Abstract
Poly(A)-specific ribonuclease (PARN) is a 3'-exoribonuclease that plays an important role in regulating the stability and maturation of RNAs. Recently, PARN has been found to regulate the maturation of the human telomerase RNA component (hTR), a noncoding RNA required for telomere elongation. Specifically, PARN cleaves the 3'-end of immature, polyadenylated hTR to form the mature, nonpolyadenylated template. Despite PARN's critical role in mediating telomere maintenance, little is known about how PARN's function is regulated by post-translational modifications. In this study, using shRNA- and CRISPR/Cas9-mediated gene silencing and knockout approaches, along with 3'-exoribonuclease activity assays and additional biochemical methods, we examined whether PARN is post-translationally modified by acetylation and what effect acetylation has on PARN's activity. We found PARN is primarily acetylated by the acetyltransferase p300 at Lys-566 and deacetylated by sirtuin1 (SIRT1). We also revealed how acetylation of PARN can decrease its enzymatic activity both in vitro, using a synthetic RNA probe, and in vivo, by quantifying endogenous levels of adenylated hTR. Furthermore, we also found that SIRT1 can regulate levels of adenylated hTR through PARN. The findings of our study uncover a mechanism by which PARN acetylation and deacetylation regulate its enzymatic activity as well as levels of mature hTR. Thus, PARN's acetylation status may play a role in regulating telomere length.
Highlights
Poly(A)-specific ribonuclease (PARN) is an RNA-processing enzyme involved in many biological processes, such as telomere maintenance and DNA damage response [1, 2]
To determine whether PARN is acetylated and which acetyltransferases catalyze the acetylation of PARN, V5-tagged PARN was co-transfected with various lysine acetyltransferase (KAT) in 293T cells
Immunoprecipitation of the V5 tag and immunoblotting with anti-acetyllysine (AcK) antibody revealed that PARN can be acetylated by the lysine acetyltransferases: ortholog of Drosophila males absent on the first (MOF), p300/cAMP response element-binding protein (CBP)-associated factor (PCAF), and p300 (Fig. 1A)
Summary
Poly(A)-specific ribonuclease (PARN) is a 39-exoribonuclease that plays an important role in regulating the stability and maturation of RNAs. Recently, PARN has been found to regulate the maturation of the human telomerase RNA component (hTR), a noncoding RNA required for telomere elongation. Acetylation of the 39-exoribonuclease CCR4-associated factor 1 (CAF1) by CBP/p300 decreases levels of polyadenylated mRNAs, which destabilizes mRNAs globally [41] These examples highlight how lysine acetylation plays a critical role in regulating the function of RNA-processing enzymes. Because little is known about PARN’s post-translational regulation, in this study, we investigated whether PARN protein is post-translationally modified by lysine acetylation and its functional relevance. We found that acetylation of PARN decreases its enzymatic activity both in vitro, using a synthetic 59-fluorescein RNA probe, and in vivo, by measuring endogenous levels of polyadenylated hTR. Our results elucidate a mechanism by which SIRT1 regulates levels of polyadenylated hTR through PARN
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