In Vivo Targeting of the Yeast Pop2 Deadenylase Subunit to Reporter Transcripts Induces Their Rapid Degradation and Generates New Decay Intermediates

Anne-Laure Finoux,Bertrand Séraphin

doi:10.1074/jbc.m600132200

Abstract

Deadenylation is the rate-limiting step of mRNA decay, yet little is known about the mechanism regulating this process. In yeast, deadenylation is mainly mediated by the Pop2-Ccr4 complex. We tested whether the selective recruitment of this deadenylase to target mRNAs was sufficient to stimulate their decay in vivo. For this purpose, the Pop2 factor was fused to a U1A RNA binding domain while U1A binding sites were inserted in untranslated regions of a reporter transcript. Analysis of the reporter fate in strains expressing the Pop2-U1A-RBD fusion demonstrated a specific activation of target mRNA decay. Increased mRNA degradation involved accumulation of deadenylated mRNAs that was not detected when the control factors Dcp2 or Pub1 were tethered to the same transcript. The rapid target mRNA degradation was also accompanied by the appearance of new decay intermediates generated by the 3' -5' trimming of the corresponding 3' -untranslated region. Interestingly, this process was not mediated by the exosome but may result from the activity of the Pop2-Ccr4 deadenylase itself. These results indicate that selective recruitment of the Pop2-Ccr4 deadenylase is sufficient to activate mRNA decay, even though this process can also be stimulated by additional mechanisms. Furthermore, deadenylase recruitment affects the downstream path of mRNA decay.

Highlights

A System to Target a Deadenylase to a Reporter mRNA—As deadenylation is the rate-limiting step for mRNA decay, it is important to understand whether this process is controlled by recruiting a constitutively active deadenylase on target substrates or whether the enzyme is present in excess but only activated locally
Because deadenylation is often the rate-limiting step for mRNA decay [6], we have developed a targeting strategy to test whether this process may be regulated by association of the main yeast deadenylase with its substrate
Consistent with this idea we have shown that the targeted deadenylase increases the rate of reporter mRNA decay

Summary

Introduction

Deadenylation is mostly mediated by the Pop2-Ccr4 complex [7, 8], which degrades poly(A) tails of substrate mRNAs with kinetics varying for each target substrate. When Pop2 was targeted to a stable PGK1 reporter mRNA, the resulting deadenylase activity was more processive and the half-life of the transcript was decreased 2-fold.

Results

Conclusion