Medulloblastoma-associated DDX3 variant selectively alters the translation response to stress Sekyung Oh 1* , Ryan A Flynn 2 , Stephen N Floor 3 , Jennifer A Doudna 3 , Howard Y Chang 2 , Yoon-Jae Cho 1,4 1 Catholic Kwandong University, South Korea, 2 Stanford University, USA, 3 University of California, Berkeley, USA, 4 Oregon Health & Science University, USA, * Correspondence: ohskjhmi@gmail.com A C D B solvent accessibility 425 Buried Solvent Exposed 25 eak 534 SG A H R V R T AR A A G I V A A 0 1 132 07 2 5 DDX3X 3 R A Hu an 40 B C D l ent ace Du le un in ing Du le un in ing ATP r l i m A B
C A FLAG-tagge B D c n truct Marker DDX3X WT DDX3X R534H r Rluc 250 150 100 75 FLAG-DDX3X C 50 37 FLAG-Rluc 25 20 15 anti-FLAG IP il er tain A B A B C C D [W. RNA biology-1] Structural and functional analysis of cancer-associated variants of the RNA helicase DDX3 Ryan A Flynn², Stephen N Floor³, Jennifer A Doudna³, Howard Y Chang², Yoon-Jae Cho⁴˙⁵, Sekyung Oh¹˙* ¹Department of Medical Sciences, Catholic Kwandong University, Incheon 22711, South Korea, ²Department of Dermatology, Stanford University, Stanford, CA 94305, USA, ³Department of Molecular and Cell Biology, Universitiy of California,
Berkeley, CA 94720, USA, ⁴Department of Neurology, Stanford University, Stanford, CA 94305, USA, ⁵Department of Pediatrics, Oregon Health and Science University, Portland, OR 97239,USA DDX3X gene, encoding a DEAD-box family RNA helicase DDX3, is frequently mutated in many cancers. The nature and scope of DDX3's interactions with RNA remain unclear, however. We here show DDX3 collaborates extensively with the translation initiation machinery through direct binding to 5'UTRs of nearly all coding RNAs, specific sites on the 18S rRNA, and multiple components of the translation initiation complex.
Impairment of translation initiation is also evident in primary medulloblastomas harboring mutations in DDX3X, further highlighting DDX3's role in this process. Arsenite-induced stress shifts DDX3 binding from the 5'UTR into the coding region of mRNAs concomitant with a general reduction of translation, and both the shift of DDX3 on mRNA and decreased translation are blunted by expression of a catalytically-impaired, cancer-associated DDX3(R534H) variant. Furthermore, despite the global repression of translation induced by arsenite, translation is preserved on select genes involved in
chromatin organization in DDX3(R534H)-expressing cells. Thus, DDX3 interacts extensively with RNA and ribosomal machinery to help remodel the translation landscape in response to stress, while cancer-related DDX3 variants adapt this response to selectively preserve translation. 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 1 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 15 • Base specific C and N labeled RNA 13 • 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 15 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 [W. RNA biology-2] NMR structural investigation of pre-miRNA-155 that is involved in human cancer development So-young Kim¹˙², Ji Yeon Shin¹, Kyeong-Mi Bang¹, Sungnam Park², Nak-Kyoon Kim¹ ¹Advanced Analysis Center, Korea Institute
of Science and Technology, Seoul 02792, Republic of Korea, ²Department of Chemistry, Korea University, Seoul 02841, Republic of Korea 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 1H and 13C resonances, all of the rest 1H and 13C resonances are being assigned. To avoid the spectral cloudiness observed in large RNA, base-specific 13C- and 15N- 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 identified.
