Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

Rädle, Bernd; Rutkowski, A.; Ruzsics, Zsolt; Friedel, Caroline C.; Koszinowski, Ulrich H.; Dölken, Lars

doi:10.3791/50195

Cited by 86 publications

(79 citation statements)

References 21 publications

(7 reference statements)

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“…Biotinylation and purification of 4TU-containing RNA 4TU labeling and purification were done as described in Cleary et al (2005) and Rädle et al (2013). RNA was eluted from the columns by adding 100 µL of 5% 2-mercaptoethanol to the columns; this was repeated 3 min later for a second elution, after which samples were immediately treated to remove DNA using RNeasy kits (QIAGEN).…”

Section: Identification Of Spliceosomal Rnasmentioning

confidence: 99%

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

Nakamoto

Lovejoy

Cygan

et al. 2017

RNA

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RNA contains over 100 modified nucleotides that are created post-transcriptionally, among which pseudouridine (Ψ) is one of the most abundant. Although it was one of the first modifications discovered, the biological role of this modification is still not fully understood. Recently, we reported that a pseudouridine synthase (TgPUS1) is necessary for differentiation of the singlecelled eukaryotic parasite Toxoplasma gondii from active to chronic infection. To better understand the biological role of pseudouridylation, we report here gel-based and deep-sequencing methods to identify TgPUS1-dependent Ψ's in Toxoplasma RNA, and the use of TgPUS1 mutants to examine the effect of this modification on mRNAs. In addition to identifying conserved sites of pseudouridylation in Toxoplasma rRNA, tRNA, and snRNA, we also report extensive pseudouridylation of Toxoplasma mRNAs, with the Ψ's being relatively depleted in the 3 ′ ′ ′ ′ ′ -UTR but enriched at position 1 of codons. We show that many Ψ's in tRNA and mRNA are dependent on the action of TgPUS1 and that TgPUS1-dependent mRNA Ψ's are enriched in developmentally regulated transcripts. RNA-seq data obtained from wild-type and TgPUS1-mutant parasites shows that genes containing a TgPUS1-dependent Ψ are relatively more abundant in mutant parasites, while pulse/chase labeling of RNA with 4-thiouracil shows that mRNAs containing TgPUS1-dependent Ψ have a modest but statistically significant increase in half-life in the mutant parasites. These data are some of the first evidence suggesting that mRNA Ψ's play an important biological role.

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Section: Identification Of Spliceosomal Rnasmentioning

confidence: 99%

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

Nakamoto

Lovejoy

Cygan

et al. 2017

RNA

View full text Add to dashboard Cite

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“…Total RNA was extracted as described above, and 60 µg was treated with TURBO DNase. 4sU biotinylation with an input mixture of human and fly total RNA (200:1) was performed as previously described (Rädle et al 2013;Gregersen et al 2014). To prepare cDNA libraries, TruSeq unstranded mRNA kit v2 was used according to the manufacturer's instructions starting from 50% of eluted 4sU-enriched RNA.…”

Section: Mrna-seq and 4su-seq Library Preparationsmentioning

confidence: 99%

DDX54 regulates transcriptome dynamics during DNA damage response

et al. 2017

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The cellular response to genotoxic stress is mediated by a well-characterized network of DNA surveillance pathways. The contribution of post-transcriptional gene regulatory networks to the DNA damage response (DDR) has not been extensively studied. Here, we systematically identified RNA-binding proteins differentially interacting with polyadenylated transcripts upon exposure of human breast carcinoma cells to ionizing radiation (IR). Interestingly, more than 260 proteins, including many nucleolar proteins, showed increased binding to poly(A) + RNA in IR-exposed cells. The functional analysis of DDX54, a candidate genotoxic stress responsive RNA helicase, revealed that this protein is an immediate-to-early DDR regulator required for the splicing efficacy of its target IR-induced pre-mRNAs. Upon IR exposure, DDX54 acts by increased interaction with a well-defined class of pre-mRNAs that harbor introns with weak acceptor splice sites, as well as by proteinprotein contacts within components of U2 snRNP and spliceosomal B complex, resulting in lower intron retention and higher processing rates of its target transcripts. Because DDX54 promotes survival after exposure to IR, its expression and/or mutation rate may impact DDR-related pathologies. Our work indicates the relevance of many uncharacterized RBPs potentially involved in the DDR.

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“…At the end of labeling, total cellular RNA was isolated using TRIzol reagent (Invitrogen). Biotinylation and purification of 4sU-tagged RNA (newly transcribed RNA) were performed as described previously (46). Following cDNA synthesis using SuperScript III reverse transcriptase (Invitrogen), hexanucleotide random primers (Invitrogen), and the RNase inhibitor RNasin (Promega), reverse transcription-quantitative PCR (qRT-PCR) was performed using TaqMan PCR and the Universal Probe Library (Roche) or SYBR green PCR.…”

Section: Cellsmentioning

confidence: 99%