A 5' Leader of Rbm3, a Cold Stress-induced mRNA, Mediates Internal Initiation of Translation with Increased Efficiency under Conditions of Mild Hypothermia.
Although mild hypothermia generally reduces protein synthesis in mammalian cells, the expression of a small number of proteins, including Rbm3, is induced under these conditions. In this study, we identify an Rbm3 mRNA with a complex 5' leader sequence containing multiple upstream open reading frames. Although these are potentially inhibitory to translation, monocistronic reporter mRNAs containing this leader were translated relatively efficiently. In addition, when tested in the intercistronic region of dicistronic mRNAs, this leader dramatically enhanced second cistron translation, both in transfected cells and in cell-free lysates, suggesting that the Rbm3 leader mediates cap-independent translation via an internal ribosome entry site (IRES). Inasmuch as Rbm3 mRNA and protein levels are both increased in cells exposed to mild hypothermia, the activity of this IRES was evaluated at a cooler temperature. Compared to 37 degrees C, IRES activity at 33 degrees C was enhanced up to 5-fold depending on the cell line. Moderate enhancements also occurred with constructs containing other viral and cellular IRESes. These effects of mild hypothermia on translation were not caused by decreased cell growth, as similar effects were not observed when cells were serum starved. The results suggest that cap-independent mechanisms may facilitate the translation of particular mRNAs during mild hypothermia.
Chappell
S A
SA
Department of Neurobiology, Scripps Research Institute and the Skaggs Institute for Chemical Biology, La Jolla, California 92037 and the Neurosciences Institute, San Diego, California 92121.
Owens
G C
GC
Mauro
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VP
D016475:3T3 Cells; D020121:5' Untranslated Regions / Q000032:analysis*; D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D018076:DNA, Complementary / Q000302:isolation & purification; D017930:Genes, Reporter; D007036:Hypothermia, Induced; D051379:Mice; D008810:Mice, Inbred C57BL; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D016601:RNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry; D017423:Sequence Analysis, RNA; D014162:Transfection
D020121:5' Untranslated Regions; D018076:DNA, Complementary; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C484384:Rbm3 protein, mouse