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-v

Cited by 12 publications

(9 citation statements)

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“…Biotinylation was performed as described previously 63 , 64 . Reaction consisted of 100 µg of RNA, 0.01 mg/ml MTSEA Biotin-XX (Biotium, 90066), 20 % DMF, 10 mM Tris (pH = 7.4), 1 mM EDTA.…”

Section: Su-seqmentioning

confidence: 99%

CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1

Hluchý

Gajdušková

Mozos

et al. 2022

Nature

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RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA-protein complex that assembles de novo on each pre-mRNA intron via an ordered assembly of intermediate complexes 1,2 . Spliceosome activation is a major control step requiring dramatic protein and RNA rearrangements leading to a catalytically active complex [1][2][3][4][5] . Splicing factor 3B subunit 1 (SF3B1) protein, a subunit of the U2 snRNP 6 , is phosphorylated during spliceosome activation 7-10 , but the responsible kinase has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N-terminus during spliceosome activation. The phosphorylation is important for association of SF3B1 with U5 and U6 snRNAs in activated spliceosome, termed B act complex, and it can be blocked by OTS964, a potent and selective inhibitor of CDK11. CDK11 inhibition prevents spliceosomal transition from the precatalytic complex B to the activated complex B act and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.

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Section: Su-seqmentioning

confidence: 99%

CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1

Hluchý

Gajdušková

Mozos

et al. 2022

Nature

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“…4SU incorporation was confirmed with dot blot analysis of 4SU labeled RNA, prior to alkylation. 4SU labeled RNA was biotinylated with EZ-link Iodoacetyl-LC-biotin (Pierce), resulting in irreversible biotinylation as described (35). Briefly, 350 µl (50 µg of RNA diluted in nuclease-free H2O) of 4SU labeled RNA was incubated with 50 µl 10x Biotinylation Buffer (100 mM Tris pH 7.4, 10 mM EDTA in nuclease-free H2O) and 100 µl of EZ-link Iodoacetyl-LC-biotin (1 mg/ml in dimethylformamide) at room temperature for 1.5 hr while rotating in dark conditions.…”

Section: Methodsmentioning

confidence: 99%

Non-redundant roles for the human mRNA decapping cofactor paralogs DCP1a and DCP1b

Vukovic,

Baranda,

Ruffin

et al. 2023

Preprint

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Eukaryotic gene expression is regulated at both the transcriptional and post-transcriptional levels, with disruption of regulation contributing significantly to human diseases. In particular, the 5' m7G mRNA cap is the central node in post-transcriptional regulation, participating in both mRNA stabilization and translation efficiency. DCP1a and DCP1b are paralogous cofactor proteins of the major mRNA cap hydrolase DCP2. As lower eukaryotes have a single DCP1 cofactor, the functional advantages gained by this evolutionary divergence remain unclear. Here we report the first functional dissection of DCP1a and DCP1b, demonstrating that DCP1a and DCP1b are distinct, non-redundant cofactors of the decapping enzyme DCP2, with unique roles in decapping complex integrity and specificity. Specifically, DCP1a is essential for decapping complex assembly and for interactions between the decapping complex and mRNA cap binding proteins. In contrast, DCP1b is essential for decapping complex interactions with the translational machinery. Additionally, DCP1a and DCP1b impact the turnover of distinct mRNAs. The observation that different ontological groups of mRNA molecules are regulated by DCP1a and DCP1b, along with their non-redundant roles in decapping complex integrity, provides the first evidence that these highly similar paralogs have qualitatively distinct functions. Furthermore, these observations implicate both DCP1a and DCP1b in transcript buffering.

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“…Dot blot analysis was performed by following published protocols (Rädle et al., 2013; Sharma et al., 2018). Briefly, RNA was applied with a biotin‐labelled DNA oligonucleotide (positive control; Integrated DNA Technologies) to a BrightStar‐Plus positively charged nylon membrane (Thermo Fisher); then, the membrane was air‐dried at room temperature for 5 min, blocked with 10% sodium dodecyl sulphate (SDS) in PBS and 1 mM EDTA at room temperature for 30 min, incubated in 10% SDS/PBS with HRP‐conjugated streptavidin (Thermo Fisher Scientific) at room temperature for 15 min, and washed for 5 min with each of three progressively declining concentrations of SDS (10%, 1%, and 0.1%) in PBS.…”

Section: Methodsmentioning

confidence: 99%

Metabolic labeling of cardiomyocyte‐derived small extracellular‐vesicle (sEV) miRNAs identifies miR‐208a in cardiac regulation of lung gene expression

Han

Yang

Zhang

et al. 2022

J of Extracellular Vesicle

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Toxoplasma gondii uracil phosphoribosyltransferase (UPRT) converts 4‐thiouracil (4TUc) into 4‐thiouridine (4TUd), which is incorporated into nascent RNAs and can be biotinylated, then labelled with streptavidin conjugates or isolated via streptavidin‐affinity methods. Here, we generated mice that expressed T. gondii UPRT only in cardiomyocytes ( CM UPRT mice) and tested our hypothesis that CM‐derived miRNAs ( CM miRs) are transferred into remote organs after myocardial infarction (MI) by small extracellular vesicles (sEV) that are released from the heart into the peripheral blood ( PB sEV). We found that 4TUd was incorporated with high specificity and sensitivity into RNAs isolated from the hearts and PB sEV of CM UPRT mice 6 h after 4TUc injection. In PB sEV, 4TUd was incorporated into CM‐specific/enriched miRs including miR‐208a, but not into miRs with other organ or tissue‐type specificities. 4TUd‐labelled miR208a was also present in lung tissues, especially lung endothelial cells (ECs), and CM‐derived miR‐208a ( CM miR‐208a) levels peaked 12 h after experimentally induced MI in PB sEV and 24 h after MI in the lung. Notably, miR‐208a is expressed from intron 29 of α myosin heavy chain (αMHC), but αMHC transcripts were nearly undetectable in the lung. When PB sEV from mice that underwent MI (MI‐ PB sEV) or sham surgery (Sham‐ PB sEV) were injected into intact mice, the expression of Tmbim6 and NLK, which are suppressed by miR‐208a and cooperatively regulate inflammation via the NF‐κB pathway, was lower in the lungs of MI‐ PB sEV–treated animals than the lungs of animals treated with Sham‐ PB sEV or saline. In MI mice, Tmbim6 and NLK were downregulated, whereas endothelial adhesion molecules and pro‐inflammatory cells were upregulated in the lung; these changes were significantly attenuated when the mice were treated with miR‐208a antagomirs prior to MI surgery. Thus, CM UPRT mice enables us to track PB sEV‐mediated transport of CM miRs and identify an miR‐208a‐mediated mechanism by which myocardial injury alters the expression of genes and inflammatory response in the lung.

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

Cited by 12 publications

References 0 publications

CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1

CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1

Non-redundant roles for the human mRNA decapping cofactor paralogs DCP1a and DCP1b

Metabolic labeling of cardiomyocyte‐derived small extracellular‐vesicle (sEV) miRNAs identifies miR‐208a in cardiac regulation of lung gene expression

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