Project description:Small extracellular vesicles (sEV) are nano-sized (40-150 nm), membrane-encapsulated vesicles that are released by malignant or pathologic and non-pathologic cells into the extracellular space and function as intercellular signaling vectors through the horizontal transfer of biologic molecules, including microRNA (miRNA) and other small non-coding RNA (ncRNA), that can alter the phenotype of recipient cells. sEV are present in essentially all extracellular biofluids, including serum, urine and saliva, and offer a new avenue for discovery and development of novel biomarkers of various disease states and exposures. The objective of this study was to determine the similarities and differences between sEV ncRNA derived from saliva, serum and urine, as well as cell-free ncRNA from serum. Saliva, urine and serum were concomitantly collected from 4 healthy donors, and sEV were isolated from each respective biofluid, along with cfRNA from serum. sEVs were isolated from the respective biofluids via differential ultracentrifugation. Small RNA-sequencing was performed on each sample, and cluster analysis was performed based on ncRNA profiles. While some similarities existed in terms of sEV ncRNA cargo across biofluids, there are also notable differences in ncRNA class and ncRNA secretion, with each sEVs in each biofluid bearing a unique ncRNA profile. We conclude that sEV ncRNA cargo varies according to biofluid, so thus should be carefully selected and interpreted when designing translational or epidemiological studies.
Project description:Purpose: To detect serum exosomal ncRNA profiles of proliferative diabetic retinopathy (PDR) by High-throughput sequencing Methods: serum exosomal non-coding RNA (ncRNA) profiles profiles of PDR and MH were generated by deep sequencing, only in once, using IlluminaHiSeq 3000. After analyzing the base composition and quality of the data, according to the analysis results of the original data, the data were filtered to remove the joint sequence and the contaminated part, and to remove low-quality base sequences.If it is paired-ended sequencing data, the filtered data should be further screened to retain the paired sequences and obtain clean data. Conclusions: Our study represents the first detailed analysis of serum exosomal transcriptomes from PDR patients by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles.
Project description:Vitamin D, a hormone that acts through the nuclear vitamin D receptor (VDR), upregulates anti-tumorigenic microRNA in prostate epithelium. This may contribute to the lower levels of aggressive prostate cancer (PCa) in patients with high serum vitamin D. Expression of other small non-coding RNAs (ncRNAs) in prostate epithelium and their potential regulation by vitamin D are uncharacterized. Laser capture microdissection (LCM) followed by small-RNA sequencing was used to identify ncRNA in the prostate epithelium of tissues from a vitamin D-supplementation trial. We compared the expression profiles to small-RNA sequencing data from primary prostate epithelial cells and publically-available benign whole prostate. Application of LCM to isolate epithelium promoted sample homogeneity and captured more diversity in ncRNA species. An abundance of PIWI-interacting RNAs (piRNAs) was detected in normal prostate epithelium, which increased under high vitamin D conditions.
Project description:Many ncRNAs serve as regulatory molecules in various physiological pathways. Distinct from protein-coding RNA expression, ncRNA expression is regulated solely by transcription and RNA processing/stability. Transcriptional regulation in ncRNA genes is thus important to be understood and is yet to be known completely. Previously, we identified that a subset of mammalian ncRNA genes is transcriptionally regulated by RNA Pol II promoter-proximal pausing and in a tissue-specific manner. In this study, human stimulus-inducible ncRNA genes were monitored to assess the function of P-TEFb, a master Pol II pausing regulator for protein-coding genes, in ncRNA transcription. Our findings indicate that the expression of many ncRNA genes is regulated by P-TEFb. Interestingly, a biphasic characteristic of P-TEFb inhibition in serum responsive ncRNA genes was observed: S2 phospho-Pol II was largely increased in the TSS (–300 to +300) whereas overall, it was decreased in the gene body (> +350) upon chemical inhibition of P-TEFb. In addition, the three representative ncRNAs, MALAT1, NEAT1, and XIST, were further analyzed for determining P-TEFb association. Taken together, we propose the transcriptional activation of many human ncRNAs utilizing pausing and releasing Pol II and the regulatory mechanism of transcriptional elongation of these genes requiring the function of P-TEFb.
Project description:Non-coding RNA (ncRNA) molecules have fundamental roles in cells and many are also stable in body fluids as extracellular RNAs. In these studies, we used RNA sequencing (RNA-seq) to investigate the profile of small non-coding RNA (sncRNA) in human serum. We analyzed more than 10 billion Illumina reads from more than 500 serum samples, included in the Norwegian population-based Janus Serum Bank (JSB). Our results suggest that many circulating RNAs in serum can be potential biomarkers and they are associated with different traits including age, sex, smoking, body size etc.
Project description:Upon Epstein-Barr virus (EBV) infection of human B lymphocytes non-coding RNAs (ncRNAs) regulate expression of viral and cellular genes. In this study, we generated a specialized cDNA library from EBV-immortalized cells and subjected it to deep sequencing. We identified 631 unique ncRNA genes, comprised of 321 potential novel differentially expressed ncRNA candidates. Subsequently, we investigated differential expression of known and potential novel ncRNA candidates by custom-designed microchips by comparing expression of ncRNA genes of EBV-immortalized versus non-infected control cells. Among the differentially expressed candidates from chip analysis, differential expression of six novel ncRNA candidates was verified by northern blot analysis. In addition, microchip analysis resulted in observation of increased expression levels of a significant number of potential ncRNA candidates that were preferentially derived from genomic loci annotated as Alu repetitive elements. Alu elements are members of the repeat subfamily of short interspersed nuclear elements (SINE) and were reported to be transcribed upon stress stimulation. While EBV infection significantly up-regulated expression of Alu-derived RNA transcripts, no significant increase in expression of these transcripts was observed under additional tested stress conditions. By employing deep sequencing followed by custom microchip analysis, we identified six novel differentially expressed ncRNAs as well as significantly increased expression levels of Alu-derived RNA transcripts. These transcripts might be involved in crucial functions upon infection by EBV.
Project description:Small open reading frame encoded peptides (SEPs), also called microproteins, play a vital role in biological processes. Plenty of their open reading frames are located on the non-coding RNA (ncRNA) range. Recent research has demonstrated that ncRNA-encoded polypeptides have essential functions and exist ubiquitously in various tissues. To better understand the role of microproteins and ncRNA-encoded polypeptide expression in different tissues, we profiled the proteomic characterization of five mouse tissues by mass spectrometry, including bottom-up, top-down, and de novo sequencing strategies. Bottom-up and top-down with database-dependent searches identified 791 microproteins in the OpenProt database. De novo sequencing obtained 309 microproteins and ten ncRNA-encoded polypeptides that were not in the SEP database. In this study, we discovered 1100 microproteins in total, including 208 ncRNA-encoded polypeptides. From the annotation of these microproteins, we found that the brain contains the largest number of neuropeptides, while the spleen contains the most immunoassociated microproteins.
Project description:RUF6 is a ncRNA gene family that is transcribed by RNA Polymerase III but actively regulates the Pol II-transcribed var virulence gene family. Understanding how RUF6 ncRNA connect to downstream effectors is lacking. We developed an RNA-directed proteomic discovery (ChIRP-MS) protocol to identify in vivo RUF6 ncRNA protein interactions. The RUF6 ncRNA interactome was purified with biotinylated antisense oligonucleotides. Quantitative label-free mass spectrometry identified several unique proteins linked to gene transcription including. Affinity purification of Pf-DDX5 identified proteins originally found by our RUF6-ChIRP protocol, validating the technique’s robustness for identifying ncRNA interactomes in P. falciparum. Our work identifies a RUF6 ncRNA protein complex that interacts with RNA Pol II to sustain var gene expression.