MiRNAs in the genomes of Bombus terrestris and Bombus impatiens
ABSTRACT: Our aims in this study were: 1) to identify the miRNAs of the bumble bees Bombus terrestris and B. impatiens; 2) to compare the total numbers of miRNAs between both bumble bee species and between them and the honey bee, Apis mellifera; and 3) to test whether the sequences and expression patterns of miRNAs were conserved between species. To investigate each of these aims we used miRNA-seq (deep sequencing of miRNA-enriched libraries) in B. terrestris, and bioinformatics prediction programs to identify miRNAs in both Bombus species. We identified 131 miRNAs in B. terrestris, and 114 in B. impatiens; of these, 17 were new miRNAs that had not previously been sequenced in any species. We found a striking level of difference in the miRNAs present between Bombus and A. mellifera, with 103 miRNAs in A. mellifera not being present in the genomes of the two bumble bees. miRNA profiles of Bombus terrestris at two developmental stages in larvae. This submission represents 'Bombus terrestris' component of study.
Project description:Honeybees are very important eusocial insects and are involved in the pollination of many plants. Queen bees and worker bees develop from the same fertilized eggs, and are thus genetically identical despite their substantial behavioural and physiological differences. The mechanism governing developmental differences between worker and queen bees has always attracted much interest. While there are several reports on mRNA expression related to caste differentiation, no systematic investigation of small RNAs has thus far been carried out. Results: Using deep sequencing we systematically profiled small RNA expression in 4th-6th day worker larvae and queen larvae (the critical stages at which the fates of workers and queens are determined), and found that 38 miRNAs were differentially expressed between worker and queen larvae. In addition, 639 mature miRNA candidates were identified in our work for the first time, of which, 526 were expressed only in workers (318) or queens (208). Conclusion: We present the first profile of honeybee small RNAs and explore the mechanism of caste differentiation between worker and queen bees. Caste-specific expression patterns and large discrepancies in small RNA profiles between worker and queen bees indicate that small RNAs may be related to the differential development of worker and queen bee larvae. Results presented here will make a valuable contribution to understanding the caste switch between worker and queen bees. Three healthy 10-frame colonies of ‘Zhenongda No.1’- a high-yielding royal jelly breed of Apis mellifera ligustica , were maintained at the Huajiachi campus of Zhejiang University. In each colony, the queen laid eggs over a period of 24 hours in one empty frame which was subsequently moved to an egg-free super-chamber. After 66 hours (less than 18 hours after hatching), we transferred 150 larvae into queen cups to rear queens in each colony and put the queen cup frames into their corresponding colonies. 40-60 worker larvae and queen larvae were collected from each colony after 4 days (73~90 h after hatching), 5 days (97~114 h after hatching) and 6 days (121~138 h after hatching). The larval samples were collected into 50 ml tubes, immediately frozen in liquid nitrogen and stored at -80C until being used for RNA extraction. After total RNA was extracted and quantified , relative equal amounts of total RNAs from each of the three sampling days were pooled into respectively worker and queen samples, and the fraction of small RNAs less than 30nt long was retained and sequenced on the Illumina/Solexa high-throughput platform (HTP).
Project description:Social caste determination in the honey bee is assumed to be determined by the dietary status of the young larvae and translated into physiological and epigenetic changes through nutrient-sensing pathways. We have employed Illumina/Solexa sequencing to examine the small RNA content in the bee larval food source, and show that worker jelly is enriched in miRNA complexity and abundance relative to royal jelly. The miRNA levels in worker jelly were 7-215 fold higher than in royal jelly, and both jellies showed dynamic changes in miRNA content during the 4th to 6th day of larval development. Adding specific miRNAs to royal jelly elicited significant changes in queen larval mRNA expression and in morphological characters of the emerging adult queen bee. We propose that miRNAs in the nurse bee secretions constitute an additional element in the regulatory control of caste determination in the honey bee. We collected worker and royal jelly of the Italian honeybee (ZND No.1, Apis mellifera ligustica) at 73~90 hours (4th-day larvae), 97~114 hours (5th-day larvae), and 121~138 hours (6th-day larvae) after hatching. After total RNA was extracted and quantified , relative equal amounts of total RNAs from each of the three sampling days were pooled into respectively worker and royal jelly samples, and the fraction of small RNAs less than 30nt long was retained and sequenced on the Illumina/Solexa high-throughput platform (HTP).
Project description:In this study we show that global levels of mature miRNAs are unaffected in C. elegans after knockdown of either subunit of CK2 (encoded by kin-3 and kin-10) by RNAi. Small RNAs were quantified from wild-type animals at L4 stage fed either vector (L4440), kin-3, kin-10, or alg-1 RNAi. Mature miRNA counts were quantified by summing the total number of reads mapping to each miRNA and normalized to the total number of mapped reads per sequencing library.
Project description:We investigated environmental determinants of caste differences in paper wasps, specifically the effects of differential nutrition. We found that nutritional restriction only partially biased wasp gene expression patterns toward being worker caste-like, which highlights the complex and multifactorial nature of environmental effects on the gene expression patterns underlying plastic phenotypes PRJNA242774; We sequenced mRNA from 16 individual 5th instar larval Polistes metricus heads from 4 groups: worker-reared (n=4), foundress-reared (n=4), restricted nutrition (n=4), and ad libitum (n=4).
Project description:To assess the efficacy of AdV-VP55 mediated degredation of host miRNAs. Small RNA profiles of HEK 293T cells treated with type 5 Adeno vectors expressing either GFP or GFP-VP55 for 24 hours
Project description:Female larvae of the honeybee (Apis mellifera) develop into either queens or workers depending on nutrition during larval development. This nutritional stimulus triggers different developmental trajectories, resulting in adults that differ in physiology, behaviour and life-span. To understand how these developmental trajectories are established we have undertaken a comprehensive analysis of differential gene expression throughout larval development. Gene expression of honeybee queen and worker larval samples was analysed at 60 hours with high-throughout sequencing
Project description:Female larvae of the honeybee (Apis mellifera) develop into either queens or workers depending on nutrition during larval development. This nutritional stimulus triggers different developmental trajectories, resulting in adults that differ in physiology, behaviour and life-span. To understand how these developmental trajectories are established we have undertaken a comprehensive analysis of differential gene expression throughout larval development. Gene expression of honeybee queen and worker larval samples was analysed at seven time points during larval development (6 hr, 12 hr, 36 hr, 60 hr, 84 hr, 108 hr and 132 hr)
Project description:We profiled and quantitated miRNAs in two skin tumors (Basal cell carcinoma and Merkel cell carcinoma) and identified tumor-specific miRNAs. We used these tumor-specific miRNAs to guide development of miRNA fluorescence in situ hybridization. 2 barcoded sequencing runs, including 40 unique samples (36 used in manuscript). The details of each sample can be found in Supplementary Tables S1 and S2.
Project description:MicroRNAs (miRNAs) are single-stranded non-coding RNAs that negatively regulate target gene expression through mRNA cleavage or translational repression. There is mounting evidence that they play critical roles in heart disease. The expression of known miRNAs in the heart has been studied at length by microarray and quantitative PCR but it is becoming evident that microRNA isoforms (isomiRs) are potentially physiologically important. It is well known that left ventricular (patho)physiology is influenced by transmural heterogeneity of cardiomyocyte phenotype, and this likely reflects underlying heterogeneity of gene expression. Given the significant role of miRNAs in regulating gene expression, knowledge of how the miRNA profile varies across the ventricular wall will be crucial to better understand the mechanisms governing transmural physiological heterogeneity. To determinine miRNA/isomiR expression profiles in the rat heart we investigated tissue from different locations across the left ventricular wall using deep sequencing. We detected significant quantities of 145 known rat miRNAs and 68 potential novel orthologs of known miRNAs, in mature, mature* and isomiR formation. Many isomiRs were detected at a higher frequency than their canonical sequence in miRBase and have different predicted targets. The most common miR-133a isomiR was more effective at targeting a construct containing a sequence from the gelsolin gene than was canonical miR-133a, as determined by dual-fluorescence assay. We identified a novel rat miR-1 homolog from a second miR-1 gene; and a novel rat miRNA similar to miR-676. We also cloned and sequenced the rat miR-486 gene which is not in miRBase (v18). Signalling pathways predicted to be targeted by the most highly detected miRNAs include Ubiquitin-mediated Proteolysis, Mitogen-Activated Protein Kinase, Regulation of Actin Cytoskeleton, Wnt signalling, Calcium Signalling, Gap junctions and Arrhythmogenic Right Ventricular Cardiomyopathy. Most miRNAs are not expressed in a gradient across the ventricular wall, with exceptions including miR-10b, miR-21, miR-99b and miR-486. The hearts of 3 male 8 month old Sprague-Dawley rats were rapidly extracted after euthanasia with sodium pentobarbital. A section of the free wall of the left ventricle was dissected into epicardium, mid-myocardium and endocardium by cutting approximately 1 mm from the epicardial and endocardial surfaces. Small RNA was extracted (miRNeasy Kit; Qiagen, Crawley UK), quantified (Nanodrop; Thermo Scientific) and quality assessed for degradation (RNA Nano Chip, Bioanalyser 2100; Aligent Technologies, Wokingham UK; only samples with a RNA integrity no. (RIN) ≥8 were carried forward) and retention of small RNA (Small RNA Chip, Bioanalyser 2100). Small RNA was preferentially ligated with adapters, reverse transcribed into cDNA and amplified with 9 individually tagged primer indices (TruSeq Small RNA Sample Preparation Kit; Illumina, Little Chesterford, UK) and a library of small RNA created for each sample. After gel purification the cDNA products were again analysed on the bioanalyser using a High Sensitivity DNA Chip and assessed for the presence and concentration of the peak corresponding to ligated and tagged miRNA (approximately 147nt). Only samples with suitable RIN values exhibiting good retention of small RNA species were used for library preparation. After pooling, the samples were sequenced by TrinSeq (Trinity Genome Sequencing Lab & Neuropsychiatric Genetics Group, Trinity College Dublin, Ireland (http://www.medicine.tcd.ie/sequencing); using TruSeq SR Cluster Kit v5 (Illumina) and the resultant data trimmed and aligned to miRBase v18 (CLC Genomics Workbench v4.0; CLC bio, Swansea UK).
Project description:Mutations of the tumor suppressor p53 lead to chemotherapy resistance and a dismal prognosis in chronic lymphocytic leukemia (CLL). Comparing miRNA/non-coding RNA expression profiles between p53 wild-type and p53 mutant samples in response to DNA damage, we exploit the impaired transcriptional activity of mutant p53 to map out p53 targets in primary CLL by small RNA sequencing. Focusing on miRNAs, we identify a set of p53-dependent miRNAs (miR-182-5p, miR-7-5p, miR-320c/d) in addition to the key p53 target miR-34a. Beyond miRNAs, the long non-coding RNAs (lncRNAs) NEAT1 and lincRNA-p21 are induced in response to DNA damage in the presence of functional p53 but not in CLL with p53 mutation. Quantification of mature miRNA, other ncRNA and mRNA expression profiles in 68 paired CLL primary samples 24h after irradiation or no treatment.