Sequencing Of Small RNA Libraries Research Articles

AthisomiRDB: A comprehensive database of Arabidopsis isomiRs.

Several pieces of evidence challenge the traditional view of miRNAs as static molecules, revealing dynamic isomiRs originating from each miRNA precursor arm. In plants, isomiRs, which result from imprecise cleavage during pre-miRNA processing and post-transcriptional alterations, serve as crucial regulators of target microRNAs (miRNAs). Despite numerous studies on Arabidopsis miRNAs, the systematic identification and annotation of isomiRs across various tissues and conditions remain limited. Due to the lack of systematically collected isomiR information, we introduce the athisomiRDB database, which houses 20 764 isomiRs from Arabidopsis small RNA-sequencing (sRNA-seq) libraries. It comprises >2700 diverse samples and allows exploration at the sample, miRNA, or isomiR levels, offering insights into the presence or absence of isomiRs. The athisomiRDB includes exclusive and ambiguous isomiRs, each with features such as transcriptional origin, variant-containing isomiRs, and identifiers for frequent single-nucleotide polymorphisms from the 1001 Genomes Project. It also provides 3' nontemplated post-transcriptional additions, isomiR-target interactions, and trait associations for each isomiR. We anticipate that athisomiRDB will play a pivotal role in unraveling the regulatory nature of the Arabidopsis miRNAome and enhancing sRNA research by leveraging isomiR profiles from extensive sRNA-seq datasets. Database URL: https://www.nipgr.ac.in/athisomiRDB.

Profiles of differential expression of miRNAs in the late stage of red blood cell preservation and their potential roles

ObjectiveTo detect the differentially expressed regulatory miRNAs in the late stage of red blood cell (RBC) preservation and predict their roles. MethodsSuspended RBCs with different storage periods of 35 day, 42 day, and 50 day were collected for routine blood tests, RNA extraction, and preparation of small RNA sequencing libraries. The constructed libraries were sequenced and the biological functions of differential miRNAs in RBCs in the late storage were analyzed by bioinformatics. ResultsRoutine indicators of RBCs in the late stage were not significantly affected by preservation time. The Pearson correlation analysis performing on RBC miRNAs with different storage days revealed that RBC miRNAs changed with the increase of storage days. RBC miRNAs from day 35 (D35), day 42 (D42) and day 50 (D50) showed significant differences (P < 0.05). Compared RBC miRNAs from D42 with these from D35, there were 690 up-regulated miRNAs and 82 down-regulated miRNAs; compared RBC miRNAs from D50 with these from D35, there were 638 up-regulated miRNAs and 123 down-regulated miRNAs; compared RBC miRNAs from D42 with these from D50, there were 271 up-regulated miRNAs and 515 down-regulated miRNAs. GO enrichment analysis of target genes of differential miRNAs were mainly involved in cell metabolism, biosynthesis, protein modification, gene expression and transcriptional regulation of biological processes. KEGG pathway enrichment analysis of miRNA target genes showed that differential miRNA target genes were closely related to pathways in cancer. ConclusionMiRNAs were differentially expressed in the late stage of RBC preservation, and may be involved in various biological processes, especially cancer.

Comparison of colostrum and milk extracellular vesicles small RNA cargo in water buffalo

Recently, much interest has been raised for the characterization of signaling molecules carried by extracellular vesicles (EVs), which are particularly enriched in milk (mEVs). Such interest is linked to the capability of EVs to cross biological barriers, resist acidification in the gastric environment, and exert modulation of the immune system, mainly through their microRNA (miRNA) content. We characterized the small-RNA cargo of colostrum EVs (colosEVs) and mEVs from Italian Mediterranean buffalo through next generation sequencing. Colostrum (first milking after birth) and milk (day 50 of lactation) were sampled from seven subjects from five farms. ColosEVs and mEVs were subjected to morphological characterization, followed by high-depth sequencing of small RNA libraries produced from total RNA. The main difference was the amount of EV in the two samples, with colostrum showing 10 to 100-fold higher content than milk. For both matrices, miRNA was the most abundant RNA species (95% for colosEVs and 96% for mEVs) and three lists were identified: colosEV-specific, mEV-specific and shared most expressed. Gene ontology (GO) enrichment analysis on miRNA targets highlighted many terms related to the epigenetic, transcriptional and translational regulations across the three lists, with a higher number of enriched terms for colosEV-specific miRNAs. Terms specific to colosEVs were related to “cell differentiation” and “microvillus assembly”, while for mEV “cardiac and blood vessel development” and “mitochondria” emergerd. Immune modulation terms were found for both sample-specific miRNAs. Overall, both matrices carry a similar molecular message in terms of biological processes potentially modulated into receiving cells, but there is significant difference in the abundance, with colostrum containing much more EVs than milk. Moreover, colosEVs carry molecules involved in signal transduction, cell cycle and immune response, as for mEVs and EVs of other previously characterized species, but with a special enrichment for miRNAs with epigenetic regulation capacities. These beneficial characteristics of colosEVs and mEVs are essential for the calf and could also be exploited for the therapeutic purposes in humans, although further studies are necessary to measure the sanitization treatment impact on EV conservation, especially in buffalo where milk is consumed almost exclusively after processing.

Analysis of Amblyomma americanum microRNAs in response to Ehrlichia chaffeensis infection and their potential role in vectorial capacity.

MicroRNAs (miRNAs) represent a subset of small noncoding RNAs and carry tremendous potential for regulating gene expression at the post-transcriptional level. They play pivotal roles in distinct cellular mechanisms including inhibition of bacterial, parasitic, and viral infections via immune response pathways. Intriguingly, pathogens have developed strategies to manipulate the host's miRNA profile, fostering environments conducive to successful infection. Therefore, changes in an arthropod host's miRNA profile in response to pathogen invasion could be critical in understanding host-pathogen dynamics. Additionally, this area of study could provide insights into discovering new targets for disease control and prevention. The main objective of the present study is to investigate the functional role of differentially expressed miRNAs upon Ehrlichia chaffeensis, a tick-borne pathogen, infection in tick vector, Amblyomma americanum. Small RNA libraries from uninfected and E. chaffeensis-infected Am. americanum midgut and salivary gland tissues were prepared using the Illumina Truseq kit. Small RNA sequencing data was analyzed using miRDeep2 and sRNAtoolbox to identify novel and known miRNAs. The differentially expressed miRNAs were validated using a quantitative PCR assay. Furthermore, a miRNA inhibitor approach was used to determine the functional role of selected miRNA candidates. The sequencing of small RNA libraries generated >147 million raw reads in all four libraries and identified a total of >250 miRNAs across the four libraries. We identified 23 and 14 differentially expressed miRNAs in salivary glands, and midgut tissues infected with E. chaffeensis, respectively. Three differentially expressed miRNAs (miR-87, miR-750, and miR-275) were further characterized to determine their roles in pathogen infection. Inhibition of target miRNAs significantly decreased the E. chaffeensis load in tick tissues, which warrants more in-depth mechanistic studies. The current study identified known and novel miRNAs and suggests that interfering with these miRNAs may impact the vectorial capacity of ticks to harbor Ehrlichia. This study identified several new miRNAs for future analysis of their functions in tick biology and tick-pathogen interaction studies.

First report of apple hammerhead viroid infecting apple trees in Montenegro.

Apple hammerhead viroid (AHVd, Pelamoviroid, Avsunviroidae) is one of the five viroids infecting apples. It has been identified on all continents except Australia since its viroid nature was confirmed (DiSerio et al. 2018; CABI and EPPO 2022). AHVd has been found in apple trees showing leaf mosaic, ringspot and dieback (Hamdi et al., 2021). Apple (Malus domestica Borkh.) and its wild relatives are traditionally grown in Montenegro. With an annual production of 7767 tons on 216 ha, it is the second most important fruit tree (after plum) in the country (Anonymous 2022). In a 2020-2022 survey, 29 apple trees exhibiting virus-like symptoms (e.g. mosaic, necrosis) were sampled throughout Montenegro, including 16 locations in eight municipalities (Podgorica, Danilovgrad, Niksic, Mojkovac, Bijelo Polje, Berane, Pljevlja and Savnik). Small RNAs were isolated using the mirVana miRNA Isolation Kit (Ambion, Life Technologies) and pooled into three bulk samples. Each bulk contained 9 to 10 samples. Libraries of sRNAs were constructed using the Ion Total RNA-Seq Kit v2 and barcoded using the Xpress RNA-Seq Barcode 1-16 Kit (Ion Torrent) according to the manufacturer's instructions. Small RNA library sequencing was performed on Illumina platform (Novogene Europe) yielding 9.9, 9.8 and 18.6 million reads in the three libraries. The CLC Genomics Workbench software was used to demultiplex the reads into pools using the 'Demultiplex Reads' tool. The online program VirusDetect (Zheng et al. 2017) was used for virus/viroid detection and identification. Besides viruses known to infect apple (apple stem grooving virus, apple stem pitting virus, apple mosaic virus), contigs mapping to AHVd were identified in all three bulks enabling full AHVd genomes reconstruction. To verify AHVd presence, all 29 apple samples were tested by reverse transcription-polymerase chain reaction (RT-PCR) using the AHVd PG13f/PG12r primers (Messmer et al. 2017). AHVd amplicons were obtained in three samples (30/21, 32/21 and 38/21) from bulk 1 and two samples (47/21 and 55/21) from bulk 2, while all samples from bulk 3 tested negative potentially due to the low titer of the pathogen or nucleotide mismatches at the 3' end of the primers. The three amplicons from bulk 1 were Sanger sequenced and partial AHVd genomes over 200 nts were obtained from two of them (30/21 and 32/21) (GenBank acc. nos. OQ863319 and OR020603). Furthermore, three full consensus AHVd genomes were assembled in Geneious Prime by mapping Sanger sequences onto contigs from Virus Detect and named 30/21, 32/21 and 38/21 (acc. nos. PP133245, -46, and -47, respectively). All three genomes exhibited conserved hammerhead motifs (Messmer et al. 2017). In BLASTn analysis, the isolate 30/21 from Montenegro shared the highest nt identity (98.8%) with the isolate SA-36 (ON564299) from Czechia, while 32/21 and 38/21 showed the highest identities (95.4% and 92.3%) with isolates SD17_2-3 (MK188691) from Canada and JF2 (ON564298) from Czechia, respectively. To the best of our knowledge, this is the first report of AHVd infecting Malus domestica in Montenegro. The AHVd-positive samples 30/21 and 32/21 originated from at least two-decade-old apple trees from Niksic, whilst 38/21 came from a 40-year-old tree from Mojkovac district, suggesting that this viroid has long been present in different parts of the country. The AHVd discovery in Montenegro should be considered in any phytosanitary regulations and pome fruit certification program in the country.

MiRNome Profiling Analysis Reveals Novel Hepatocellular Carcinoma Diagnostic, Prognostic and Treatment-Related Candidate Biomarkers: Post hoc Analysis of SORAMIC Trial

Introduction: Early diagnosis of hepatocellular carcinoma (HCC) as well as evaluation of prognosis and prediction of treatment efficacy remains challenging due to the missing specific non-invasive biomarkers. The aim of this study was to identify disease-specific microRNA (miRNA) patterns for diagnosis, prediction of prognosis, and treatment response in patients with HCC. Methods: The study population included 42 HCC patients from SORAMIC clinical trial: 22 patients received sorafenib monotherapy, 20 patients underwent 90Y radioembolization in combination with sorafenib. 20 individuals were included in the control group. HCC patients underwent collection of plasma samples before and 7–9 weeks after the beginning of the treatment. Isolation of circulating miRNAs, preparation of small RNA sequencing libraries and next-generation sequencing were performed. Association analysis for novel diagnostic, prognostic, and treatment-related candidate biomarkers was performed. Results: A total of 42 differentially expressed (16 up-regulated and 26 down-regulated) miRNAs were identified comparing baseline and control group plasma samples. hsa-miR-215-5p and hsa-miR-192-5p were down-regulated, while hsa-miR-483-5p and hsa-miR-23b-3p were up-regulated comparing baseline and 7–9 weeks post-sorafenib monotherapy samples. hsa-miR-215-5p was the sole down-regulated miRNA in the same combination therapy comparison. hsa-miR-183-5p, hsa-miR-28-3p, and hsa-miR-1246 were found to be significantly up-regulated comparing non-responders versus responders to sorafenib. High hsa-miR-215-5p expression was significantly associated with worse HCC patients’ prognosis. Conclusions: Systematic miRNA profiling of highly characterized samples from SORAMIC study revealed a subset of potential miRNA biomarkers for HCC diagnosis and prognosis of sorafenib-treated patients’ survival.

Small RNA-Seq to Unveil the miRNA Expression Patterns and Identify the Target Genes in Panax ginseng.

Panax ginseng, renowned for its medicinal properties, relies on adventitious roots and hairy roots as crucial sources for the production of ginsenosides. Despite the widespread utilization of ginseng, investigations into its miRNAs have remained scarce. To address this gap, two samples of ginseng adventitious roots and ginseng hairy roots were collected, and subsequent construction and sequencing of small RNA libraries of ginseng adventitious roots and hairy roots were performed using the Illumina HiSeq X Ten platform. The analysis of the sequencing data unveiled total miRNAs 2432. The miR166 and miR396 were the most highly expressed miRNA families in ginseng. The miRNA expression analysis results were used to validate the qRT-PCR. Target genes of miRNA were predicted and GO function annotation and KEGG pathway analysis were performed on target genes. It was found that miRNAs are mainly involved in synthetic pathways and biological processes in plants, which include metabolic and bioregulatory processes. The plant miRNAs enriched KEGG pathways are associated with some metabolism, especially amino acid metabolism and carbohydrate metabolism. These results provide valuable insights miRNAs and their roles in metabolic processes in ginseng.

Differentially Expressed Conserved Plant Vegetative Phase-Change-Related microRNAs in Mature and Rejuvenated Silver Birch In Vitro Propagated Tissues.

In plants, phase change from the juvenile stage to maturity involves physiological and anatomical changes, which are initiated and controlled by evolutionary highly conserved microRNAs. This process is of particular significance for the in vitro propagation of woody plant species, as individuals or tissues that have undergone the transition to vegetative maturity are recalcitrant to propagation. Conserved miRNAs differentially expressed between juvenile (including rejuvenated) and mature silver birch tissues were identified using high-throughput sequencing of small RNA libraries. Expression of some miR156 isoforms was high in juvenile tissues and has been previously reported to regulate phase transitions in a range of species. Additional miRNAs, such as miR394 and miR396, that were previously reported to be highly expressed in juvenile woody plant tissues were also differentially expressed in this study. However, expression of miR172, previously reported to be highly expressed in mature tissues, was low in all sample types in this study. The obtained results will provide insight for further investigation of the molecular mechanisms regulating vegetative phase change in silver birch and other perennial woody plant species, by analysing a wider range of genotypes, tissue types and maturation stages. This knowledge can potentially assist in identification of rejuvenated material at an earlier stage than currently possible, increasing the efficiency of silver birch in vitro propagation.

Characterization of microRNAs and Target Genes in Musa acuminata subsp. burmannicoides, var. Calcutta 4 during Interaction with Pseudocercospora musae

Endogenous microRNAs (miRNAs) are small non-coding RNAs that perform post-transcriptional regulatory roles across diverse cellular processes, including defence responses to biotic stresses. Pseudocercospora musae, the causal agent of Sigatoka leaf spot disease in banana (Musa spp.), is an important fungal pathogen of the plant. Illumina HiSeq 2500 sequencing of small RNA libraries derived from leaf material in Musa acuminata subsp. burmannicoides, var. Calcutta 4 (resistant) after inoculation with fungal conidiospores and equivalent non-inoculated controls revealed 202 conserved miRNAs from 30 miR-families together with 24 predicted novel miRNAs. Conserved members included those from families miRNA156, miRNA166, miRNA171, miRNA396, miRNA167, miRNA172, miRNA160, miRNA164, miRNA168, miRNA159, miRNA169, miRNA393, miRNA535, miRNA482, miRNA2118, and miRNA397, all known to be involved in plant immune responses. Gene ontology (GO) analysis of gene targets indicated molecular activity terms related to defence responses that included nucleotide binding, oxidoreductase activity, and protein kinase activity. Biological process terms associated with defence included response to hormone and response to oxidative stress. DNA binding and transcription factor activity also indicated the involvement of miRNA target genes in the regulation of gene expression during defence responses. sRNA-seq expression data for miRNAs and RNAseq data for target genes were validated using stem-loop quantitative real-time PCR (qRT-PCR). For the 11 conserved miRNAs selected based on family abundance and known involvement in plant defence responses, the data revealed a frequent negative correlation of expression between miRNAs and target host genes. This examination provides novel information on miRNA-mediated host defence responses, applicable in genetic engineering for the control of Sigatoka leaf spot disease.

MiRador: a fast and precise tool for the prediction of plant miRNAs.

Plant microRNAs (miRNAs) are short, noncoding RNA molecules that restrict gene expression via posttranscriptional regulation and function in several essential pathways, including development, growth, and stress responses. Accurately identifying miRNAs in populations of small RNA sequencing libraries is a computationally intensive process that has resulted in the misidentification of inaccurately annotated miRNA sequences. In recent years, criteria for miRNA annotation have been refined with the aim to reduce these misannotations. Here, we describe miRador, a miRNA identification tool that utilizes the most up-to-date, community-established criteria for accurate identification of miRNAs in plants. We combined target prediction and Parallel Analysis of RNA Ends (PARE) data to assess the precision of the miRNAs identified by miRador. We compared miRador to other commonly used miRNA prediction tools and found that miRador is at least as precise as other prediction tools while being substantially faster than other tools. miRador should be broadly useful for the plant community to identify and annotate miRNAs in plant genomes.

RNA Sequencing Unveils Very Small RNAs With Potential Regulatory Functions in Bacteria.

RNA sequencing (RNA-seq) is the gold standard for the discovery of small non-coding RNAs. Following a long-standing approach, reads shorter than 16 nucleotides (nt) are removed from the small RNA sequencing libraries or datasets. The serendipitous discovery of an eukaryotic 12 nt-long RNA species capable of modulating the microRNA from which they derive prompted us to challenge this dogma and, by expanding the window of RNA sizes down to 8 nt, to confirm the existence of functional very small RNAs (vsRNAs <16 nt). Here we report the detailed profiling of vsRNAs in Escherichia coli, E. coli-derived outer membrane vesicles (OMVs) and five other bacterial strains (Pseudomonas aeruginosa PA7, P. aeruginosa PAO1, Salmonella enterica serovar Typhimurium 14028S, Legionella pneumophila JR32 Philadelphia-1 and Staphylococcus aureus HG001). vsRNAs of 8–15 nt in length [RNAs (8-15 nt)] were found to be more abundant than RNAs of 16–30 nt in length [RNAs (16–30 nt)]. vsRNA biotypes were distinct and varied within and across bacterial species and accounted for one third of reads identified in the 8–30 nt window. The tRNA-derived fragments (tRFs) have appeared as a major biotype among the vsRNAs, notably Ile-tRF and Ala-tRF, and were selectively loaded in OMVs. tRF-derived vsRNAs appear to be thermodynamically stable with at least 2 G-C basepairs and stem-loop structure. The analyzed tRF-derived vsRNAs are predicted to target several human host mRNAs with diverse functions. Bacterial vsRNAs and OMV-derived vsRNAs could be novel players likely modulating the intricate relationship between pathogens and their hosts.

Atrophic gastritis and gastric cancer tissue miRNome analysis reveals hsa-miR-129-1 and hsa-miR-196a as potential early diagnostic biomarkers.

BACKGROUNDGastric cancer (GC) is one of the most frequently diagnosed tumor globally. In most cases, GC develops in a stepwise manner from chronic gastritis or atrophic gastritis (AG) to cancer. One of the major issues in clinical settings of GC is diagnosis at advanced disease stages resulting in poor prognosis. MicroRNAs (miRNAs) are small noncoding molecules that play an essential role in a variety of fundamental biological processes. However, clinical potential of miRNA profiling in the gastric cancerogenesis, especially in premalignant GC cases, remains unclear.AIMTo evaluate the AG and GC tissue miRNomes and identify specific miRNAs’ potential for clinical applications (e.g., non-invasive diagnostics).METHODSStudy included a total of 125 subjects: Controls (CON), AG, and GC patients. All study subjects were recruited at the Departments of Surgery or Gastroenterology, Hospital of Lithuanian University of Health Sciences and divided into the profiling (n = 60) and validation (n = 65) cohorts. Total RNA isolated from tissue samples was used for preparation of small RNA sequencing libraries and profiled using next-generation sequencing (NGS). Based on NGS data, deregulated miRNAs hsa-miR-129-1-3p and hsa-miR-196a-5p were analyzed in plasma samples of independent cohort consisting of CON, AG, and GC patients. Expression level of hsa-miR-129-1-3p and hsa-miR-196a-5p was determined using the quantitative real-time polymerase chain reaction and 2-ΔΔCt method.RESULTSResults of tissue analysis revealed 20 differentially expressed miRNAs in AG group compared to CON group, 129 deregulated miRNAs in GC compared to CON, and 99 altered miRNAs comparing GC and AG groups. Only 2 miRNAs (hsa-miR-129-1-3p and hsa-miR-196a-5p) were identified to be step-wise deregulated in healthy-premalignant-malignant sequence. Area under the curve (AUC)-receiver operating characteristic analysis revealed that expression level of hsa-miR-196a-5p is significant for discrimination of CON vs AG, CON vs GC and AG vs GC and resulted in AUCs: 88.0%, 93.1% and 66.3%, respectively. Compar-ing results in tissue and plasma samples, hsa-miR-129-1-3p was significantly down-regulated in GC compared to AG (P = 0.0021 and P = 0.024, tissue and plasma, respectively). Moreover, analysis revealed that hsa-miR-215-3p/5p and hsa-miR-934 were significantly deregulated in GC based on Helicobacter pylori (H. pylori) infection status [log2 fold change (FC) = -4.52, P-adjusted = 0.02; log2FC = -4.00, P-adjusted = 0.02; log2FC = 6.09, P-adjusted = 0.02, respectively].CONCLUSIONComprehensive miRNome study provides evidence for gradual deregulation of hsa-miR-196a-5p and hsa-miR-129-1-3p in gastric carcinogenesis and found hsa-miR-215-3p/5p and hsa-miR-934 to be significantly deregulated in H. pylori carrying GC patients.

P036 MicroRNA composition of colon crypt-top and crypt-bottom epithelial cells in Ulcerative Colitis

Abstract Background Ulcerative colitis (UC) is a chronic relapsing large intestine condition of inflammatory origin1. One of the most common features of UC is the injury of the intestinal barrier, which is composed primarily of colonic epithelial cells (CEpCs)2. The aim of this study was to identify UC-induced miRNA markers in CEpCs by determining the miRNA expression profile changes in crypt-top and crypt-bottom CEpC populations during active (aUC) and inactive (iUC) UC. Methods Crypt-top and crypt-bottom CEpCs were sorted from biopsies of healthy control (HC) individuals (n=19), patients with aUC (n=17) and iUC (n=15) using FACS technology. Total RNA was extracted, small RNA sequencing libraries were prepared and sequenced using Illumina platform. Sequencing data was processed with nextflow-core/smrnaseq pipeline. Differential expression, correlation, miRNA-target interactions, gene set enrichment analyses and data visualisation were performed using Rstudio software packages DESeq2, isomiRs, multimiR, SingleCellExperiment, clusterProfiler, ReactomePA, etc. The miRNAs with an adjusted p-value &amp;lt; 0.05, and absolute value of log2 fold change &amp;gt; 1 were considered to be significantly differentially expressed. Results 432 unique miRNAs were identified in samples. Changes of expression profile during aUC were identified in crypt-bottom CEpCs (compared to: (i) HC - 23 miRNAs, (ii) iUC - 22 miRNAs), as well as in crypt-top CEpCs (compared to: (i) HC - 28 miRNAs, (ii) iUC - 9 miRNAs). Also, 7 miRNAs were differentially expressed in crypt-bottom CEpCs and 3 miRNAs in crypt-top CEpCs during iUC compared to HC. 5 miRNAs were identified to be differentially expressed during aUC, 2 miRNAs - during iUC, and 11 miRNAs in HC when comparing expression profiles of crypt-bottom and crypt-bottom CEpCs. We also identified 16 and 14 miRNAs which expression in crypt-bottom and crypt-top CEpCs moderately (0.5&amp;lt;rho&amp;lt;0.7) correlated with Mayo score, respectively. Finally, the gene sets of pathways revealed the involvement of several miRNAs in biological processes and molecular functions associated with UC pathogenesis. Conclusion The changes of expression profiles of miRNAs revealed that crypt-top and crypt-bottom CEpCs respond to inflammation differently, the expression of these miRNAs reflects disease activity and modulates the processes of UC pathogenesis. References

Integrated Analysis of Small RNA, Transcriptome, and Degradome Sequencing Reveals the MiR156, MiR5488 and MiR399 are Involved in the Regulation of Male Sterility in PTGMS Rice.

A photoperiod- and thermo-sensitive genic male sterile (PTGMS) line is the basic material for two-hybrid rice and is an important genetic breeding resource. Peiai64S (PA64S) is an important germplasm resource of PTGMS rice, and it has been applied to two-line hybrid rice systems in China. Pollen fertility in PA64S is regulated by the temperature and photoperiod, but the mechanism of the fertility transition is unclear. In this study, we obtained the male fertile plant PA64S(F) and the male sterile plant PA64S(S) by controlling different temperatures under long light conditions and used the male fertile and sterile plants to investigate the role of microRNAs (miRNAs) in regulating male fertility in rice. We performed the small RNA library sequencing of anthers from PA64S(S) and PA64S(F). A total of 196 miRNAs were identified—166 known miRNAs among 27 miRNA families and 30 novel miRNAs. In the transcriptome analysis, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes revealed significant enrichment in the synthesis and metabolism of fatty acids and some secondary metabolism pathways such as fatty acid metabolism and phenylalanine metabolism. With a comprehensive analysis of miRNA, transcriptome, and degradome sequencing, we identified that 13 pairs of miRNA/target genes regulated male fertility in rice by responding to temperature change, among which the miR156, miR5488, and miR399 affect the male fertility of PA64S by influencing SPLs, the lignin synthesis of anther walls, and the flavonoid metabolism pathway. The results provide a new understanding of PTGMS rice, which will help us better understand the potential regulatory mechanisms of male sterility in the future.

MiR-193b-5p protects BRL-3A cells from acrylamide-induced cell cycle arrest by targeting FoxO3

Acrylamide (AA), an important by-product of the Maillard reaction, has been reported to be genotoxic and carcinogenic. The present study employed miRNAs to investigate the toxic mechanism of AA and their role against AA toxicity. Deep sequencing of small RNA libraries was performed and miR-193b-5p was applied for further study. AA significantly reduced the level of miR-193b-5p and its ectopic expression promoted cell cycle G1/S transition and cell proliferation by upregulating the cyclin-dependent kinase regulator Cyclin D1 and downregulating the cyclin-dependent kinase inhibitor p21, while miR-193b-5p inhibitor led to the opposite results. Dual luciferase assay demonstrated miR-193b-5p regulated the expression of FoxO3 by directly targeting the FoxO3 3′-untranslated region (3′-UTR). Knockdown of FoxO3 induced cell cycle G1/S transition and cell proliferation, which was suppressed by the inhibition of miR-193b-5p but promoted by miR-193b-5p mimics. MiR-193b-5p inhibitor strengthened the effect of FoxO3, contrary to the effect of miR-193b-5p mimics. In conclusion, miR-193b-5p acted as a regulator of cell cycle G1/S transition and cell proliferation by targeting FoxO3 to mediate the expression of p21 and Cyclin D1.

Dynamics of miRNA mediated regulation of legume symbiosis.

Symbiotic nitrogen fixation in legume nodules is important in soils with low nitrogen availability. The initiation and sustainability of symbiosis require cellular reprogramming that involves the miRNA-mediated inhibition or activation of specific nodulation genes. The high-throughput sequencing of small RNA libraries has identified miRNAs and their targets, which are the major players in the post-transcriptional gene regulation (PTGS) of the different stages of legume-rhizobia symbiosis ranging from bacterial colonization and organogenesis to symbiotic nitrogen fixation. Here, we present an overview of information obtained from the miRNA libraries from nodulating tissues that have been sequenced to date. The functional analysis of miRNAs has revealed roles in phytohormone homeostasis and spatio-temporal regulation, as well as the mobility of miRNAs and their functions in shoot to root signalling that affects diverse functions, including bacterial entry, meristem division and differentiation, nitrogen fixation and senescence. Furthermore, small RNA fragments of rhizobial origin repress complementary plant mRNAs. We also consider the roles of miRNAs in determinate or indeterminate nodules. Taken together, this overview confirms that miRNAs are master regulators of the legume-rhizobia symbiosis.

Mitoribosome Profiling from Human Cell Culture: A High Resolution View of Mitochondrial Translation.

Ribosome profiling (Ribo-Seq) is a technique that allows genome-wide, quantitative analysis of translation. In recent years, it has found multiple applications in studies of translation in diverse organisms, tracking protein synthesis with single codon resolution. Traditional protocols applied for generating Ribo-Seq libraries from mammalian cell cultures are not suitable to study mitochondrial translation due to differences between eukaryotic cytosolic and mitochondrial ribosomes. Here, we present an adapted protocol enriching for mitoribosome footprints. In addition, we describe the preparation of small RNA sequencing libraries from the resultant mitochondrial ribosomal protected fragments (mtRPFs).

Differential Expression of Maize and Teosinte microRNAs under Submergence, Drought, and Alternated Stress.

Submergence and drought stresses are the main constraints to crop production worldwide. MicroRNAs (miRNAs) are known to play a major role in plant response to various stresses. In this study, we analyzed the expression of maize and teosinte miRNAs by high-throughput sequencing of small RNA libraries in maize and its ancestor teosinte (Zea mays ssp. parviglumis), under submergence, drought, and alternated stress. We found that the expression patterns of 67 miRNA sequences representing 23 miRNA families in maize and other plants were regulated by submergence or drought. miR159a, miR166b, miR167c, and miR169c were downregulated by submergence in both plants but more severely in maize. miR156k and miR164e were upregulated by drought in teosinte but downregulated in maize. Small RNA profiling of teosinte subject to alternate treatments with drought and submergence revealed that submergence as the first stress attenuated the response to drought, while drought being the first stress did not alter the response to submergence. The miRNAs identified herein, and their potential targets, indicate that control of development, growth, and response to oxidative stress could be crucial for adaptation and that there exists evolutionary divergence between these two subspecies in miRNA response to abiotic stresses.

Analysis of microRNA expression profiles dynamic in different life stages of Haemaphysalis longicornis ticks by deep sequencing of small RNA libraries

The three-host tick Haemaphysalis longicornis is an obligate blood-sucking ectoparasite. In life-stage transitions, microRNAs (miRNAs) show a variety of expression changes. To investigate these changes, deep sequencing technology was applied to identify the conserved and potentially novel miRNAs expressed during the different life stages of H. longicornis. Total RNA from eggs, unfed larvae, unfed nymphs and unfed adults was extracted for deep sequence analysis. Deep sequencing on a Hiseq 4000 generated a total of 111,192,069 reads, grouped into four small RNA (sRNA) libraries, one for each of the four developmental stages of H. longicornis. Among these sequences, 78 conserved and 55 potentially novel miRNAs were identified, including stage-specific and differentially expressed miRNAs. Gene ontology (GO) analysis indicated significantly enriched GO terms related to cell proliferation and differentiation, including specific terms for the processes of development, growth, metabolism, regulation of biological functions, reproduction, and membrane enzyme regular activity. Kyoto Encyclopedia of Gene and Genomes (KEGG) analysis revealed a significant enrichment of the insulin, notch, Hippo, and Wnt signaling pathways for growth and development. Our data highlight the abundance of miRNA changes (conserved and potentially novel) in the different life stages of H. longicornis. In particular, stage-specific miRNAs, as observed, are essential regulators for the development of H. longicornis.

Depletion of erythropoietic miR-486-5p and miR-451a improves detectability of rare microRNAs in peripheral blood-derived small RNA sequencing libraries.

Erythroid-specific miR-451a and miR-486-5p are two of the most dominant microRNAs (miRNAs) in human peripheral blood. In small RNA sequencing libraries, their overabundance reduces diversity as well as complexity and consequently causes negative effects such as missing detectability and inaccurate quantification of low abundant miRNAs. Here we present a simple, cost-effective and easy to implement hybridization-based method to deplete these two erythropoietic miRNAs from blood-derived RNA samples. By utilization of blocking oligonucleotides, this method provides a highly efficient and specific depletion of miR-486-5p and miR-451a, which leads to a considerable increase of measured expression as well as detectability of low abundant miRNA species. The blocking oligos are compatible with common 5′ ligation-dependent small RNA library preparation protocols, including commercially available kits, such as Illumina TruSeq and Perkin Elmer NEXTflex. Furthermore, the here described method and oligo design principle can be easily adapted to target many other miRNA molecules, depending on context and research question.