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: smh85@cam.ac.uk

[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