The presence of modified nucleosides in extracellular fluids leads to the specific incorporation of 5-chlorocytidine into RNA and modulates the transcription and translation

Abstract

Myeloperoxidase (MPO) is able to promote several kinds of damage and is involved in mechanisms leading to various diseases such as atherosclerosis or cancers. An example of these damages is the chlorination of nucleic acids, which is considered as a specific marker of the MPO activity. Since 5-chlorocytidine has been recently shown in healthy donor plasmas, this study aimed at discovering if these circulating modified nucleosides could be incorporated into RNA and DNA and if their presence impacts the ability of enzymes involved in the incorporation, transcription, and translation processes. Experimentations, which were carried out in vitro with endothelial and prostatic cells, showed a large penetration of all chloronucleosides but an exclusive incorporation of 5-chlorocytidine into RNA. However, no incorporation into DNA was observed. This specific incorporation is accompanied by an important reduction of translation yield. Although, in vitro, DNA polymerase processed in the presence of chloronucleosides but more slowly than in control conditions, ribonucleotide reductase could not reduce chloronucleotides prior to the replication. This reduction seems to be a limiting step, protecting DNA from chloronucleoside incorporation. This study shows the capacity of transcription enzyme to specifically incorporate 5-chlorocytidine into RNA and the loss of capacity-complete or partial-of different enzymes, involved in replication, transcription or translation, in the presence of chloronucleosides. Questions remain about the long-term impact of such specific incorporation in the RNA and such decrease of protein production on the cell viability and function.

8-Chloroadenosine induces apoptosis in human coronary artery endothelial cells through the activation of the unfolded protein response

“…The chlorinated ribose nucleotides 8ClA, 8ClG and 5ClC are incorporated into cellular RNA, whereas the chlorinated deoxyribose nucleotides 8CldA, 8CldG and 5CldC, are incorporated into DNA of HCAEC. The lack of incorporation of the chlorinated ribose nucleosides into DNA supports previous data showing that ribonucleotide reductase displays a lack of affinity for chlorinated nucleotides [14,24]. However, this may be cell type specific, as there is evidence for the incorporation of 8ClA into the DNA of human gliomal cell lines for example [39].…”

“…There is increasing evidence for the formation of chlorinated nucleosides in different inflammatory pathologies [9,[13], [14], [15]]. This is attributed to the overproduction of the MPO-derived oxidant HOCl, which reacts readily with RNA and DNA, forming a range of stable chlorinated products [2].…”

“…However, this may be cell type specific, as there is evidence for the incorporation of 8ClA into the DNA of human gliomal cell lines for example [39]. In general, the accumulation of each chlorinated nucleoside increased with incubation time, reaching a maximum level after 24–48 h. It has been shown previously that 5Cl( d )C and 8Cl( d )G can penetrate cells to accumulate in the nucleotide pool of the EA.hy926 endothelial cell line, though in this case, only incorporation of 5ClC was evident in the RNA [14]. This contrasts with our findings in HCAEC, and may reflect the difference between primary cells used in the present study, compared to an immortalised cell line.…”

“…The reaction of HOCl with RNA and DNA forms both unstable chloramines (N–Cl species) [7] and stable chlorinated products, including 8-chloro-adenosine (8ClA), 8-chloro-guanosine (8ClG), 5-chloro-cytidine (5ClC), 8-chloro-2′-deoxyadenosine (8CldA), 8-chloro-2′-deoxyguanosine (8CldG), 5-chloro-2′-deoxycytidine (5CldC) and 5-chloro-uracil (5ClU) [[8], [9], [10], [11], [12]]. A number of these stable products have been detected in elevated amounts in the urine, plasma, inflammatory fluids or the diseased tissues of patients with different pathological conditions [9,13,14], including atherosclerosis [15]. Although chlorinated nucleosides are recognised as biomarkers, it is not well understood as to whether these materials can also play a role in promoting the progression of chronic inflammatory disease.…”

Stealth fluorescence labeling for live microscopy imaging of mRNA delivery

“…Satisfactorily, we observed stable protein expression levels when increasing the tC O content of the transcripts, ranging from 80%-137% of the non-labeled control (Figure 5c). This result suggests that tC O , unlike other sugar- 57 or base-modified 64 nucleotide analogues, does not impair ribosomal processing in cell-free systems.…”