Small RNA Deep Sequencing Data Research Articles

Viral and host small RNA transcriptome analysis of SARS-CoV-1 and SARS-CoV-2-infected human cells reveals novel viral short RNAs

RNA viruses have been shown to express various short RNAs, some of which have regulatory roles during replication, transcription, and translation of viral genomes. However, short viral RNAs generated from SARS-CoV-1 and SARS-CoV-2 genomic RNAs remained largely unexplored, possibly due limitations of the widely used library preparation methods for small RNA deep sequencing and corresponding data processing. By analyzing publicly available small RNA sequencing datasets, we observed that human Calu-3 cells infected by SARS-CoV-1 or SARS-CoV-2 accumulate multiple previously unreported short viral RNAs. In addition, we verified the presence of the five most abundant SARS-CoV-2 short viral RNAs in SARS-CoV-2-infected human lung adenocarcinoma cells by quantitative PCR. Interestingly, the copy number of the observed SARS-CoV-2 short viral RNAs dramatically exceeded the expression of previously reported viral microRNAs in the same cells. We hypothesize that the reported SARS-CoV-2 short viral RNAs could serve as biomarkers for early infection stages due to their high abundance. Furthermore, unlike SARS-CoV-1, the SARS-CoV-2 infection induced significant (Benjamini-Hochberg-corrected p-value <0.05) deregulation of Y-RNA, transfer RNA, vault RNA, as well as more than 300 endogenous short RNAs that aligned predominantly to human protein-coding and long noncoding RNA transcripts. In particular, more than 20-fold upregulation of reads derived from Y-RNA (and several transfer RNAs) have been documented in RNA-seq datasets from SARS-CoV-2 infected cells. Finally, a significant proportion of short RNAs derived from full-length viral genomes also aligned to various human genome (hg38) sequences, suggesting opportunities to investigate regulatory roles of short viral RNAs during infection. Further characterization of the small RNA landscape of both viral and host genomes is clearly warranted to improve our understanding of molecular events related to infection and to design more efficient strategies for therapeutic interventions as well as early diagnosis.

Identification of MicroRNAs and Natural Antisense Transcript-Originated Endogenous siRNAs from Small-RNA Deep Sequencing Data.

Next Generation Sequencing (NGS) is becoming a routine experimental technology. It has been a great success in recent years to profile small-RNA species using NGS. Indeed, a large quantity of small-RNA profiling data has been generated from NGS, and computational methods have been developed to process and analyze NGS data for the purpose of identification of novel and expressed small noncoding RNAs and analysis of their roles in nearly all biological processes and pathways in eukaryotes. We discuss here the computational procedures and major steps for identification of microRNAs and natural antisense transcript-originated small interfering RNAs (nat-siRNAs) from NGS small-RNA profiling data.

Differential expression of piRNAs in reprogrammed pluripotent stem cells from mouse embryonic fibroblasts.

piRNAs are a large class of small noncoding RNA that interact with an animal-specific class of Argonaute proteins, P-element induced wimpy proteins. piRNAs were initially discovered in mouse testes to be a fundamental component of spermatogenesis. Outside of the germline, piRNAs were found to function in embryogenesis, development, regeneration and cancer cells. However, despite a decade of scrutiny, functional understanding of this class of small RNAs remains very limited. To determine whether there are piRNAs present and involved in the cellular reprogramming process, we extracted piwi-interacting RNA (piRNA) signatures from a small RNA deep sequencing data set of mouse embryonic fibroblasts (MEFs), mouse embryonic stem cells (mESCs) and reprogrammed stem cells by three different technologies. We successfully identified three piRNA families specifically expressed in these reprogrammed stem cells. Meanwhile, there were almost no piRNAs observed in MEFs and mESCs. Further analysis indicated that these piRNAs may associate with the reprogramming process but not cellular pluripotency. Target gene prediction suggested that at least one of piRNAs, piR-mmu-64162, may take part in the reprogramming process by regulating cell senescence. Overall, we firstly identified the potential reprogramming associated piRNAs, shedding new light on piRNA functions.

Oasis 2: improved online analysis of small RNA-seq data

BackgroundSmall RNA molecules play important roles in many biological processes and their dysregulation or dysfunction can cause disease. The current method of choice for genome-wide sRNA expression profiling is deep sequencing.ResultsHere we present Oasis 2, which is a new main release of the Oasis web application for the detection, differential expression, and classification of small RNAs in deep sequencing data. Compared to its predecessor Oasis, Oasis 2 features a novel and speed-optimized sRNA detection module that supports the identification of small RNAs in any organism with higher accuracy. Next to the improved detection of small RNAs in a target organism, the software now also recognizes potential cross-species miRNAs and viral and bacterial sRNAs in infected samples. In addition, novel miRNAs can now be queried and visualized interactively, providing essential information for over 700 high-quality miRNA predictions across 14 organisms. Robust biomarker signatures can now be obtained using the novel enhanced classification module.ConclusionsOasis 2 enables biologists and medical researchers to rapidly analyze and query small RNA deep sequencing data with improved precision, recall, and speed, in an interactive and user-friendly environment.Availability and ImplementationOasis 2 is implemented in Java, J2EE, mysql, Python, R, PHP and JavaScript. It is freely available at https://oasis.dzne.de

Computational Approaches to tRNA-Derived Small RNAs.

tRNA-derived small RNAs (tDRs) are a group of small, non-coding RNAs derived from transfer RNAs (tRNAs). They can be classified as tRNA halves and tRNA-derived small RNA fragments (tRFs). Accumulating experimental evidence suggests their functional roles in cells and in various biological processes. Advances in next-generation sequencing (NGS) techniques allow a large amount of small RNA deep-sequencing data to be generated. To investigate tDRs from these data, software to identify tDRs and databases to retrieve or manage tDR data have been devised. In this review, we summarized the tools and databases for tDR identification and collection, with the aim of helping researchers choose the best tools for their analysis and inspiring the invention or improvement of tools in the field.

Using Small RNA Deep Sequencing Data to Detect Human Viruses.

Small RNA sequencing (sRNA-seq) can be used to detect viruses in infected hosts without the necessity to have any prior knowledge or specialized sample preparation. The sRNA-seq method was initially used for viral detection and identification in plants and then in invertebrates and fungi. However, it is still controversial to use sRNA-seq in the detection of mammalian or human viruses. In this study, we used 931 sRNA-seq runs of data from the NCBI SRA database to detect and identify viruses in human cells or tissues, particularly from some clinical samples. Six viruses including HPV-18, HBV, HCV, HIV-1, SMRV, and EBV were detected from 36 runs of data. Four viruses were consistent with the annotations from the previous studies. HIV-1 was found in clinical samples without the HIV-positive reports, and SMRV was found in Diffuse Large B-Cell Lymphoma cells for the first time. In conclusion, these results suggest the sRNA-seq can be used to detect viruses in mammals and humans.

Unravelling the complexity of microRNA-mediated gene regulation in black pepper (Piper nigrum L.) using high-throughput small RNA profiling

Analysis of high-throughput small RNA deep sequencing data, in combination with black pepper transcriptome sequences revealed microRNA-mediated gene regulation in black pepper ( Piper nigrum L.). Black pepper is an important spice crop and its berries are used worldwide as a natural food additive that contributes unique flavour to foods. In the present study to characterize microRNAs from black pepper, we generated a small RNA library from black pepper leaf and sequenced it by Illumina high-throughput sequencing technology. MicroRNAs belonging to a total of 303 conserved miRNA families were identified from the sRNAome data. Subsequent analysis from recently sequenced black pepper transcriptome confirmed precursor sequences of 50 conserved miRNAs and four potential novel miRNA candidates. Stem-loop qRT-PCR experiments demonstrated differential expression of eight conserved miRNAs in black pepper. Computational analysis of targets of the miRNAs showed 223 potential black pepper unigene targets that encode diverse transcription factors and enzymes involved in plant development, disease resistance, metabolic and signalling pathways. RLM-RACE experiments further mapped miRNA-mediated cleavage at five of the mRNA targets. In addition, miRNA isoforms corresponding to 18 miRNA families were also identified from black pepper. This study presents the first large-scale identification of microRNAs from black pepper and provides the foundation for the future studies of miRNA-mediated gene regulation of stress responses and diverse metabolic processes in black pepper.

Oasis: online analysis of small RNA deep sequencing data.

Summary: Oasis is a web application that allows for the fast and flexible online analysis of small-RNA-seq (sRNA-seq) data. It was designed for the end user in the lab, providing an easy-to-use web frontend including video tutorials, demo data and best practice step-by-step guidelines on how to analyze sRNA-seq data. Oasis’ exclusive selling points are a differential expression module that allows for the multivariate analysis of samples, a classification module for robust biomarker detection and an advanced programming interface that supports the batch submission of jobs. Both modules include the analysis of novel miRNAs, miRNA targets and functional analyses including GO and pathway enrichment. Oasis generates downloadable interactive web reports for easy visualization, exploration and analysis of data on a local system. Finally, Oasis’ modular workflow enables for the rapid (re-) analysis of data.Availability and implementation: Oasis is implemented in Python, R, Java, PHP, C++ and JavaScript. It is freely available at http://oasis.dzne.de.Contact: stefan.bonn@dzne.deSupplementary information: Supplementary data are available at Bioinformatics online.

An Efficient Strategy of Screening for Pathogens in Wild-Caught Ticks and Mosquitoes by Reusing Small RNA Deep Sequencing Data

This paper explored our hypothesis that sRNA (18∼30 bp) deep sequencing technique can be used as an efficient strategy to identify microorganisms other than viruses, such as prokaryotic and eukaryotic pathogens. In the study, the clean reads derived from the sRNA deep sequencing data of wild-caught ticks and mosquitoes were compared against the NCBI nucleotide collection (non-redundant nt database) using Blastn. The blast results were then analyzed with in-house Python scripts. An empirical formula was proposed to identify the putative pathogens. Results showed that not only viruses but also prokaryotic and eukaryotic species of interest can be screened out and were subsequently confirmed with experiments. Specially, a novel Rickettsia spp. was indicated to exist in Haemaphysalis longicornis ticks collected in Beijing. Our study demonstrated the reuse of sRNA deep sequencing data would have the potential to trace the origin of pathogens or discover novel agents of emerging/re-emerging infectious diseases.

Identification of Novel MicroRNAs in Primates by Using the Synteny Information and Small RNA Deep Sequencing Data

Current technologies that are used for genome-wide microRNA (miRNA) prediction are mainly based on BLAST tool. They often produce a large number of false positives. Here, we describe an effective approach for identifying orthologous pre-miRNAs in several primates based on syntenic information. Some of them have been validated by small RNA high throughput sequencing data. This approach uses the synteny information and experimentally validated miRNAs of human, and incorporates currently available algorithms and tools to identify the pre-miRNAs in five other primates. First, we identified 929 potential pre-miRNAs in the marmoset in which miRNAs have not yet been reported. Then, we predicted the miRNAs in other primates, and we successfully re-identified most of the published miRNAs and found 721, 979, 650 and 639 new potential pre-miRNAs in chimpanzee, gorilla, orangutan and rhesus macaque, respectively. Furthermore, the miRNA transcriptome in the four primates have been re-analyzed and some novel predicted miRNAs have been supported by the small RNA sequencing data. Finally, we analyzed the potential functions of those validated miRNAs and explored the regulatory elements and transcription factors of some validated miRNA genes of interest. The results show that our approach can effectively identify novel miRNAs and some miRNAs that supported by small RNA sequencing data maybe play roles in the nervous system.

Identification of miRNAs and miRNA-mediated regulatory pathways in Carica papaya

Plant microRNAs (miRNAs) post-transcriptionally regulate target gene expression to modulate growth and development and biotic and abiotic stress responses. By analyzing small RNA deep sequencing data in combination with the genome sequence, we identified 75 conserved miRNAs and 11 novel miRNAs. Their target genes were also predicted. For most conserved miRNAs, the miRNA-target pairs were conserved across plant species. In addition to these conserved miRNA-target pairs, we also identified some papaya-specific miRNA-target regulatory pathways. Both miR168 and miR530 target the Argonaute 1 gene, indicating a second autoregulatory mechanism for miRNA regulation. A non-conserved miRNA was mapped within an intron of Dicer-like 1 (DCL1), suggesting a conserved homeostatic autoregulatory mechanism for DCL1 expression. A 21-nt miRNA triggers secondary siRNA production from its target genes, nucleotide-binding site leucine-rich repeat protein genes. Certain phased-miRNAs were processed from their conserved miRNA precursors, indicating a putative miRNA evolution mechanism. In addition, we identified a Carica papaya-specific miRNA that targets an ethylene receptor gene, implying its function in the ethylene signaling pathway. This work will also advance our understanding of miRNA functions and evolution in plants.

A genome-wide survey of microRNA truncation and 3′ nucleotide addition events in larch (Larix leptolepis)

MicroRNAs (miRNAs) play essential roles in numerous developmental and metabolic processes in animals and plants. Although the framework of miRNA biogenesis and function is established, the mechanism of miRNA degradation or modification remains to be investigated in plants. Mature miRNAs may be truncated or added nucleotides to generate variants. A detailed analysis of small RNA deep sequencing data sets resulted in the cloning of a large number of variants derived from larch miRNAs. Many 5'- and/or 3'-end truncated versions of miRNAs suggested that larch miRNAs might be degraded through either 5'-3' or 3'-5'. The relative abundance of variants truncated from 3'-end was higher than that of 5'-end for most miRNAs. The addition of adenine, uridine, and cytidine to the 3'-end of miRNAs was globally present, and the subtle variability in isomiR abundance might be regulated and biologically meaningful. It is the first report for cytidine addition in plant, and our examination of published small RNA deep sequencing data sets of Arabidopsis, rice, and moss suggests that cytidine addition to miRNA 3'-end exists broadly in plants. In addition, the nucleotide addition might be associated with 3'-5' miRNA degradation. Our results provide valuable information for a genome-wide survey of miRNA truncation and modification in larch or plants.

Noncanonical microRNAs and endogenous siRNAs in normal and psoriatic human skin.

Noncanonical microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs) are key gene regulators in eukaryotes. Noncanonical miRNAs, which bypass part of the canonical miRNA biogenesis pathway, can originate from a variety of genomic loci, which include small nucleolar RNAs (snoRNAs), transfer RNAs (tRNAs) and introns, whereas endo-siRNAs can arise from repetitive elements, some of which are transposable. The roles of noncanonical miRNAs and endo-siRNAs in complex diseases have yet to be characterized. To investigate their potential expression and function in psoriasis, we carried out a comprehensive, genome-wide search for noncanonical miRNAs and endo-siRNAs in small RNA deep-sequencing data sets from normal and psoriatic human skin. By analyzing more than 670 million qualified reads from 67 small RNA libraries, we identified 21 novel, noncanonical miRNAs (3 snoRNA-derived and 2 tRNA-derived miRNAs and 16 miRtrons) and 39 novel endo-siRNAs that were expressed in skin. The expression of four novel small RNAs was validated by qRT-PCR in human skin, and their Argonaute association was confirmed by co-immunoprecipitation of ectopic small RNAs in HEK293 cells. Fifteen noncanonical miRNAs or endo-siRNAs were significantly differentially expressed in psoriatic-involved versus normal skin, including an Alu-short interspersed element-derived siRNA which was 17-fold up-regulated in psoriatic-involved skin. These and other differentially expressed small noncoding RNAs may function as regulators of gene expression in skin and potentially play a role in psoriasis pathogenesis.

Identification of conserved and novel microRNAs in Aquilaria sinensis based on small RNA sequencing and transcriptome sequence data

Agarwood is in great demand for its high value in medicine, incense, and perfume across Asia, Middle East, and Europe. As agarwood is formed only when the Aquilaria trees are wounded or infected by some microbes, overharvesting and habitat loss are threatening some populations of agarwood-producing species. Aquilaria sinensis is such a significant economic tree species. To promote the production efficiency and protect the resource of A. sinensis, it would be critical to reveal the regulation mechanisms of stress-induced agarwood formation. MicroRNAs (miRNAs), a key gene expression regulator involved in various plant stress response and metabolic processes, might function in agarwood formation, but no report concerning miRNAs in Aquilaria is available. In this study, the small RNA high-throughput sequencing and 454 transcriptome data were adopted to identify both conserved and novel miRNAs in A. sinensis. Deep sequencing showed that the small RNA (sRNA) population of A. sinensis was complex and the length of sRNAs varied. By in silico analysis of the small RNA deep sequencing data and transcriptome data, we discovered 27 novel miRNAs in A. sinensis. Based on the mature miRNA sequence conservation, we identified 74 putative conserved miRNAs from A. sinensis and 10 of them were confirmed with hairpin forming precursor. Interestingly, a novel miRNA sequence was determined to be the miRNA* of asi-miR408, but with accumulation much higher than asi-miR408. The expression levels of ten stress-responsive miRNAs were examined during the time-course after wound treatment. Eight were shown to be wound-responsive. This not only shows the existence of miRNAs in this Asian economically significant tree species but also indicated its critical role in stress-induced agarwood formation. The highly accumulated miRNA* of asi-miR408 implied miRNA*s would be functional as well as miRNAs in plants.

Human box C/D snoRNA processing conservation across multiple cell types

Small nucleolar RNAs (snoRNAs) function mainly as guides for the post-transcriptional modification of ribosomal RNAs (rRNAs). In recent years, several studies have identified a wealth of small fragments (<35 nt) derived from snoRNAs (termed sdRNAs) that stably accumulate in the cell, some of which may regulate splicing or translation. A comparison of human small RNA deep sequencing data sets reveals that box C/D sdRNA accumulation patterns are conserved across multiple cell types although the ratio of the abundance of different sdRNAs from a given snoRNA varies. sdRNA profiles of many snoRNAs are specific and resemble the cleavage profiles of miRNAs. Many do not show characteristics of general RNA degradation, as seen for the accumulation of small fragments derived from snRNA or rRNA. While 53% of the sdRNAs contain an snoRNA box C motif and boxes D and D′ are also common in sdRNAs (54%), relatively few (12%) contain a full snoRNA guide region. One box C/D snoRNA, HBII-180C, was analysed in greater detail, revealing the presence of C′ box-containing sdRNAs complementary to several pre-messenger RNAs (pre-mRNAs) including FGFR3. Functional analyses demonstrated that this region of HBII-180C can influence the alternative splicing of FGFR3 pre-mRNA, supporting a role for some snoRNAs in the regulation of splicing.

Identification of novel and known miRNAs in deep-sequencing data with miRDeep2.

miRNAs comprise an abundant class of small non-coding RNAs that play important roles in a wide range of biological processes by post-transcriptional regulation of a large fraction of animal genes. High-throughput sequencing machines and the availability of completely sequenced genomes make it possible to reliably identify miRNAs with computational methods. This unit documents how to use the miRDeep2 software package to identify novel and known microRNAs in small RNA deep-sequencing data. Moreover, the usage of miRDeep2 to profile miRNA expression across samples is illustrated.

Analysis of A to I editing of miRNA in macrophages exposed to Salmonella

The main mediator of the lipopolysaccharide (LPS) response in macrophages is activation of Toll-like receptor 4 (TLR4). This generates interferon-beta (INF-beta) production that stimulates increased expression of the RNA editing enzyme ADAR1. To determine if there is an increase in RNA editing in mature miRNA in response to TLR4 activation upon Salmonella infection of macrophages we analyzed small RNA deep sequencing data. Interestingly, we found that direct infection of macrophage cell lines with Salmonella does not result in an increase of edited mature miRNA. Thus, despite elevated levels of ADAR1 during TLR4 activation of macrophages mediated by Salmonella infection, ADAR1 does not result in redirection of miRNA. The most common consequence of ADAR activity on miRNA is a reduction in the mature miRNA level due to interference with miRNA processing of pri-miRNA. However, we found very few miRNAs with reductions in level, and no significant difference between miRNAs previously reported to be edited and those reported to be not edited. In particular, we did not see significant decrease in mir-22 and mir-142, nor editing of pri-mir-22 or pri-mir-142 in infected RAW macrophages. Thus, ADAR1 has very little, if any, effect on the miRNA machinery following TL4 activation by Salmonella infection.

Dynamic isomiR regulation in Drosophila development

Several recent reports have demonstrated that microRNAs (miRNAs) can exhibit heterogeneous ends and post-transcriptional nontemplate 3' end additions of uridines or adenosines. Using two small RNA deep-sequencing data sets, we show here that these miRNA isoforms (isomiRs) are differentially expressed across Drosophila melanogaster development and tissues. Specifically, we demonstrate that: (1) nontemplate nucleotide additions of adenosines to miRNA 3' ends are highly abundant in early development; (2) a subset of miRNAs with nontemplate 3' Us are expressed in adult tissues; and (3) the size of at least eight "mature" (unmodified) miRNAs varies in a life-cycle or tissue-specific manner. These results suggest that subtle variability in isomiR expression, which is widely thought to be the result of inexact Dicer processing, is regulated and biologically meaningful. Indeed, a subset of the miRNAs enriched for 3' adenosine additions during early embryonic development, including miR-282 and miR-312, show enrichment for target sites in developmental genes that are expressed during late embryogenesis, suggesting that nontemplate additions increase miRNA stability or strengthen miRNA:target interactions. This work suggests that isomiR expression is an important aspect of miRNA biology, which warrants further investigation.

Identification of Novel miRNAs and miRNA Dependent Developmental Shifts of Gene Expression in Arabidopsis thaliana

microRNAs (miRNAs) are small, endogenous RNAs of 20∼25 nucleotides, processed from stem-loop regions of longer RNA precursors. Plant miRNAs act as negative regulators of target mRNAs predominately by slicing target transcripts, and a number of miRNAs play important roles in development. We analyzed a number of published datasets from Arabidopsis thaliana to characterize novel miRNAs, novel miRNA targets, and miRNA-regulated developmental changes in gene expression. These data include microarray profiling data and small RNA (sRNA) deep sequencing data derived from miRNA biogenesis/transport mutants, microarray profiling data of mRNAs in a developmental series, and computational predictions of conserved genomic stem-loop structures. Our conservative analyses identified five novel mature miRNAs and seven miRNA targets, including one novel target gene. Two complementary miRNAs that target distinct mRNAs were encoded by one gene. We found that genes targeted by known miRNAs, and genes up-regulated or down-regulated in miRNA mutant inflorescences, are highly expressed in the wild type inflorescence. In addition, transcripts upregulated within the mutant inflorescences were abundant in wild type leaves and shoot meristems and low in pollen and seed. Downregulated transcripts were abundant in wild type pollen and seed and low in shoot meristems, roots and leaves. Thus, disrupting miRNA function causes the inflorescence transcriptome to resemble the leaf and meristem and to differ from pollen and seed. Applications of our computational approach to other species and the use of more liberal criteria than reported here will further expand the number of identified miRNAs and miRNA targets. Our findings suggest that miRNAs have a global role in promoting vegetative to reproductive transitions in A. thaliana.

Multiple distinct small RNAs originate from the same microRNA precursors

BackgroundMicroRNAs (miRNAs), which originate from precursor transcripts with stem-loop structures, are essential gene expression regulators in eukaryotes.ResultsWe report 19 miRNA precursors in Arabidopsis that can yield multiple distinct miRNA-like RNAs in addition to miRNAs and miRNA*s. These miRNA precursor-derived miRNA-like RNAs are often arranged in phase and form duplexes with an approximately two-nucleotide 3'-end overhang. Their production depends on the same biogenesis pathway as their sibling miRNAs and does not require RNA-dependent RNA polymerases or RNA polymerase IV. These miRNA-like RNAs are methylated, and many of them are associated with Argonaute proteins. Some of the miRNA-like RNAs are differentially expressed in response to bacterial challenges, and some are more abundant than the cognate miRNAs. Computational and expression analyses demonstrate that some of these miRNA-like RNAs are potentially functional and they target protein-coding genes for silencing. The function of some of these miRNA-like RNAs was further supported by their target cleavage products from the published small RNA degradome data. Our systematic examination of public small-RNA deep sequencing data from four additional plant species (Oryza sativa, Physcomitrella patens, Medicago truncatula and Populus trichocarpa) and four animals (Homo sapiens, Mus musculus, Caenorhabditis elegans and Drosophila) shows that such miRNA-like RNAs exist broadly in eukaryotes.ConclusionsWe demonstrate that multiple miRNAs could derive from miRNA precursors by sequential processing of Dicer or Dicer-like proteins. Our results suggest that the pool of miRNAs is larger than was previously recognized, and miRNA-mediated gene regulation may be broader and more complex than previously thought.