Differential Stability of Thymidylate Synthase 3′-Untranslated Region Polymorphic Variants Regulated by AUF1

Myriam Gorospe,Kotb Abdelmohsen,Ashish Lal,Jennifer L. Martindale,Robert D. Ladner,Rudolf Pullmann

doi:10.1074/jbc.m600282200

Myriam Gorospe, Kotb Abdelmohsen + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m600282200

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Abstract

A 6-nucleotide insertion (I)/deletion (D) polymorphism in the 3'-untranslated region of the thymidylate synthase gene was shown to influence mRNA stability, but the molecular basis of this effect has not been elucidated. Here, studies of both endogenous and ectopically expressed thymidylate synthase alleles revealed that the mRNA-binding, decay-promoting protein AUF1 has higher affinity for allele D mRNA. AUF1 overexpression preferentially suppressed D allele mRNA levels, whereas AUF1 silencing selectively elevated D allele mRNA levels. Our results illustrate the functional consequences of ribonucleoprotein associations involving a polymorphic RNA sequence and uncover a novel mechanism of action for non-coding RNA polymorphisms.

Highlights

Post-transcriptional gene regulatory processes, including altered mRNA turnover, have emerged as key mechanisms controlling gene expression in physiologic and pathologic situations [1]
A polymorphism consisting of the deletion (D)/ insertion (I) of a 6-bp stretch (TTAAAG) in the thymidylate synthase (TS) 3Ј-untranslated regions (UTRs) was recently shown to appear with a frequency for the D allele of ϳ30 – 40% in Caucasians [17]
To measure the half-life of chimeric TS alleles, cells were treated with 4 ␮g/ml actinomycin D to block de novo transcription, RNA was collected at the times indicated, and reverse transcription (RT)-qPCR was performed to measure the levels of Luc(D) and Luc(I) mRNAs

Summary

Introduction

Post-transcriptional gene regulatory processes, including altered mRNA turnover, have emerged as key mechanisms controlling gene expression in physiologic and pathologic situations [1]. To measure the half-life of chimeric TS alleles, cells were treated with 4 ␮g/ml actinomycin D to block de novo transcription, RNA was collected at the times indicated, and RT-qPCR was performed to measure the levels of Luc(D) and Luc(I) mRNAs. These values were normalized to the amount of GAPDH mRNA in each sample and represented the T7 RNA polymerase promoter sequence was added to the as the percentage of mRNA levels present at time 0, before 5Ј-end of all fragments.

Results

Conclusion