Internal ribosome entry site-mediated translation of Smad5 in vivo: requirement for a nuclear event.
Smad5 is thought to relay signals of the bone morphogenetic protein pathway. The 5' untranslated region (5'UTR) of human Smad5 mRNA is long, has the potential to form secondary structures and contains five AUG codons. Here we show that the 5'UTR of Smad5 contains an internal ribosome entry site (IRES) located within 100 nt of the 3' end of the 5'UTR. The Smad5 IRES was 4-8-fold more active than the poliovirus IRES in C2C12 cells, which have osteoblastic differentiation ability, but was 5-10-fold less active than the poliovirus IRES in 293T cells. When an in vitro transcript of a dicistronic Smad5 IRES construct was transfected into C2C12 cells, the Smad5 IRES was not able to stimulate the translation of the downstream cistron, although the cap-dependent translation of the upstream cistron was efficient. In contrast, the poliovirus IRES in a dicistronic in vitro transcript was able to stimulate the translation of the downstream cistron to a similar extent as in the case of transfection of the corresponding dicistronic DNA construct. These results suggest that Smad5 IRES activity displays cell specificity and that some as yet unidentified nuclear event may be required for efficient Smad5 IRES-driven translation initiation.
Shiroki
Kazuko
K
Institute for Biomolecular Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan.
Ohsawa
Chieko
C
Sugi
Natuki
N
Wakiyama
Motoaki
M
Miura
Kin-ichiro
K
Watanabe
Manabu
M
Suzuki
Yutaka
Y
Sugano
Sumio
S
D016475:3T3 Cells; D020121:5' Untranslated Regions / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009865:Oocytes; D010006:Osteoblasts / Q000166:cytology; D010750:Phosphoproteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D017384:Sequence Deletion; D051902:Smad5 Protein; D015534:Trans-Activators / Q000235:genetics*; D014158:Transcription, Genetic; D014981:Xenopus
D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C497797:SMAD5 protein, human; D051902:Smad5 Protein; C497798:Smad5 protein, mouse; D015534:Trans-Activators; D008156:Luciferases