Elucidation of a C-Rich Signature Motif in Target mRNAs of RNA-Binding Protein TIAR

Abstract

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 ϳ2,209 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.

Translational coregulation of 5′TOP mRNAs by TIA-1 and TIAR

“…Thus, TIA-1 and TIAR exist in complex with 59TOP mRNAs, as well as with a subset of other cellular mRNAs. This is consistent with previous observations from global analyses of TIA-1-and TIAR-associated mRNAs (Lopez de Silanes et al 2005;Mazan-Mamczarz et al 2006;Kim et al 2007).…”

“…lanes 6,7 and lane 5, input samples are shown in lanes 1-4). In addition, mRNAs for calmodulin 2 and b-actin, which lack 59TOPs but have previously been described to bear TIA-1-and TIARbinding sites in their 39 UTRs (Lopez de Silanes et al 2005;Mazan-Mamczarz et al 2006;Kim et al 2007), served as positive controls, and both copurify with TIA-1 and TIAR as expected (Fig. 1A, middle panels).…”

“…Based on previous studies implicating TIA-1 and TIAR in translational repression (Piecyk et al 2000 Kim et al 2007) and in binding to pyrimidine-rich sequences (Dember et al 1996;Lopez de Silanes et al 2005;Mazan-Mamczarz et al 2006;Kim et al 2007Kim et al , 2011, we speculated that TIA-1 and TIAR may play a role in the control of 59TOP mRNA translation. To first determine whether TIA-1 and TIAR exist in complex with 59TOP mRNAs, we established human HEK293 cell lines stably expressing Flagtagged TIA-1 or TIAR under control of tetracycline-inducible promoters.…”

“…This allowed ligation into the MluI-HindIII sites of pcTET2Bwt and pcTET2Bwt-3MS2. To create the reporter constructs containing TIA-1-or TIAR-binding sites in the 39 UTR of b-globin mRNA, the following sequences for TIA-1 (59-TTCTTCTTTACTTAAGTTATGTAATAGTTGAA AGGAA-39) (Lopez de Silanes et al 2005;Mazan-Mamczarz et al 2006) and TIAR (59-TTGCCACCTCCTTGCTCCTGCCCAGAC AG-39) (Kim et al 2007) were inserted into the NotI site of pcTET2-Bwt. To create the reporter construct containing the 59TOP element, including downstream sequences, from human rpL32 mRNA in the 39 UTR of b-globin mRNA, the sequence 59-CCCTTCTCTCTCGGCGGCTGCCTACGGAGGTGGCAGCC ATCTCCTTCTCGGCT-39 was inserted between NotI and XbaI sites of pcTET2-Bwt.…”

Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged

“…Among the earliest functions attributed to TIA-1 and TIAR was the ability to bind mRNAs and recruit them to cytoplasmic bodies (known as stress granules) in response to cellular stress such as heat shock (Kedersha et al 1999). Both proteins have also been found to bind AU-rich elements (AREs) and act as translational repressors (Piecyk et al 2000;Kawai et al 2006;Kim et al 2007). TIA-1 and TIAR have been shown to be regulators of the inflammatory response that function by silencing translation of key mediators of inflammation, such as TNF-a and COX-2 (Piecyk et al 2000;Lopez de Silanes et al 2005a).…”