Identification of microRNAs in Macaca fascicularis (Cynomolgus Monkey) by Homology Search and Experimental Validation by Small RNA-Seq and RT-qPCR Using Kidney Cortex Tissues

MicroRNAs (miRNAs) present in tissues and biofluids are emerging as sensitive and specific safety biomarkers. MiRNAs have not been thoroughly described in M. fascicularis, an animal model used in pharmaceutical industry especially in drug safety evaluation. Here we investigated the miRNAs in M. fascicularis. For Macaca mulatta, a closely related species of M. fascicularis, 619 stem-loop precursor miRNAs (pre-miRNAs) and 914 mature miRNAs are available in miRBase version 21. Using M. mulatta miRNAs as a reference list and homology search tools, we identified 604 pre-miRNAs and 913 mature miRNAs in the genome of M. fascicularis. In order to validate the miRNAs identified by homology search we attempted to sequence miRNAs expressed in kidney cortex from M. fascicularis. MiRNAs expressed in kidney cortex may indeed be released in urine upon kidney cortex damage and be potentially used to monitor drug induced kidney injury. Hence small RNA sequencing libraries were prepared using kidney cortex tissues obtained from three naive M. fascicularis and sequenced. Analysis of sequencing data indicated that 432 out of 913 mature miRNAs were expressed in kidney cortex tissues. Assigning these 432 miRNAs to pre-miRNAs revealed that 273 were expressed from both the -5p and -3p arms of 150 pre-miRNAs and 159 miRNAs expressed from either the -5p or -3p arm of 176 pre-miRNAs. Mapping sequencing reads to pre-miRNAs also facilitated the detection of twenty-two new miRNAs. To substantiate miRNAs identified by small RNA sequencing, 313 miRNAs were examined by RT-qPCR. Expression of 262 miRNAs in kidney cortex tissues ware confirmed by TaqMan microRNA RT-qPCR assays. Analysis of kidney cortex miRNA targeted genes suggested that they play important role in kidney development and function. Data presented in this study may serve as a valuable resource to assess the renal safety biomarker potential of miRNAs in Cynomolgus monkeys.     

Identification and comparative analysis of the miRNA expression profiles from four tissues of Micropterus salmoides using deep sequencing

In the present study, four small RNA libraries were constructed from an M. salmoides population and sequenced using deep sequencing technology. A total of 9,888,822; 8,519,365; 20,566,198; and 15,762,254 raw reads representing 666,097; 755,711; 978,923; and 840,175 unique sequences were obtained from the spleen, liver, kidney, and muscle libraries, respectively. As a result, 509 known miRNAs belonging to 143 families and 1157 novel miRNAs were identified. The miRNAs displayed diverse expression levels among the four libraries, among which most of the known miRNAs were expressed at higher levels than the novel miRNAs. Furthermore, stem-loop qRT-PCR was applied to validate and profile the expression of the differentially expressed miRNAs in the four different tissues, which revealed that some miRNAs showed tissue specific expression. The identification of miRNAs in M. salmoides will provide new information and enhance our understanding of the functions of miRNAs in regulating biological processes.     

Combined Small RNA and Degradome Sequencing Reveals Novel MiRNAs and Their Targets in the High-Yield Mutant Wheat Strain Yunong 3114

Wheat is one of the main food sources worldwide; large amount studies have been conducted to improve wheat production. MicroRNAs (miRNAs) with about 20–30 nucleotide are a class of regulatory small RNAs (sRNAs), which could regulate gene expression through sequence-specific base pairing with target mRNAs, playing important roles in plant growth. An ideal plant architecture (IPA) is crucial to enhance yield in bread wheat. In this study, the high-yield wheat strain Yunong 3114 was EMS-mutagenesis from the wild-type strain Yunong 201, exhibiting a preferable plant structure compared with the wild-type strain. We constructed small RNA and degradome libraries from Yunong 201 and Yunong 3114, and performed small RNA sequencing of these libraries in order identify miRNAs and their targets related to IPA in wheat. Totally, we identified 488 known and 837 novel miRNAs from Yunong 3114 and 391 known and 533 novel miRNAs from Yunong 201. The number of miRNAs in the mutant increased. A total of 37 known and 432 putative novel miRNAs were specifically expressed in the mutant strain; furthermore, 23 known and 159 putative novel miRNAs were specifically expressed in the wild-type strain. A total of 150 known and 100 novel miRNAs were differentially expressed between mutant and wild-type strains. Among these differentially expressed novel miRNAs, 4 and 8 predict novel miRNAs were evidenced by degradome sequencing and showed up-regulated and down-regulated expressions in the mutant strain Yunong 3114, respectively. Targeted gene annotation and previous results indicated that this set of miRNAs is related to plant structure. Our results further suggested that miRNAs may be necessary to obtain an optimal wheat structure.     

Identification of microRNAs from Plutella xylostella larvae associated with parasitization by Diadegma semiclausum

MicroRNAs (miRNAs) as small non-coding RNAs play important roles in many biological processes such as development, cell signaling and immune response. Small RNA deep sequencing technology provided an opportunity for a thorough survey of miRNAs in a global key pest Plutella xylostella as well as comparative analysis of miRNA expression profile of the insect in association with parasitization by Diadegma semiclausum. Combining the deep sequencing data and bioinformatics, 235 miRNAs were identified from P. xylostella. Differential expression of host cellular miRNAs in response to parasitism was examined by making small RNA libraries from parasitized and naive second instar larvae of P. xylostella. Bantam, miR-276*, miR-10, miR-31 and miR-184 were detected as five most abundant miRNAs in both libraries and 96 miRNAs were identified that were differentially expressed after parasitization. Bantam*, miR-184 and miR-281* were significantly down-regulated and two miRNAs miR-279b and miR-2944b* were highly induced in parasitized larvae. Interestingly, high copy numbers and differential expression of several miRNA passenger strands (miRNA*) suggest their potential roles in host-parasitoid interaction. In conclusion, expression profiling of miRNAs provided insights into their possible involvement in insect immune response to parasitism and offer an important resource for further studies.     

A deeply conserved,noncanonical miRNA hosted by ribosomal DNA

Advances in small RNA sequencing technologies and comparative genomics have fueled comprehensive microRNA (miRNA) gene annotations in humans and model organisms. Although new miRNAs continue to be discovered in recent years, these have universally been lowly expressed, recently evolved, and of debatable endogenous activity, leading to the general assumption that virtually all biologically important miRNAs have been identified. Here, we analyzed small RNAs that emanate from the highly repetitive rDNA arrays of Drosophila. In addition to endo-siRNAs derived from sense and antisense strands of the pre-rRNA sequence, we unexpectedly identified a novel, deeply conserved, noncanonical miRNA. Although this miRNA is widely expressed, this miRNA was not identified by previous studies due to bioinformatics filters removing such repetitive sequences. Deep-sequencing data provide clear evidence for specific processing with precisely defined 5′ and 3′ ends. Furthermore, we demonstrate that the mature miRNA species is incorporated in the effector complexes and has detectable trans regulatory activity. Processing of this miRNA requires Dicer-1, whereas the Drosha–Pasha complex is dispensable. The miRNA hairpin sequence is located in the internal transcribed spacer 1 region of rDNA and is highly conserved among Dipteran species that were separated from their common ancestor ∼100 million years ago. Our results suggest that biologically active miRNA genes may remain unidentified even in well-studied organisms.     

Identification of microRNAs involved in drought stress responses in early-maturing cotton by high-throughput sequencing

Drought stress is one of the most important abiotic stresses. Cotton is classified as drought tolerant crop but the regulatory mechanism is unknown. MicroRNAs (miRNAs) have been implicated important roles in stress responses in many plants. However, the study of miRNAs in cotton responsive to drought stress is limited, especially in early-maturing cotton. In this study, we performed deep sequencing of small RNAs to identify known and novel miRNAs involved in the regulation of drought stress and understand the expression profile of miRNAs in early-maturing cotton. Three cotton small RNA libraries: non-stressed Shizao1 (early-maturing cotton variety) library (NSS), drought-stressed Shizao1 library (DSS) and non-stressed Jimian958 (medium-maturing cotton variety) library (NSJ) were constructed for deep sequencing. As a result, we identified a total of 64 known and 67 novel miRNAs in the 3 libraries and 88 of them were dramatically differentially expressed (greater than twofold) during drought stress. In addition, we found the expression of 41 miRNAs increased or reduced more than twofold in early-maturing cotton variety compared with that in medium-maturing cotton variety. Our results significantly increased the number of miRNAs in cotton and revealed for the first time the expression profile of miRNAs for early-maturing cotton.     

Tracking miRNA precursor metabolic products and processing sites through completely analyzing high-throughput sequencing data

The small non-coding important regulatory molecules, microRNAs (miRNAs), have been widely and deeply studied especially combining high-throughput sequencing technologies. Here, we attempted to track detailed miRNA precursor metabolic products and gain further insight into pre-miRNA processing by completely analyzing high-throughput sequencing data. Highly expressed miRNA precursors could be entirely covered by various short RNAs and small RNA fragments with a hierarchical distribution. miRNAs and some miRNA* regions were detected quite abundant short RNAs as expected, while other regions of precursors were found shorter RNAs or small fragments with fewer sequence counts. Furthermore, we developed a method to analyze relative expression levels of special RNA classes according to divergence of 5′ and 3′ ends, respectively. Generally, there were several quite abundant RNA classes from a given miRNA locus, which suggested dominant cleavage sites of Drosha and Dicer during pre-miRNA processing. Compared with 3′ end, dominant cleavage site in 5′ end always focused on a specific position, which ensured conservation of the identity of miRNA (5′-seed sequence, nucleotides 2–8). Overall, a comprehensive analysis of sequencing data can be used to track pre-miRNA metabolic products and mechanism of pre-miRNA processing and metabolism.     

An expression atlas of miRNAs in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. MiRNA expression often exhibits spatial and temporal specificity. However, genome-wide miRNA expression patterns in different organs during development of Arabidopsis thaliana have not yet been systemically investigated. In this study, we sequenced small RNA libraries generated from 27 different organ/tissue types, which cover the entire life cycle of Arabidopsis. Analysis of the sequencing data revealed that most miRNAs are ubiquitously expressed, whereas a small set of miRNAs display highly specific expression patterns. In addition, different miRNA members within the same family have distinct spatial and temporal expression patterns. Moreover, we found that some miRNAs are produced from different arms of their hairpin precursors at different developmental stages. This work provides new insights into the regulation of miRNA biogenesis and a rich resource for future investigation of miRNA functions in Arabidopsis.     

Small RNA and Degradome Sequencing Reveal Complex Roles of miRNAs and Their Targets in Developing Wheat Grains

Plant microRNAs (miRNAs) have been shown to play critical roles in plant development. In this study, we employed small RNA combined with degradome sequencing to survey development-related miRNAs and their validated targets during wheat grain development. A total of 186 known miRNAs and 37 novel miRNAs were identified in four small RNA libraries. Moreover, a miRNA-like long hairpin locus was first identified to produce 21~22-nt phased siRNAs that act in trans to cleave target mRNAs. A comparison of the miRNAomes revealed that 55 miRNA families were differentially expressed during the grain development. Predicted and validated targets of these development-related miRNAs are involved in different cellular responses and metabolic processes including cell proliferation, auxin signaling, nutrient metabolism and gene expression. This study provides insight into the complex roles of miRNAs and their targets in regulating wheat grain development.     

An expression atlas of miRNAs in Arabidopsis thaliana

MicroRNAs(miRNAs) are small non-coding RNAs that regulate a variety of biological processes. miRNA expression often exhibits spatial and temporal specificity. However, genome-wide miRNA expression patterns in different organs during development of Arabidopsis thaliana have not yet been systemically investigated. In this study, we sequenced small RNA libraries generated from 27 different organ/tissue types, which cover the entire life cycle of Arabidopsis. Analysis of the sequencing data revealed that most miRNAs are ubiquitously expressed, whereas a small set of miRNAs display highly specific expression patterns. In addition, different miRNA members within the same family have distinct spatial and temporal expression patterns. Moreover, we found that some miRNAs are produced from different arms of their hairpin precursors at different developmental stages. This work provides new insights into the regulation of miRNA biogenesis and a rich resource for future investigation of miRNA functions in Arabidopsis.