High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay

Dölken, Lars; Ruzsics, Zsolt; Rädle, Bernd; Friedel, Caroline C.; Zimmer, Ralf; Mages, Jörg; Hoffmann, Reinhard; Dickinson, Paul; Forster, Thorsten; Ghazal, Peter; Koszinowski, Ulrich H.

doi:10.1261/rna.1136108

Cited by 395 publications

(581 citation statements)

References 26 publications

(42 reference statements)

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“…Total cellular RNA was prepared with TRIzol (Invitrogen). Newly synthesized RNA obtained with 4-thiouracil labeling of cells at 250 μM in culture medium for 60 min was affinity-purified as described (54). RNA samples were amplified and labeled using the Affymetrix One-Cycle Target Labeling Kit and were hybridized to Affymetrix MG 430 2.0 arrays.…”

Section: Methodsmentioning

confidence: 99%

Nontransformed, GM-CSF–dependent macrophage lines are a unique model to study tissue macrophage functions

Fejér

Wegner

Győry

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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131

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Significance Macrophages—cells crucially involved in defense against infections—exhibit, depending on their anatomical location, distinct biological properties. Studies of the underlying mechanisms are of scientific and clinical interest, but are hampered by the difficulty of obtaining primary tissue macrophages in sufficient numbers and purity. Here, we report the generation of nontransformed murine macrophages, which are similar to alveolar macrophages and can be grown continuously without change of phenotype and in unlimited amounts. Such macrophages helped us to recognize several innate immune properties of alveolar macrophages that are involved in the pathogenesis of infectious lung inflammation.

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Section: Methodsmentioning

confidence: 99%

Nontransformed, GM-CSF–dependent macrophage lines are a unique model to study tissue macrophage functions

Fejér

Wegner

Győry

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

131

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“…NFkB and AP-1 are transcription factors regulating transcription initiation, whereas p38 is a kinase that has been shown to regulate mRNA stability by phosphorylating RNA-binding proteins such as tristetraproline (3). Numerous studies have profiled TNF-induced gene expression and mRNA stabilization in different cells by using steady-state RNA and the transcription inhibitor actinomycin D. However, actinomycin D induces cellular stress responses involving p53 (32) and have been shown to introduce artifacts in mRNA stability determinations (16,33). In this study, we developed Bru-Seq and BruChase-Seq to profile the transcriptome and RNA stabilome of unperturbed human skin fibroblasts at homeostasis and after induction of the proinflammatory response by TNF treatment.…”

Section: Coordinated and Complex Regulation Of The Transcriptome And Rnamentioning

confidence: 99%

Coordinated regulation of synthesis and stability of RNA during the acute TNF-induced proinflammatory response

Paulsen

Veloso

Prasad

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

117

178

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Steady-state gene expression is a coordination of synthesis and decay of RNA through epigenetic regulation, transcription factors, micro RNAs (miRNAs), and RNA-binding proteins. Here, we present bromouride labeling and sequencing (Bru-Seq) and bromouridine pulse-chase and sequencing (BruChase-Seq) to assess genomewide changes to RNA synthesis and stability in human fibroblasts at homeostasis and after exposure to the proinflammatory tumor necrosis factor (TNF). The inflammatory response in human cells involves rapid and dramatic changes in gene expression, and the Bru-Seq and BruChase-Seq techniques revealed a coordinated and complex regulation of gene expression both at the transcriptional and posttranscriptional levels. The combinatory analysis of both RNA synthesis and stability using Bru-Seq and BruChase-Seq allows for a much deeper understanding of mechanisms of gene regulation than afforded by the analysis of steady-state total RNA and should be useful in many biological settings.T he acute inflammatory response is critical for the defense against infections and in the healing of damaged tissues (1). The orchestration of the reprogramming of gene expression associated with the acute inflammatory response is complex and involves both transcriptional and posttranscriptional regulation (2-5). Conventional exploration of gene expression using total RNA does not fully capture this complexity because it does not provide insight into the contribution of nascent RNA synthesis or RNA decay to steady-state RNA changes. A number of different approaches have recently been developed to assess nascent RNA synthesis in cells such as global run-on and sequencing (GROSeq) (6), native elongating transcript sequencing (NET-Seq) (7), nascent RNA sequencing (Nascent-Seq) (8), and metabolic labeling of nascent RNA by using microarrays (9) or RNA-Seq (10, 11). By comparing the data obtained with metabolically labeled nascent RNA with the steady-state RNA levels, the rates of degradation of all transcripts can be computationally estimated. The stability of steady-state RNA can also be estimated from the decay rate of steady-state RNA after transcription inhibition (12)(13)(14) or by immunoprecipitation of metabolically labeled steady-state RNA after different chase periods (15, 16). These approaches work well when the system is at homeostasis, but not when conditions are altered by environmental stimuli or stress, such as the induction of the acute inflammatory response, when the rates of decay of transcripts are expected to change (10,11).In this study, we present Bru-Seq and BruChase-Seq based on bromouridine pulse labeling of nascent RNA followed by chases in uridine to obtain RNA populations of specific ages. The Brulabeled RNA is then immunocaptured followed by deep sequencing. These techniques allowed us to assess changes in the rates of both synthesis and degradation of RNA globally after the activation of the proinflammatory response by TNF. Our results provide a comprehensive and complex picture of the contribution of tr...

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“…For thiouridine labeling, cells were incubated with 500 mM thiouridine for the indicated times. RNA was then isolated with Tri Reagent (Sigma) and the labeled RNA purified by coupling with biotin, capture on streptavidin paramagnetic particles (Promega), and elution with b mercaptoethanol (Dolken et al 2008). The eluates were precipitated with RNase free glycogen (Roche) as a carrier and dissolved in water.…”

Section: Reagentsmentioning

confidence: 99%

Inhibition of polyadenylation reduces inflammatory gene induction

Kondrashov

Meijer

Barthet-Barateig

et al. 2012

RNA

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Cordycepin (39 deoxyadenosine) has long been used in the study of in vitro assembled polyadenylation complexes, because it terminates the poly(A) tail and arrests the cleavage complex. It is derived from caterpillar fungi, which are highly prized in Chinese traditional medicine. Here we show that cordycepin specifically inhibits the induction of inflammatory mRNAs by cytokines in human airway smooth muscle cells without affecting the expression of control mRNAs. Cordycepin treatment results in shorter poly(A) tails, and a reduction in the efficiency of mRNA cleavage and transcription termination is observed, indicating that the effects of cordycepin on 39 processing in cells are similar to those described in in vitro reactions. For the CCL2 and CXCL1 mRNAs, the effects of cordycepin are post-transcriptional, with the mRNA disappearing during or immediately after nuclear export. In contrast, although the recruitment of RNA polymerase II to the IL8 promoter is also unaffected, the levels of nascent transcript are reduced, indicating a defect in transcription elongation. We show that a reporter construct with 39 sequences from a histone gene is unaffected by cordycepin, while CXCL1 sequences confer cordycepin sensitivity to the reporter, demonstrating that polyadenylation is indeed required for the effect of cordycepin on gene expression. In addition, treatment with another polyadenyation inhibitor and knockdown of poly(A) polymerase a also specifically reduced the induction of inflammatory mRNAs. These data demonstrate that there are differences in the 39 processing of inflammatory and housekeeping genes and identify polyadenylation as a novel target for anti-inflammatory drugs.

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High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay

Cited by 395 publications

References 26 publications

Nontransformed, GM-CSF–dependent macrophage lines are a unique model to study tissue macrophage functions

Nontransformed, GM-CSF–dependent macrophage lines are a unique model to study tissue macrophage functions

Coordinated regulation of synthesis and stability of RNA during the acute TNF-induced proinflammatory response

Inhibition of polyadenylation reduces inflammatory gene induction

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