Functional Significance for a Heterogenous Ribonucleoprotein A18 Signature RNA Motif in the 3′-Untranslated Region of Ataxia Telangiectasia Mutated and Rad3-related (ATR) Transcript

Ruiqing Yang,France Carrier,Fred E Indig,Ming Zhan,Narasimha Rao Nalabothula,Qingyuan Yang

doi:10.1074/jbc.m109.013128

Abstract

The predominantly nuclear heterogenous ribonucleoprotein A18 (hnRNP A18) translocates to the cytosol in response to cellular stress and increases translation by specifically binding to the 3'-untranslated region (UTR) of several mRNA transcripts and the eukaryotic initiation factor 4G. Here, we identified a 51-nucleotide motif that is present 11.49 times more often in the 3'-UTR of hnRNP A18 mRNA targets than in the UniGene data base. This motif was identified by computational analysis of primary sequences and secondary structures of hnRNP A18 mRNA targets against the unaligned sequences. Band shift analyses indicate that the motif is sufficient to confer binding to hnRNP A18. A search of the entire UniGene data base indicates that the hnRNP A18 motif is also present in the 3'-UTR of the ataxia telangiectasia mutated and Rad3-related (ATR) mRNA. Validation of the predicted hnRNP A18 motif is provided by amplification of endogenous ATR transcript on polysomal fractions immunoprecipitated with hnRNP A18. Moreover, overexpression of hnRNP A18 results in increased ATR protein levels and increased phosphorylation of Chk1, a preferred ATR substrate, in response to UV radiation. In addition, our data indicate that inhibition of casein kinase II or GSK3beta significantly reduced hnRNP A18 cytosolic translocation in response to UV radiation. To our knowledge, this constitutes the first demonstration of a post-transcriptional regulatory mechanism for ATR activity. hnRNP A18 could thus become a new target to trigger ATR activity as back-up stress response mechanisms to functionally compensate for absent or defective responders.

Highlights

ATR2 belongs to a family of protein serine-threonine kinases whose catalytic domains share evolutionary relationship with
HnRNP A18 Translocation to the Cytosol in Response to Cellular Stress Is Phosphorylation-dependent—We have previously shown that the predominantly nuclear hnRNP A18 is translocated to the cytosol in response to UV radiation (11)
F, relative expression of GFP-hnRNP A18 expressed as a ratio (%) of cytoplasmic over total GFPhnRNP A18. t test was used to calculate statistical significance between UV and casein kinase 2 (CK2) inhibitor ϩ UV; UV and GSK3␤ inhibitor ϩ UV; CK2 inhibitor ϩ UV and GSK3␤ inhibitor ϩ UV

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Treatments—The human colorectal carcinoma RKO cells were grown and maintained as described before (11). The RKO cells were either stably (11) or transiently transfected with GFP-hnRNP A18 (11) and hnRNP A18 phospho mimetic mutant with Lipofectamine LTX (Invitrogen). Computational Analysis—The hnRNP A18 binding motif was identified from the 3Ј-UTR of the experimental data collected in Ref. 11. The 2923 motifs were modeled by the Stochastic context-free grammar (SCFG) algorithm and the SCFG enhanced with maximum likelihood Both models were searched against the experimental data set (3Ј-UTR). The top 100 motifs were further searched against the entire UniGene 3Ј-UTR data set. The final motif was chosen, which is high in the ratio and highly frequent in the experimental data set. Increasing amounts of recombinant GST-hnRNP A18 were incubated with radiolabeled hnRNP A18 RNA binding motif 1, ATR 3Ј-UTR (nucleotide 150 –199), R1 or R2 probe, and tRNA (50 ␮g/ml) as nonspecific competitor. All of the antibodies were rabbit polyclonal and were used at 1:1000 dilutions

RESULTS

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DISCUSSION