circRNA biogenesis competes with pre-mRNA splicing, Mol. Cell, vol.56, pp.55-66, 2014. ,
Nucleophosmin-anaplastic, 1998. ,
Chromosomal translocations induced at specified loci in human stem cells, Proc. Natl. Acad. Sci. USA, vol.106, pp.10620-10625, 2009. ,
circRNA_100290 plays a role in oral cancer by functioning as a sponge of the miR-29 family, Oncogene, vol.36, pp.4551-4561, 2017. ,
Targeted genomic rearrangements using CRISPR/Cas technology, Nat. Commun, vol.5, p.3728, 2014. ,
Mis-splicing yields circular RNA molecules, FASEB J, vol.7, pp.155-160, 1993. ,
The RNA binding protein quaking regulates formation of circRNAs, Cell, vol.160, pp.1125-1134, 2015. ,
Mll fusions generated by Cre-loxP-mediated de novo translocations can induce lineage reassignment in tumorigenesis, EMBO J, vol.24, pp.3136-3146, 2005. ,
FUS affects circular RNA expression in murine embryonic stem cell-derived motor neurons, Nat. Commun, vol.8, p.14741, 2017. ,
, , 2007.
, , 2007.
, , 2013.
Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma, Science, vol.263, pp.1281-1284, 1994. ,
Translation of CircRNAs, Mol. Cell, vol.66, pp.9-21, 2017. ,
, Cancer translocations in human cells induced by zinc finger and TALE nucleases, 2013.
, Genome Res, vol.23, pp.1182-1193
Creating cancer translocations in human cells using Cas9 DSBs and nCas9 paired nicks, Methods Enzymol, vol.546, pp.251-271, 2014. ,
Circular RNAs in the mammalian brain are highly abundant, conserved, and dynamically expressed, Mol. Cell, vol.58, pp.870-885, 2015. ,
TEL-AML1 corrupts hematopoietic stem cells to persist in the bone marrow and initiate leukemia, Cell Stem Cell, vol.5, pp.43-53, 2009. ,
Roles of circular RNAs in neurologic disease, Front. Mol. Neurosci, vol.9, p.25, 2016. ,
Engineering human tumour-associated chromosomal translocations with the RNAguided CRISPR-Cas9 system, ALK-positive anaplastic large cell lymphoma with the variant RNF213-, ATIC-and TPM3-ALK fusions is characterized by copy number gain of the rearranged ALK gene, vol.5, pp.1605-1616, 2014. ,
, , 2002.
, Anaplastic lymphoma kinase (ALK) activates Stat3 and protects hematopoietic cells from cell death, Oncogene, vol.21, pp.1038-1047
Multilevel dysregulation of STAT3 activation in anaplastic lymphoma kinase-positive T/null-cell lymphoma, J. Immunol, vol.168, pp.466-474, 2002. ,
Diverse alternative back-splicing and alternative splicing landscape of circular RNAs, Genome Res, vol.26, pp.1277-1287, 2016. ,
, Circular intronic long noncoding RNAs, 2013.
, Mol. Cell, vol.51, pp.19-29, 2018.
, IL-3 starved and wild-type Ba/F3 cells were then seeded at 10
For detection of translocation junctions from pooled cells expressing CRISPR/Cas9, we performed a nested PCR on 50 ng of genomic DNA as previously described, Genomic DNA from cells was extracted using E.Z.N.A Tissue DNA Kit (Omega Biotek), 2014. ,
Briefly one-round PCR was performed on four serial dilutions of DNA from transfected cells, 2014. ,
, RT-PCR and cDNA amplification of fusion transcript 5 days after transfection, RNA was extracted using Nucleospin RNA Kit
, RT-PCR was performed using ProtoScript® II First Strand cDNA Synthesis Kit (NEB) according to manufacturer's instructions. Primers sequences are listed in Table S4
PCR product was cloned between NheI and EcoRI sites in pLJM1-EGFP plasmid (Addgene Plasmid 19319). pLJM1-NPM1-ALK vector was then transfected in RPEheating at 95°C for 10 minutes. First-round PCR was performed on lysate with primers used for translocation junction detection (listed below). Second-round PCR was performed with SYBR green in real-time PCR conditions, NPM1-ALK cDNA overexpression NPM1-ALK cDNA was amplified by PCR from SUDHLI cells using primers containing NheI and EcoRI sites, 2009. ,
, X-100, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, and 1 mM DTT
Thermo Fisher Scientific). Typically, 30 µg of protein extracts were run on an 8% (w/v) Tris-HCl SDS PAGE gel. For SUDHL1 control cell line, 10 µg of protein extract were used. Gel was blotted, and probed with Phospho-ALK (Tyr1604), ALK (31F12), Phospho-STAT3 (Tyr705), Stat3, Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), p44/42 MAPK (ERK1/2), Phospho-AKT (Thr308), Phospho-AKT (Ser473), and AKT antibodies from Cell Signaling. Murine NPM1-ALK protein expression was assessed with ALK (M19) antibody, with addition of Complete cocktail protease inhibitor tablets (Roche) and Halt Protease and Phosphatase Inhibitor Cocktail ,
, Other antibodies were used according to manufacturer's recommendations. Secondary antibodies were HRP-conjugated, and blot developed with ECL prime (GE Healthcare Life)
, Murine Alk and Npm1 genes were labeled using homemade probes from BAC RP23-100F5 and RP23-74P19. Mll and Af9 genes were labeled using homemade probes from BAC RP23-23P9 and RP23-440D4. Metaphase spreading, probe labeling, hybridization, washing and fluorescence detection were performed according to standard procedures, homemade NPM1 probe from BAC RP11-546B8