2020
DOI: 10.1002/cpch.89
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Visualizing RNA Cytidine Acetyltransferase Activity by Northern Blotting

Abstract: Cytidine acetyltransferases are an emerging class of nucleic‐acid‐modifying enzymes responsible for the establishment of N4‐acetylcytidine (ac4C) in RNA. In contrast to histone acetyltransferases, whose activity is commonly studied by western blotting, relatively few methods exist for quickly assessing the activity of cytidine acetyltransferases from a biological sample of interest or the distribution of ac4C across different RNA species. In this protocol, we describe a method for analysis of cellular cytidine… Show more

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Cited by 4 publications
(6 citation statements)
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“…RNA was transferred onto Amersham Hybond-N+ membranes (GE Healthcare) using a downward capillary method as described previously. 45 After transfer, membranes were crosslinked three times at 150 mJ/ cm 2 in a UV 254nm Stratalinker 2400 (Stratagene). Membranes were then blocked with 5% nonfat milk in 0.1% TBST for 30 min at room temperature and washed three times at 5 min each in 0.1% TBST.…”
Section: ■ Materials and Methodsmentioning
confidence: 99%
See 1 more Smart Citation
“…RNA was transferred onto Amersham Hybond-N+ membranes (GE Healthcare) using a downward capillary method as described previously. 45 After transfer, membranes were crosslinked three times at 150 mJ/ cm 2 in a UV 254nm Stratalinker 2400 (Stratagene). Membranes were then blocked with 5% nonfat milk in 0.1% TBST for 30 min at room temperature and washed three times at 5 min each in 0.1% TBST.…”
Section: ■ Materials and Methodsmentioning
confidence: 99%
“…Loading controls were analyzed by imaging ethidium bromide before transfer. RNA was transferred onto Amersham Hybond-N+ membranes (GE Healthcare) using a downward capillary method as described previously . After transfer, membranes were crosslinked three times at 150 mJ/cm 2 in a UV 254nm Stratalinker 2400 (Stratagene).…”
Section: Methodsmentioning
confidence: 99%
“…This emphasizes the need for orthogonal validation when establishing the presence of novel ac 4 C sites and also poses important mechanistic considerations as to their function when putatively detected. The conclusion that the dominant sites of eukaryotic ac 4 C reside in rRNA and tRNA validates prior work and is also intuitive based on antiac 4 C northern blotting analysis . This led us to explore how ac 4 C-seq could enable mechanism and discovery efforts in these more quantitatively abundant contexts.…”
Section: Ac4c-seq Defines the Dominant Sites Of Ac4c In Eukaryotic Rnamentioning
confidence: 99%
“…This approach yielded a number of unexpected discoveries. Sequencing and antiac 4 C northern blotting , specified nematodes ( C . elegans ) as a model organism that evolutionarily dispenses with rRNA acetylation (Figure b).…”
Section: Profiling Rna-mediated Rna Acetylation Across Evolutionmentioning
confidence: 99%
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