Differential expression of snoRNAs during ATDC5 chondrogenic differentiation
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ABSTRACT: Previously we have shown that the snoRNA RMRP is differentially expressed during chondrogenic differentiation and interference with its function led to important changes in the outcome of chondrogenic differentiation with consequences for ribosomal RNA levels and human disease. To identify additional snoRNAs that may play a role in chondrogenic differentiation, we performed small RNA sequencing (<200 nt) in ATDC5 chondrogenic differentiation at day 0, 7 and 14.
Project description:Background: MicroRNAs (miRNAs) are a class of single-stranded non-coding small RNAs (sRNAs) that are 20-24 nucleotides (nt) in length. Extensive studies have indicated that miRNAs play important roles in plant growth, development, and stress responses. With more copper (Cu) and copper-containing compounds used as bactericides and fungicides in viticulture, Cu stress has become one of the serious environmental problems that affect plant growth and development. In order to uncover the hidden response mechanisms of Cu stress, many Cu-responsive miRNAs have been detected in several plant species. However, there have been few reports about the grapevine miRNAs in response to Cu. Results: Here, two small RNA libraries were constructed from Cu-treated and water-treated (control) leaves of 'Summer Black' grapevine. Following high-throughput sequencing and filtering, 158 known vvi-miRNAs and 98 novel vvi-miRNAs were identified in the two libraries. Among these, 24 could only be detected in the treatment, and 63 were only detected in the control. Additionally, 100 known vvi-miRNAs were found to be clearly responsive to Cu, among which 96 were down-regulated and four were up-regulated; 47 novel vvi-miRNAs were found to be clearly responsive to Cu, among which 35 were down-regulated and 12 were up-regulated. Subsequently, expression patterns of a set of Cu-responsive vvi-miRNAs were validated by quantitative real-time PCR (qRT-PCR). There existed some consistency in expression levels of Cu-responsive vvi-miRNAs between high-throughput sequencing and qRT-PCR assays. In addition, 92 putative targets for 79 known vvi-miRNAs and 51 putative targets for 22 novel vvi-miRNAs were predicted, and most of the targets are involved in multiple biological processes including transcriptional regulation and response to biotic and abiotic stresses. Conclusions: In this study, 147 Cu-responsive vvi-miRNAs were identified using high-throughput sequencing, and their target genes were predicted, which will be helpful to understanding the molecular mechanisms of miRNAs in response to Cu stress. Furthermore, this work can also provide a foundation for further study of the networks of miRNAs involved in grapevine plant growth and breeding some Cu-tolerant grapevine cultivars. Mixed 'Summer Black' grapevine young leaves (2 weeks old), large leaves (5 weeks old), and old leaves (9 week old) in randomly-selected plants from both the Cu-treated and control groups were collected for high-throughput sequencing. Subsequently, we carried out the analysis of Solexa sequencing data, and performed the research of regulatory modes of grapevine miRNAs on their target genes during Cu stress.
Project description:Preparation of exosomes isolated from semen contain a substantial amount of RNA, mostly from 20 to 100 nucleotides in length. We sequenced separately 20-40 and 40-100 nucleotide fractions of RNA from exosomes isolated from semenal fluid from six healthy donors. We found various classes of small non-coding RNA, including mature microRNA and piwi-RNA, as well as abundant Y RNAs and tRNAs present in both full length and fragmented forms. Specific RNAs were consistently present in all donors. For example, fifteen (of ~2,600 known) microRNAs constituted over 80% of mature microRNA in SE. Additionally, tRNA fragments were strongly enriched for 5M-bM-^@M-^Y-ends of 18-19 or 30-34 nucleotides in length. Size-fractionated small RNA profiles from exosomes isolated from the seminal fluid of six healthy donors
Project description:Setaria viridis, the wild ancestor of millet, exhibits strong repression of crown root growth in drought. We compare in gene expression in the S. viridis crown between drought vs watered treatments. RNA from Lower region (crown) or Upper region (stem) of watered (W) or drought (D) treated Setaria plants were harvested at 6 or 9 days after sowing; there are 8 samples per biological repeat, 3 biological repeats.
Project description:We previously reported widespread differential expression of long non-protein-coding RNAs (ncRNAs) in response to virus infection. Here, we expanded the study through small RNA transcriptome sequencing analysis of the host response to both severe acute respiratory syndrome coronavirus (SARS-CoV) and influenza virus infections across four founder mouse strains of the Collaborative Cross, a recombinant inbred mouse resource for mapping complex traits. We observed differential expression of over 200 small RNAs of diverse classes during infection. A majority of identified microRNAs (miRNAs) showed divergent changes in expression across mouse strains with respect to SARS-CoV and influenza virus infections and responded differently to a highly pathogenic reconstructed 1918 virus compared to a minimally pathogenic seasonal influenza virus isolate. Novel insights into miRNA expression changes, including the association with pathogenic outcomes and large differences between in vivo and in vitro experimental systems, were further elucidated by a survey of selected miRNAs across diverse virus infections. The small RNAs identified also included many non-miRNA small RNAs, such as small nucleolar RNAs (snoRNAs), in addition to nonannotated small RNAs. An integrative sequencing analysis of both small RNAs and long transcripts from the same samples showed that the results revealing differential expression of miRNAs during infection were largely due to transcriptional regulation and that the predicted miRNA-mRNA network could modulate global host responses to virus infection in a combinatorial fashion. These findings represent the first integrated sequencing analysis of the response of host small RNAs to virus infection and show that small RNAs are an integrated component of complex networks involved in regulating the host response to infection. IMPORTANCE: Most studies examining the host transcriptional response to infection focus only on protein-coding genes. However, mammalian genomes transcribe many short and long non-protein-coding RNAs (ncRNAs). With the advent of deepsequencing technologies, systematic transcriptome analysis of the host response, including analysis of ncRNAs of different sizes, is now possible. Using this approach, we recently discovered widespread differential expression of host long (>200 nucleotide[nt]) ncRNAs in response to virus infection. Here, the samples described in the previous report were again used, but we sequenced another fraction of the transcriptome to study very short (about 20 to 30 nt) ncRNAs. We demonstrated that virus infection also altered expression of many short ncRNAs of diverse classes. Putting the results of the two studies together, we show that small RNAs may also play an important role in regulating the host response to virus infection. The small RNA transcriptome deep sequencing analysis was performed on lung samples from our previously published study (Unique signatures of long noncoding RNA expression in response to virus infection and altered innate immune signaling , X Peng, MBio. 2010 Oct 26;1(5). pii: e00206-10.). We infected four of the eight founder mouse strains used in generating the Collaborative Cross, a recombinant inbred mouse resource for mapping complex traits (41). These strains included 129S1/SvImJ (129/S1), WSB/EiJ (WSB), PWK/PhJ (PWK), and CAST/EiJ (CAST) mice. Ten-week-old mice were intranasally infected with phosphate-buffered saline (PBS) alone or with 1X10^5 PFU of mouse adapted severe acute respiratory syndrome coronavirus (SARS-CoV; rMA15), or 500 PFU of influenza A virus strain A/Pr/8/34 (H1N1; PR8). To match the previous whole-transcriptome analysis, we performed small RNA transcriptome sequencing analysis on the same eight samples from mice with SARS-CoV infections, including one SARS-CoV rMA15-infected mouse and one matched mock-infected mouse from each of the four strains at 2 days postinfection (dpi). In addition, we sequenced the small RNA transcriptome for 12 samples obtained from influenza virus infected mice, including two PR8-infected mice and one matched mockinfected mouse from each of the four strains at 2 dpi.
Project description:The present study was designed to find out the transcriptional system that is connected with the development of leaves and production of metabolites during photosynthesis. We examine the cellular expression by use the up-to-date RNA sequencing expertise to carry out a complete analysis of the transcriptional profile of V. vinifera leaves at four different developmental stages. Total RNA extracted from 4 samples with three replication for each and compared within all samples.
Project description:We aimed to identify miRNAs and pathways specifically deregulated in adolescent and young adult (AYA) T-ALL patients. Small RNA-seq showed no major differences between AYA and pediatric T-ALL, but it revealed downregulation of miR-143-3p in T-ALL patients. Prediction algorithms identified several known and putative oncogenes targeted by this miRNA, including KRAS, FGF1, and FGF9. Pathway analysis indicated signaling pathways related to cell growth and proliferation, including FGFR signaling and PI3K-AKT signaling, with the majority of genes overrepresented in these pathways being predicted targets of hsa-miR-143-3p. By luciferase reporter assays, we validated direct interactions of this miRNA with KRAS, FGF1 and FGF9. In cell proliferation assays, we showed reduction of cell growth upon miR-143-3p overexpression in two T-ALL cell lines. Our study is the first description of the miRNA transcriptome in AYA T-ALL patients and the first report on tumor suppressor potential of miR-143-3p in T-ALL. Downregulation of this miRNA in T-ALL patients might contribute to enhanced growth and viability of leukemic cells. We also discuss the potential role of miR-143-3p in FGFR signaling. Although this requires more extensive validation, it might be an interesting direction, since FGFR inhibition proved promising in preclinical studies in various cancers.
Project description:Tuberculosis is an infectious disease, with latent infection with Mycobacterium tuberculosis (M.tb) affecting 1/3 of humanity. It can remain quiescent for long time, making eradication very difficult. To do so, M.tb need to carefully orchestrate its gene expression to survive immune response and starvation but still be able to reactivate to enable transmission to new hosts. Here we used whole transcriptome deep sequencing to compare gene expression between wild type M.tb and a strain with its whiB6, a gene encoding a putative redox-sensing transcription factor, disrupted by a transposon. We found several genes associated with dormancy such as hspX, fdxA and narK2 upregulated in the transposon mutant, indicating that WhiB6 may be a repressor of such genes. The results suggest that WhiB6 may be a complement to the dosS/dosR system in regulating genes important for dormancy. Triplicate cultures of a mutant with its whiB6 gene disrupted by a transposon and wild-type M. tuberculosis CDC1551 were grown in 7H9 media. RNA was extracted and depleted from rRNA. Global gene expression was measured using AB SOLID RNA sequencing.
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:The relative importance of gene expression regulation at the mRNA versus the protein-level is a matter of ongoing debate. We present an in-depth analysis of a cervical cancer cell line responding to protein misfolding stress induced by dithiothreitol treatment, quantifying the dynamics of mRNA and protein concentrations for >3,500 genes over a time-course 30 hours. While the early phase of the experiment (<two hours) was marked by apoptosis, surviving cells were marked by a strong response to unfolded proteins and stress of the endoplasmatic reticulum, specifically during the intermediate phase (two to eight hours). Using statistical time series analysis, we detected significant changes in the regulatory parameters at the RNA-level, caused by transcription and mRNA degradation, and at the protein-level, caused by translation and protein degradation. mRNA- and protein-level regulation were of equal importance (XX and YYY%, respectively), but displayed different magnitudes and dynamics: mRNA fold changes were much smaller on average than those of the proteins. While our method did not capture immediate changes, we found the strongest regulatory response between two and eight hours after the treatment. mRNA-level regulation showed a spike around this time interval, while protein-level regulation was delayed and continued at slower pace until the end of the experiment.
Project description:We report the application of small RNA sequencing technology for high-throughput profiling of microRNA molecules (miRNAs) in blood leukocytes of Parkinson's Disease (PD) patients. Expression changes which may contribute to PD pathogenesis and reaction to treatment were identified in RNA preparations from blood leukocyte samples collected from PD patients prior to and following DBS neurosurgery on stimulation and following a short one hour cessation of the electrical stimulation (which enabled dissociation of the surgery effects from the electrical stimulation alone) and from matched healthy control volunteers, all under signed informed consents. Examination of four states: Parkinson's Disease (PD) patients 1 day prior to DBS, PD patients a few weeks after DBS neurosurgery (upon symptoms stabilization) both on and following one hour electrical stimulation cessation, and age-matched healthy control volunteers.