (PCa) is the most common non-skin cancer among American men. MicroRNAs (miRNAs) are critical post-transcriptional regulators and involved in prostate cancer tumorigenesis. The aim of this study is to identify a PCa-specific expression profile of miRNAs from plasma to guide prostate cancer diagnosis and therapeutic treatment. Methods: Plasma was collected from 5 PCa patients and 5 normal men. Circulating RNA was extracted from 1) 200ul plasma or 2) the pellets of anti-Ago2 immunoprecipitations from 500ul plasma using the miRNeasy Serum/Plasma kit (Qiagen), with addition of glycogen as a carrier. Small RNA libraries were constructed using the NEBNext Multiplex Small RNA Prep Set for Illumina® (New England. BioLabs). The cDNA library fragments were purified by Blue Pippin (Sage Science) for extraction of 140-160 bp size fraction containing small RNA inserts. Equimolar amounts of cDNA library samples were pooled and were sequenced in a single flowcell on an Illumina HiSeq2500 with 50 cycle kit and rapid run model. Bioinformatics analysis: Adaptor was firstly removed from the raw reads, and the sequences were mapped to several small RNA databases by using bowtie1 with 1 mismatch. The multiple aligned reads were weighted to the mapped small RNAs based on their unique mapped reads counts. We then calculated the RPM (reads per million) as indicator of the expression levels of the small RNA. To get confident analysis results, we discarded the small RNAs whose averages of the raw reads counts in cancer and normal groups are smaller than 25 and only focus on the mature microRNAs. The moderate t-test is applied to find the differently expressed (DE) microRNAs between normal and cancer group. Results: Two major small RNA classes identified from total plasma are miRNA (47.7%) and yRNA (35.0%). The percentage of miRNA increased to 85.3% by Ago2-IP method. The compositions of categories of small RNAs in cancer and normal samples are similar. We identified 28 and 22 differential miRNAs (>2 fold change between cancer and normal group) by total plasma and Ago-2 IP methods respectively. Conclusions: We discovered a unique expression profile of miRNA detectable in the plasma from prostate cancer patients. Extracted RNA from the pellets of anti-Ago2 immunoprecipitations can enhance the detection of miRNA. Expand study to confirm these findings are needed. Citation Format: Xianghua Liu, Andrew Hunter, Qing Zhang, Quanyuan He, Annemarie Benton, Gerri Ortiz, Vaishali Pannu, Nick Xiao, Mark Miglarese, David Spetzler. Small non-coding RNA profiling from prostate cancer plasma by deep sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4437. doi:10.1158/1538-7445.AM2017-4437

Micro-RNAs are approximately 21-25-nucleotide-long noncoding RNAs that regulate gene expression primarily at the post-transcriptional level in animals. Here, we report that micro-RNA-1 (miR-1), abundant in the cardiac and smooth muscles, is expressed in the lung and is down-regulated in human primary lung cancer tissues and cell lines. In situ hybridization demonstrated localization of miR-1 in bronchial epithelial cells. The tumor suppressor C/EBPalpha, frequently suppressed in lung cancer, reactivated miR-1 expression in the lung cancer cells. Repressed miR-1 was also activated in lung cancer cells upon treatment with a histone deacetylase inhibitor. These observations led us to examine the antitumorigenic potential of miR-1 in lung cancer cells. Expression of miR-1 in nonexpressing A549 and H1299 cells reversed their tumorigenic properties, such as growth, replication potential, motility/migration, clonogenic survival, and tumor formation in nude mice. Exogenous miR-1 significantly reduced expression of oncogenic targets, such as MET, a receptor tyrosine kinase, and Pim-1, a Ser/Thr kinase, frequently up-regulated in lung cancer. Similarly, the levels of two additional targets, FoxP1, a transcription factor with oncogeneic property, and HDAC4 that represses differentiation-promoting genes, were reduced in miR-1-expressing cells. Conversely, depletion of miR-1 facilitated N417 cell growth with concomitant elevation of these targets. Further, ectopic miR-1 induced apoptosis in A549 cells in response to the potent anticancer drug doxorubicin. Enhanced activation of caspases 3 and 7, cleavage of their substrate PARP-1, and depletion of anti-apoptotic Mcl-1 contributed to the sensitivity of miR-1-expressing cells to doxorubicin. Thus, miR-1 has potential therapeutic application against lung cancers.