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A transcriptome-wide atlas of RNP compositions reveals diverse classes of mRNAs and lncRNAs

ABSTRACT: Aim: To determine how different classes of transcript (e.g. lncRNAs and mRNAs) are defined in the cell. Approach: We determined the transcriptome-wide targets of key RNA packaging, maturation, export and turnover factors. We used the CRAC technique, whereby RNA:protein interactions are fixed by UV irradiation of yeast cultures, and RNA:protein complexes obtained via a stringent multi-step purification. A mild RNase treatment fragments the bound RNAs, which are then used as templates for RT-PCR, prior to sequencing. This approach enabled us to compare the maturation and turnover pathways of mRNAs and lncRNAs. Results: Our data reveal that mRNA and lncRNA maturation pathways diverge prior to nuclear export, and 3' end processing emerges as a key step in determining transcript fate. Our analyses also reveal when and where the tested proteins bind to mRNAs, and thus offer much insight into the dynamic assembly of mRNPs. Analyses of reads with non-genome-encoded A-tails enabled us to distinguish proteins bound to stable poly(A) tails on full-length mRNAs, and to short oligo(A)4-5 tails on nuclear surveillance intermediates. This lead to the identification of a novel class of promoter-proximal ncRNAs, that we suggest arise from early termination within protein-coding genes.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE46742 | GEO | 2013/08/29

SECONDARY ACCESSION(S): PRJNA202221

REPOSITORIES: GEO

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Project description:Long noncoding RNAs (lncRNAs) have emerged as important regulators of diverse cellular processes, but their roles in the developing immune system are poorly understood. In this study, we analysed lncRNA expression during human B-cell development by array-based expression profiling of eleven distinct flow-sorted B-cell subsets, comprising pre-B1, pre-B2, immature, naive, memory, and plasma cells from bone marrow biopsies (n=7), and naive, centroblast, centrocyte, memory, and plasmablast cells from tonsil tissue samples (n=6), respectively. A remapping strategy was used to assign the array probes to 37630 gene-level probe sets, reflecting the most recent updates in genomic and transcriptomic databases, which enabled expression profiling of 19579 long noncoding RNAs, comprising 3947 antisense RNAs, 5277 lincRNAs, 7625 pseudogenes, and 2730 additional lncRNAs. As a first step towards inferring the functions of the identified lncRNAs in developing B-cells, we analysed their co-expression with well-characterized protein-coding genes, a method known as â??guilt by associationâ??. By using weighted gene co-expression network analysis, we identified 272 lincRNAs, 471 antisense RNAs, 376 pseudogene RNAs, and 64 lncRNAs within seven sub-networks associated with distinct stages of B-cell development, such as early B-cell development, B-cell proliferation, affinity maturation of antibody, and terminal differentiation. These data provide an important resource for future studies on the functions of lncRNAs in development of the adaptive immune response, and the pathogenesis of B-cell malignancies that originate from distinct B-cell subpopulations. Tonsils were collected from six patients (homo sapiens) during routine tonsillectomy. Mononuclear cells were isolated from tonsils and prepared for multiparametric flow cytometry using an optimized and validated protocol. B-cell subsets of naive, centroblasts, centrocytes, menory and plasmablast cells were isolated and a total of 30 gene expression profiles were generated using the HuEx-1_0-st-v2-micro array chip from Affymetrix.

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A transcriptome-wide atlas of RNP compositions reveals diverse classes of mRNAs and lncRNAs

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