metabolism of 5-methylcytosine in rna of 5-methylcytosine in rna sabrina m. huber, pieter van delft,...
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Synthesis of stable isotope labelled standards (SILs)
hm5C + 3 Da
m5C + 4 Da
C + 3 Da
UHPLC-MS/MS nucleoside quantification method
Purified RNA is spiked with a known amount of stable isotope-labelled (SIL) standards and enzymatically digested to ribonucleosides. These are separated on an ultra-high performance liquid chromatography (UHPLC) column and quantified using tandem mass spectrometry (MS/MS).
cells
tissue
RNA isolation
enzymatic RNA digestion
SIL spiking
UHPLC-MS/MS
NNH2
ON
O
OHOH
HO
D3C
D
3.8 4.0 4.2 4.4 4.60
250
500
750
1000
Time [min]3.8 4.0 4.2 4.4 4.60
250
500
750
1000
Time [min]3.8 4.0 4.2 4.4 4.60
250
500
750
1000
Time [min]
N
NH2
ON
H3C
HR
N
NH2
ON
D3C
DR
N
NH2
ON
HO
R
m5C:m/z = 258 → 126.06619 ± 5 ppm
m5C + 4 Da:m/z = 262 → 130.09130 ± 5 ppm
hm5C:m/z = 274 → 142.06110 ± 5 ppm
NHO
ONO
OHOH
HO
NNH2
ONO
OHOH
HO
H3C
NH
N
NO
NH2NO
OHOH
HO
NNH2
ON
O
OHOH
HO
D3C
D
4.33 min4.04 min4.04 min
Synthesis of [2-13C,1,3-15N2]-cytidine (C + 3 Da), 5-methyl-D3-[6-D]-cytidine (m5C + 4 Da), and 5-hydroxymethyl-[2-13C,1,3-15N2]-cytidine (hm5C + 3 Da). Asterisks (*) represent a 15N or 13C isotopologue.
Introduction
More than 150 modified nucleosides exist in RNA, but the precise function and significance of nearly all of them remain unknown.[1] 5-methylcytosine (m5C) is one of the most abundant modifications and can influence RNA stability, processing, and protein binding.[2-4] We probe the dynamics of m5C and its metabolic derivatives in RNA to better understand the biological role of modified cytosine residues.
Metabolism of 5-methylcytosine in RNA Sabrina M. Huber, Pieter van Delft, Eric A. Miska, Shankar Balasubramanian Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK
Contact: [email protected]
[1] M. A. Machnicka et al., Nucleic Acids Res. 2013, 41, D262 – 267; [2] M. Schaefer et al., Genes Dev. 2010, 24, 1590 – 1595; [3] S. Hussain et al., Cell Reports 2013, 4, 255 – 261; [4] T. Amort et al., RNA Biology 2013, 10, 1 – 7.
Bibliography
hm5C is pervasive across all three domains of life
hm5C was detected in total RNA from various model organisms. Its levels are species dependent and up to a 1000-fold lower than those of m5C.
hm5C in RNA may be evolutionary conserved RNA N
NH2
ON
O
OHO
O
13CD
D
D
NNH2
ON
O
OHO
O
13CD
HO
D
NNH2
ON
O
OHO
O
13C
O
DCO2H
NH2213CD3S
[Ox] [Ox]
In vivo oxidation of m5C to hm5C and f5C
Upon feeding mice with L-methionine-(methyl-13C,D3), 13C and D isotope incorporation was observed into m5C, 5-hydroxymethylcytosine (hm5C) and 5-formylcytidine (f5C), establishing them as metabolic derivatives of m5C.
Transcription-independent formation of hm5C
Freshly transcribed cellular RNA was labelled with 15N2-cytidine and methylation and hydroxymethylation thereof was marked with 13C and D isotopes. While m5C is established co-transcriptionally, its oxidation to hm5C boosts at a specific stage during the cell cycle.
! purification of specific RNA types and quantification of their modifications " polyA RNA, tRNA & rRNA, small RNAs
! identification of proteins binding specifically to m5C- or hm5C-modified RNA
Future perspectives
0 5 10 150
17
34
51
68
15N213CD3-m5C
15N2-C
15N213CD2-hm5C
time [h]
labe
lling
effi
cien
cy [%
]
Poster: S. M. Huber et al., ChemBioChem 2015, 16, 752 – 755.
N
NH2
ON
H3C N
NH2
ON
HO
m5C hm5C
[Ox]
?structure
stability
protein binding
processing???
M. jannas
chii
E. coli
A. thali
ana
C. eleg
ans
Mouse b
rain
0.0001
0.001
0.01
0.1
1
10m5C hm5C
% C
mod
/ ( C
+ m
5 C +
hm
5 C)
H2N NH2
O
HH
ONH
NH
O
O O
OBzBzO
BzO
N
NH2
ONO
OHOH
HO
polyphosphoricacid*
***
** N
NH
O
O*
**
* **
N
NH2
ONO
OO
HO * **HO N
NH2
ONO
OHOH
HO * **HO
NH
NH
O
O
D3C
D
O
OHOH
HO N
NH
NH2
O
D3C
D
O
OBzBzO
BzO N
N
NH2
O***
O
OBzBzO
BzO OAc
1. TMSCl, HMDS
, TMSOTf2.
NN
NPO
3
2. NH4OH
1.
NaOMe
1. H2SO4, acetone2. paraformaldehyde, KOH
90 % TFA
NaOMe