Page 3 - W. RNA biology

NMR structural investigation of pre-miRNA-155
that is involved in human cancer development

So-young Kim , Ji Yeon Shin , Kyeong-Mi Bang , Sungnam Park , Nak-Kyoon Kim 1,*
1
1
2
1,2
1. Advanced Analysis Center, Korea Institute of Science and Technology, Hwarang-ro 14-gil 5 Seongbuk-gu Seoul, Republic of Korea, *nkkim@kist.re.kr
2. Department of Chemistry, Korea University, Korea, 145 Anam-ro Seongbuk-gu Seoul, Republic of Korea
BACKGROUND Abstract
MicroRNAs (miRNAs) are short, non-coding single stranded RNAs (ssRNAs) found
in eukaryotic cells and some viruses. Pre-miRNAs are processed into mature
miRNA duplexes by an enzyme called Dicer. MiRNA-155 is one of the miRNAs that
suppresses apoptosis in human cancer, causing breast, pancreatic and lung
cancer. Previously, we showed that a synthetic peptide bound to the major groove
of pre-miRNA-155 inhibit the Dicer mediated maturation of the pre-miRNA-155.
Therefore, it is important to understand the structural characteristics of pre-miRNA-
155, which are important for recognition of the interacting peptide. Accordingly,
NMR spectroscopy was used to obtain sophisticated tertiary structures of pre-
miRNA-155 in solution. At present, in addition to the previously assigned H and
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 A process from pre-miRNA 13 1 13
to mature-miRNA by Dicer C resonances, all of the rest H and C resonances are being assigned. To avoid
the spectral cloudiness observed in large RNA, base-specific 13 C- and 15 N- labeled
RNAs were synthesized and a series of filtered/edited NOE experiments were
examined. With NOE based distance information, an exact structure of the pre-
miRNA-155 will be calculated, and the specific characteristics of miRNA-155 will be
Peptide is located in the major groove identified.
of the apical stem-loop region of pre-
miRNA-155
Binding of peptide to pre-miRNA-155 AIM
monitored by 1H-15N HSQC
Chemical shift perturbations upon  To determine and characterize the 3D solution structure of pre-miRNA-155
addition of peptide to pre-miRNA-155  To elucidate the specific tertiary interaction between pre-miRNA-155 and the
indicates that peptide bind to the peptide
apical loop of pre-miRNA-155
METHODS
 Nonlabeled, uniformly and base-specifically 13 C, N-labeled, truncated pre-miRNA-155 was prepared in vitro by transcription with T7
15
polymerase using synthetic DNA templates.
 All NMR spectra were acquired with a Bruker Avance Ⅲ HD 800MH spectrometer equipped with a cryogenic probe.
 NMR Spectra were processed with Bruker Topspin 3.5 pl 7 and analyzed with Sparky 3.114 (University of California, San Fransisco,CA)
RESULTS

 2D NOESY spectrum of pre-miRNA-155

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• Base specific C and N labeled RNA
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• 2D HCCH-COSY spectra provide sequential correlation of
H1’-H4’ of the sugar ring
Representative 3D HCCH-TOCSY spectra with A-labeled
pre-miRNA 155 (w(H)-w2(C), and w2(c)-w3(H))
• 2D TOCSY : H5 and H6
peaks of C and U •Sugar carbons
• 2D COSY : AU and GC (C1',C2’,C3’,C4’)
13 C, N- labeled RNA, & sugar protons
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H2’, H3’ assignments (H1',H2’,H3’,H4')
• 2D 11echo NOESY : in 3D HCCHTOCSY
sequential assignment of •Like this way, all residues
imino (NH) protons in
helical region of RNA have been assigned.
W1-W2 dimension W2-W3 dimension
CONCLUSION ACKNOWLEDGEMENTS

• The 80% out of the total resonance assignment in NOESY has been completed. This study was supported financially
• Aromatic H8/H6/H2 have been assigned completely. by KIST (2V08170)
• Sugar proton & carbon ( C1’H1’,C2’H2’, C3’H3’, C4’H4’ ) assignment have been
assigned completely. Contact information
• NOE based distance calculation is under process for RNA structure determination. *nkkim@kist.re.kr