In-vivo targeted tagging of RNA isolates cell specific transcriptional responses to environmental stimuli and identifies liver-to-adipose RNA transfer

J. Darr; M. Lassi; Archana Tomar; R. Gerlini; F. Scheid; M. Hrabe de Angelis; M. Witting; R. Teperino

n we could not detect any signs of 157 DNA damage or apoptosis in the liver during the different treatment regimens as demonstrated by 158 staining for phosph-P53 and cleaved Caspase-3, supporting HD5EU as a non-toxic agent (Sup To validate that nuclear staining evident in-vitro and in-vivo following HD5EU treatment is indeed 177 indicative of 5EU incorporation into transcribing RNA, we adopted the mass-spectrometry method 178 described by Su et. al [41] . Using a column with a smaller inner diameter and lower flow rates to 179 improve the response of individual nucleotides, we were able to identify a wide range of 180 unmodified and modified nucleotides (Sup. Table 1 ). 5EU (m/z 269.0768) was well separated quantify 5EU incorporation into both short (less than 200bp) and long RNA isolated from the liver 195 of HD5EU treated mice, 2 hours following the administration of the compound (Figure 3f-g) . 8h 196 following HD5EU administration 5EU was still detectable in long RNA (though it could not be 197 accurately quantified as it was below quantification limit), whilst only a moderate reduction was 198 detected in short RNA. 199 Taken together, these results confirm that HD5EU is metabolized in a CYP3A4 dependent manner 200 to 5EU, which is then incorporated into transcribing RNA. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/670398 doi: bioRxiv preprint kidney of saline treated control mice (Sup. Figure 3a -e). In addition, following a 2 hour treatment 221 with HD5EU we failed to generate libraries following biotinylation and pull-down from plasma 222 and from additional tissues of the HD5EU-treated mice, this in contrast to liver and kidney where 223 expected library amplicons were generated (Sup. Figure 3a- To further assess the levels of non-specific RNA pull-down, we prepared a 10:1 mixture of non-229 labeled small RNAs from S. Cerevisiae with labeled small RNAs derived from mouse liver. reprogramming. 237 We continued to examine if in-vivo labeling enriches for transcriptional programs of specific 238 cellular populations within complex tissues in-vivo, such as the proximal renal epithelial cells in 239 the kidney and hepatocytes in liver, and whether detection of environmentally induced 240 transcriptional reprogramming is possible. To this end we fed mice with high fat (HFD) or control 241 low fat (LFD) diets for two weeks. Following this acute HFD exposure, which is expected to alter 242 the transcriptional program in the liver [42], we administered HD5EU 2 hours before sacrificing. 243 We then continued to generate poly-A RNA libraries from kidney and liver input and pull-down 244 RNA.