Biology subjects

Hagelskamp, F.

Publications and source records attributed to Hagelskamp, F..

2 recordsLinked to original sources

bioRxiv2025

ALKBH1 activity in vitro and human cell lines by isotope dilution mass spectrometry

AlkB homolog 1 (ALKBH1) is a member of the AlkB family of Fe(II) and -ketoglutarate (-KG)-dependent dioxygenases, known for its enzymatic activity on various nucleic acid substrates. Its known targets include N1-methyladenosine (m1A), N6-methyladenosine (m6A), N3-methylcytidine (m3C), and 5-methylcytosine (m5C) in RNA, as well as N6-methyladenine (N6-mA, 6mA) in DNA and the histone protein H2A. Moreover, dysregulation or dysfunction of ALKBH1 has been implicated in a broad spectrum of human diseases. In order to shed further light on the substrate scope and role of ALKBH1, we used quantitative mass spectrometry to assess its activity in vitro and in human cell lines. To study ALKBH1 activity on defined substrates in vitro, we enzymatically generated tRNAs specifically carrying the m3C32 (tRNA-ThrUGU and tRNA-SerUCN) and i6A37 (tRNA-SerUCN) modifications. Here we show that ALKBH1 reduces m3C, m1A and m5C in vitro in total extracts of tRNAs, but does not impact on rRNA modifications in human cell lines. However, upon overexpression or siRNA-mediated knock-down of alkbh1 in human cell lines no impact on the modification of total tRNA extracts or specifically enriched RNAs could be observed. In addition, varying the glucose and fetal bovine serum (FBS) concentration in the growth medium of HEK293T cells, in combination with alkbh1 siRNA-mediated knock-down, also shows no impact on the tRNA modification spectra. Based on our data, we conclude that in human cells lines grown under optimal conditions ALKBH1 does not play an important role in the demethylation of tRNAs.

biochemistry↗

bioRxiv2020

Cell culture NAIL-MS allows insight into human RNA modification dynamics in vivo

In the last years, studies about the dynamics of RNA modifications are among the most controversially discussed. As the main reason, we have identified the unavailability of a technique which allows to follow the temporal dynamics of RNA transcripts in human cell culture. Here, we present a NAIL-MS (nucleic acid isotope labeling coupled mass spectrometry) scheme for efficient stable isotope labeling in both RNA and DNA (>95% within 7 days) in common human cell lines and growth media. Validation experiments reveal that the labeling procedure itself does neither interfere with the isotope dilution MS quantification nor with RNA modification density. We design pulse chase NAIL-MS experiments and apply the new tool to study the temporal placement of modified nucleosides in e.g. tRNAPhe and 18S rRNA. In existing RNAs, we observe a constant loss of modified nucleosides over time which is masked by a post-transcriptional methylation mechanism and thus not detectable without NAIL-MS. During alkylation stress, NAIL-MS reveals an adaptation of tRNA modifications in new transcripts but not existing transcripts. Overall, we present a fast and reliable stable isotope labeling strategy which allows a more detailed study of RNA modification dynamics in human cell culture. With cell culture NAIL-MS it is finally possible to study the speed of both modification and demethylation reactions inside human cells. Thus it will be possible to study the impact of external stimuli and stress on human RNA modification kinetics and processing of mature RNA.

biochemistry↗