Transcript Dynamics of Proinflammatory Genes Revealed by Sequence Analysis of Subcellular RNA Fractions

Abstract

SUMMARY Macrophages respond to inflammatory stimuli by modulating the expression of hundreds of genes in a defined temporal cascade, with diverse transcriptional and post-transcriptional mechanisms contributing to the regulatory network. We examined pro-inflammatory gene regulation in activated macrophages by performing RNA-Seq with fractionated chromatin-associated, nucleoplasmic, and cytoplasmic transcripts. This methodological approach allowed us to separate the synthesis of nascent transcripts from transcript processing and the accumulation of mature mRNAs. In addition to documenting the sub-cellular locations of coding and non-coding transcripts, the results provide a high-resolution view of the relationship between defined promoter and chromatin properties and the temporal regulation of diverse classes of co-expressed genes. The data also reveal a striking accumulation of full-length yet incompletely spliced transcripts in the chromatin fraction, suggesting that splicing often occurs after transcription has been completed, with transcripts retained on the chromatin until fully spliced.

Isolation of the protein and RNA content of active sites of transcription from mammalian cells

“…Similarly, there exist many methods for isolating and characterizing newly-synthesized RNA, for example "GRO-seq" 15 , "mNET-seq" 16,17 , "chromatin RNA-seq" 18 , "poly(A)-depleted RNA-seq" 19 , "nascent-seq" 20 , and the metabolic tagging of newly-made RNA using 4-thiouridine 21,22 . Each of these has its own particular shortcomings, and all are relatively laborious and/or require a high sequencing depth; most also focus on particular parts of the transcriptome (for a comparison of RNA-seq methods, see Table 1).…”

Inducible RasGEF1B circular RNA is a positive regulator of ICAM-1 in the TLR4/LPS pathway

“…17 We successfully identified a total of 1916 circRNAs across different subcellular fractions and treatment conditions (Table S1). We then compared the performance and validity of our annotation-based pipeline with published pipeline.…”

“…17 The sequences of all possible circular splice junctions within the same gene based on annotated exons (the ENSEMBL63 annotation and the mm9 version of the mouse genome were used) were compiled, retaining RL15 bp on each side of the junctions (equivalent to requiring at minimal length of 15bp for spliced alignment overhangs) where RL is the read length. The circular junction sequences were then combined with the sequences of the full-length annotated transcripts and a Bowtie index was created, which was used to align reads that do not map to the whole genome sequence.…”

“…17 While mouse macrophages produce thousands of distinct circRNAs species at different time points after LPS stimulation, we focused on one of them, mouse circRasGEF1B (mcircRasGEF1B), which we demonstrate to function as a positive regulator of LPS response and to be conserved between mouse and human. We showed that several TLR pathways regulate the expression of mcircRasGEF1B, including TLR4, TLR9, TLR3 and TLR2/TLR1.…”

Toward an Understanding of the Gene-Specific and Global Logic of Inducible Gene Transcription

“…Our laboratory has relied extensively on the fourth method, in which chromatin is first separated from the cytoplasm and nucleoplasm of a cell population by biochemical fractionation (Bhatt et al 2012). RNAs that purify with the chromatin fraction include recently transcribed RNAs and RNAs being actively transcribed.…”