rp, ribosomal protein 60S 47 rpl 40S 32 rps rp mrna rpl30 rps14 rpl12 rpl3 rps13 rp mrna nonsense-mediated mrna decay (NMD) C. elegans smg-2 smg-2 mrna 50 rp 7 rp 4 rpl 4 rp NMD mrna 6 rp mrna rp mrna rpl10a 40 80% rp mrna rp mrna 8 rp mrna mrna rp rp rp smg-2 rp rp RT-PCR rp rp 1. rp rpl-1 rpl-1 1 2 DNA Gateway system (Invitrogen) BP pentr-l1/r5 pentr-l5-(+2)ggs6-mcherry-l2 pentr-l5-ggs6-egfp-l2 pdest-eft-3p pdest-myo-3p Gateway system LR II Plus L10ARE rpl-1 DEL Quickchange (Stratagene) RPL-1 RPL-30 cdna Entry cdna PCR pentr-d/topo (Invitrogen) pdest-eft-3p GST pdest-cold-gst Gateway system LR 2. N2 rpl-1 (LEICA M205 FA) CCD (LEICA DFC310FX) 3. GST-RPL-1 GST-RPL-30 pcold-gst-rpl-1 pcold-gst-rpl-30 BL21 (DE3) plyss (invitrogen) LB Glutathione Sepharose 4B
N2 DNA rpl-1 32 P RNA [α 32 P]UTP T7 RNA polymerase (Takara) in vitro 6%RNA RNA (20 mm HEPES-KOH (ph7.9) 320 mm KCl 5% glycerol 1% Triton X-100 1 mm DTT 0.1 mm PMSF) GST-RPL-1 GST-RPL-30 100 ng/μl trna 50 ng/μl BSA 20 30 4% phosphoimage analyzer (FLA-3000G, Fuji Film) 4. RPL10A HEK293T DNA RPL10A 3 5 pentr-d/topo Destination pdest-cdna3 Gateway system RPL10A DEL Quickchange RPL10A RPL26 Entry cdna pentr-d/topo Destination pdest-me18s Gateway system 5. HeLa NIH 3T3 10% - () DMEM ( ) 100 μg/ml emetin (SIGMA) 6 FuGENE HD (Roche)24 6. RT-PCR RNA RNA I super( ) RQ1 DNase I (Promega) RNeasy Mini (QIAGEN) (BECKMAN COULTER) PrimeScript (TaKaRa) oligo dt (TaKaRa)cDNA ExTaq (TaKaRa) PCR PCR 2100 expert (Agilent) 1 rp 6 mrna 8 rp mrna RT-PCR rp 7 rp mrna 7 rp 8 rp mrna 1 rp rp 2 rpl-1 2 8 rp 8 rp 8 rp rpl-1 2 40 rpl-1 rpl-1 1 (WT ) rpl-1 1 2 RFP GFP 2 N2 WT RFP GFP WT WT RT-PCR 2 WT rpl-1
WT rpl-1 RFP rpl-26-30 GFP RPL-1 cdna GFP RPL-30 cdna WT rpl-1 39 DEL DEL RFP GFP RPL-1 rpl-1 rpl-1 2 rpl-1 RPL10A (L10ARE) 3. RPL-1 L10ARE rpl-1 RPL-1 mrna rpl-1 1 2 4 GST-RPL-1 GST-RPL-1 3 1 2 4 RPL-1 rpl-1 mrna 3 3 L10ARE WT 5 DEL 6 GST-RPL-1 5 6 GST-RPL-30 RPL-1 L10ARE 4. RPL10A rpl-1 L10ARE rpl-1 RPL10A mrna NMD NMD mrna emetin HeLa mrna RPL10A mrna emetin 2 RT-PCR 3 mrna GT-AG HEK293T NIH3T3 RPL10A rpl-1 NMD mrna RPL10A rpl-1 RPL10A WT NIH3T3 RFP RT-PCR RPL10A RPL26 L10ARE RPL10A DEL RFP RPL10A RPL26 rpl-1 RPL10A 5. rp RPL-1 L10ARE RPL-1
mrna smg-2 rpl-1 rpl-26 rpl-30 RNA polya+ RNA 3 rp rp RPL-1 6. 8 rp 2 40 rpl-1 rpl-1 L10ARE RPL-1 L10ARE rpl-1 mrna RPL-1 mrna L10ARE rpl-1 RPL10A NMD L10ARE 1. Kuroyanagi H, Takei S, Suzuki Y. Comprehensive analysis of mutually exclusive alternative splicing in C. elegans. Worm. 3: e28459, 2014. 2.. New Technology. Medical Science Digest 39: 502-503, 2013. 3. Hidehito Kuroyanagi. Switch-like regulation of tissue-specific alternative pre-mrna processing patterns revealed by customized fluorescence reporters. Worm 2: e23834, 2013. 4 Kuroyanagi H, Watanabe Y, Hagiwara M. CELF family RNA-binding protein UNC-75 regulates two sets of mutually exclusive exons of the unc-32 gene in neuron-specific manners in Caenorhabditis elegans. PLoS Genetics. 9: e1003337, 2013. DOI: 10.1371/journal.pgen.1003337. 5. Kuroyanagi H, Watanabe Y, Suzuki Y, Hagiwara M. Position-dependent and neuron-specific splicing regulation by the CELF family RNA-binding protein UNC-75 in Caenorhabditis elegans. Nucleic Acids Research. 41: 4015-4025, 2013. doi: 10.1093/nar/gkt097. 6. Iwasa H, Maimaiti S, Kuroyanagi H, Kawano S, Inami T, Ikeda M, Nakagawa K, Hata Y. Yes-associated protein homolog, YAP-1, is involved in the thermotolerance and aging in the nematode Caenorhabditis elegans. Experimental Cell Research. 319: 931 945, 2013. doi: 10.1016/j.yexcr.2013.01.020. 7. Iwasa H, Kuroyanagi H, Maimaiti S, Ikeda M, Nakagawa K, Hata Y. Characterization of RSF-1, the Caenorhabditis elegans homolog of the Ras-association domain family protein 1. Experimental Cell Research. 319: 1 11, 2013. doi: 10.1016/j.yexcr.2012.10.008. 8. Ohno G, Ono K, Togo M, Watanabe Y, Ono S, Hagiwara M, Kuroyanagi H. Muscle-Specific Splicing Factors ASD-2 and SUP-12 Cooperatively Switch Alternative Pre-mRNA Processing Patterns of the ADF/Cofilin Gene in Caenorhabditis elegans. PLoS Genetics. 8: e1002991, 2012. doi: 10.1371/journal.pgen.1002991. 1.. mrna. 169 2013 11 9 2.. rp. 2013 9 28 3.. mrna. 85 2013 9 19-21 4.,. rp 15 RNA 2013 7 24-26 5. Takei, S., & KUROYANAGI, H. Ribosomal Protein L1 regulates alternative splicing of its own pre-mrna. 19th International C. elegans Meeting. 2013 6 26-30. 6. Genta Ohno, Kanako Ono, Marina Togo, Yohei Watanabe, Shoichiro Ono, Masatoshi Hagiwara, Hidehito Kuroyanagi. Muscle-Specific Splicing Factors ASD-2 and SUP-12 Cooperatively
Switch Alternative Pre-mRNA Processing Patterns of the ADF/Cofilin Gene in C. elegans. Cold Spring Harbor Asia conferences on RNA Biology. 2012 10 8-12. 7.,,,. RNA II LST-3 mrna 14 RNA 2012 7 18-20 http://www.tmd.ac.jp/end/index.html (1) KUROYANAGI Hidehito 30323702