Project description:Purpose: mRNA translation into protein is highly regulated, but the role of mRNA isoforms, noncoding RNAs (ncRNAs), and genetic variants has yet to be systematically studied. Using high-throughput sequencing (RNA-seq), we have measured cellular levels of mRNAs and ncRNAs, and their isoforms, in lymphoblast cell lines (LCL) and in polysomal fractions, the latter shown to yield strong correlations of mRNAs with expressed protein levels. Analysis of allelic RNA ratios at heterozygous SNPs served to reveal genetic factors in ribosomal loading. Methods: RNA-seq was performed on cytosolic extracts and polysomal fractions (3 ribosomes or more) from three lymphoblastoid cell lines. As each RNA fraction was amplified (NuGen kit), and relative contributions from various RNA classes differed between cytosol and polysomes, the fraction of any given RNA species loaded onto polysomes was difficult to quantitate. Therefore, we focused on relative recovery of the various RNA classes and rank order of single RNAs compared to total RNA. Results: RNA-seq of coding and non-coding RNAs (including microRNAs) in three LCLs revealed significant differences in polysomal loading of individual RNAs and isoforms, and between RNA classes. Moreover, correlated distribution between protein-coding and non-coding RNAs suggests possible interactions between them. Allele-selective RNA recruitment revealed strong genetic influence on polysomal loading for multiple RNAs. Allelic effects can be attributed to generation of different RNA isoforms before polysomal loading or to differential loading onto polysomes, the latter defining a direct genetic effect on translation. Several variants and genes identified by this approach are also associated with RNA expression and clinical phenotypes in various databases. Conclusions: These results provide a novel approach using complete transcriptome RNA-seq to study polysomal RNA recruitment and regulatory variants affecting protein translation. cells from 3 samples were grown to 5x105 cells/mL density in T75 tissue culture flask and harvested, total RNA and polysome bound RNA was sequenced by Ion Proton
Project description:Purpose: mRNA translation into protein is highly regulated, but the role of mRNA isoforms, noncoding RNAs (ncRNAs), and genetic variants has yet to be systematically studied. Using high-throughput sequencing (RNA-seq), we have measured cellular levels of mRNAs and ncRNAs, and their isoforms, in lymphoblast cell lines (LCL) and in polysomal fractions, the latter shown to yield strong correlations of mRNAs with expressed protein levels. Analysis of allelic RNA ratios at heterozygous SNPs served to reveal genetic factors in ribosomal loading. Methods: RNA-seq was performed on cytosolic extracts and polysomal fractions (3 ribosomes or more) from three lymphoblastoid cell lines. As each RNA fraction was amplified (NuGen kit), and relative contributions from various RNA classes differed between cytosol and polysomes, the fraction of any given RNA species loaded onto polysomes was difficult to quantitate. Therefore, we focused on relative recovery of the various RNA classes and rank order of single RNAs compared to total RNA. Results: RNA-seq of coding and non-coding RNAs (including microRNAs) in three LCLs revealed significant differences in polysomal loading of individual RNAs and isoforms, and between RNA classes. Moreover, correlated distribution between protein-coding and non-coding RNAs suggests possible interactions between them. Allele-selective RNA recruitment revealed strong genetic influence on polysomal loading for multiple RNAs. Allelic effects can be attributed to generation of different RNA isoforms before polysomal loading or to differential loading onto polysomes, the latter defining a direct genetic effect on translation. Several variants and genes identified by this approach are also associated with RNA expression and clinical phenotypes in various databases. Conclusions: These results provide a novel approach using complete transcriptome RNA-seq to study polysomal RNA recruitment and regulatory variants affecting protein translation.
2015-06-30 | GSE66135 | GEO
Project description:Monosomes and polysomes bind functionally distinct classes of protein-coding and noncoding RNAs
Project description:Single-cell RNA-sequencing of mouse fibroblasts for the identification and functional characterization of non-coding RNAs. Non-coding RNAs were assigned putative functions based on single-cell expression patters, e.g. phase specific expression during the cell cycle. Additionally, allele-level resolution was used to characterize coordinated and mutually exclusive expressions of noncoding RNAs against nearby protein coding genes.
Project description:We performed ribosome profiling of polysomes (Poly-RIBOseq) to monitor translation of RNAs associated with polysomes in brain extracts from a mouse model Keywords: ribosome profiling, translation, polysome profiling, Poly-RIBOseq
Project description:Methyl-7-guanosine (m7G) “capping” of coding and some noncoding RNAs is critical for their maturation and subsequent activity. Here, we discovered that eukaryotic translation initiation factor 4E(eIF4E), itself a cap-binding protein, drives the expression of the capping machinery and the increased capping efficiency of ∼100 coding and noncoding RNAs. This dataset collects transcriptomic data for quantitative cap immunoprecipitation (CapIP) assay in eIF4E-Flag or vector stable U2Os cells.
Project description:Exon junction complexes (EJCs) deposited on spliced mRNAs play multifunctional roles in the regulation of gene expression. Whereas the formation and components of EJCs are well characterized, the underlying molecular mechanisms for gene regulation remain poorly understood. Here we find that a eukaryotic translation initiation factor (eIF) 4A3, a core component of EJC directly interacts with eIF3g, a subunit of eIF3 complex. This interaction serves as a linker between the EJC and eIF3 complex, consequently driving an internal ribosomal recruitment. Accordingly, artificially tethered EJC component or cellular EJC deposited on mRNA after splicing promotes internal initiation of translation in a way that is resistant to cellular stress induced by serum starvation. We also demonstrate that translatable endogenous or reporter circular RNAs depend on EJC for their association with polysomes. Our results uncover an internal initiation driven by EJC, expanding the protein-coding potential of human transcriptome including circular RNAs.
Project description:Interventions: Case series:Nil
Primary outcome(s): intestinal microecological disorders;blood non-coding RNAs and immune status
Study Design: Randomized parallel controlled trial
Project description:To understand the role of long non-coding RNAs and interaction with coding RNAs in bladder urothelial cell carcinoma (BUCC), we performed genome-wide screening long non-coding RNAs and coding RNAs expression on primary BUCC tissues and normal tissues using long non-coding RNA array (Agilent plateform (GPL13825). By comparing these two groups, significantly differentially expressed lncRNAs and coding RNAs were identified. We further identifed a subset of long noncoding RNAs and their correlation with neighboring coding genes using bioinformatic tools. This analysis provides foundamental understaning of transcriptomic landscape changing during bladder carcinogenesis. 12 BUCC primary tumors and 3 normal tissues were used for long noncoding RNA array experiments which including long non-coding RNAs and coding RNAs. The differential expression of subset of long noncoding RNAs and their interaction with coding RNAs in BUCC compared with normal tissue will be identified with comtational analysis.
Project description:The MHC region encodes HLA genes and is the most complex region in the human genome. The extensive polymorphic nature of the HLA hinders accurate localization and functional assessment of disease risk loci within this region. Using targeted capture sequencing and constructing individualized genomes for transcriptome alignment, we identified 908 novel transcripts within the human MHC region. These include 593 novel isoforms of known genes, 137 antisense strand RNAs, 119 novel long intergenic noncoding RNAs, and 5 transcripts of 3 novel putative protein-coding human endogenous retrovirus genes. We revealed allele-dependent expression imbalance involving 88% of all heterozygous transcribed single nucleotide polymorphisms throughout the MHC transcriptome. Among these variants, we show that the genetic variant associated with Behc ̧et’s disease in the HLA-B/MICA region, which tags HLA-B*51, is within novel long intergenic noncoding RNA transcripts that are exclusively expressed from the haplotype with the protective but not the disease risk allele. Further, we showed that the transcriptome within the MHC region can be defined by 14 distinct coexpression clusters, with evidence of coregulation by unique transcription factors in at least 9 of these clusters. Our data suggest a very complex regulatory map of the human MHC, and can help uncover functional consequences of disease risk loci in this region.