Elucidation of a C-rich signature motif in target mRNAs of RNA-binding protein TIAR.
The RNA-binding protein TIAR (related to TIA-1 [T-cell-restricted intracellular antigen 1]) was shown to associate with subsets of mRNAs bearing U-rich sequences in their 3' untranslated regions. TIAR can function as a translational repressor, particularly in response to cytotoxic agents. Using unstressed colon cancer cells, collections of mRNAs associated with TIAR were isolated by immunoprecipitation (IP) of (TIAR-RNA) ribonucleoprotein (RNP) complexes, identified by microarray analysis, and used to elucidate a common signature motif present among TIAR target transcripts. The predicted TIAR motif was an unexpectedly cytosine-rich, 28- to 32-nucleotide-long element forming a stem and a loop of variable size with an additional side loop. The ability of TIAR to bind an RNA oligonucleotide with a representative C-rich TIAR motif sequence was verified in vitro using surface plasmon resonance. By this analysis, TIAR containing two or three RNA recognition domains (TIAR12 and TIAR123) showed low but significant binding to the C-rich sequence. In vivo, insertion of the C-rich motif into a heterologous reporter strongly suppressed its translation in cultured cells. Using this signature motif, an additional approximately 2,209 UniGene targets were identified (2.0% of the total UniGene database). A subset of specific mRNAs were validated by RNP IP analysis. Interestingly, in response to treatment with short-wavelength UV light (UVC), a stress agent causing DNA damage, each of these target mRNAs bearing C-rich motifs dissociated from TIAR. In turn, expression of the encoded proteins was elevated in a TIAR-dependent manner. In sum, we report the identification of a C-rich signature motif present in TIAR target mRNAs whose association with TIAR decreases following exposure to a stress-causing agent.
Department of Biochemistry and Molecular Biology, Monash University, Victoria, Australia.
Henry S
HS
Kim
Yuki
Y
Kuwano
Ming
M
Zhan
Rudolf
R
Pullmann
Krystyna
K
Mazan-Mamczarz
Huai
H
Li
Nancy
N
Kedersha
Paul
P
Anderson
Matthew C J
MC
Wilce
Myriam
M
Gorospe
Jacqueline A
JA
Wilce
D000818:Animals; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms; D004249:DNA Damage; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020411:Oligonucleotide Array Sequence Analysis; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D014466:Ultraviolet Rays
D000954:Antigens, Surface; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C076751:TIAL1 protein, human