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Dcps Is a Transcript-Specific Modulator of RNA in Mammalian Cells

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Dcps Is a Transcript-Specific Modulator of RNA in Mammalian Cells Downloaded from rnajournal.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press DcpS is a transcript-specific modulator of RNA in mammalian cells MI ZHOU,1 SOPHIE BAIL,1 HEATHER L. PLASTERER,2 JAMES RUSCHE,2 and MEGERDITCH KILEDJIAN1 1Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854, USA 2Repligen Corporation, Waltham, Massachusetts 02453, USA ABSTRACT The scavenger decapping enzyme DcpS is a multifunctional protein initially identified by its property to hydrolyze the resulting cap structure following 3′ end mRNA decay. In Saccharomyces cerevisiae, the DcpS homolog Dcs1 is an obligate cofactor for the 5′-3′ exoribonuclease Xrn1 while the Caenorhabditis elegans homolog Dcs-1, facilitates Xrn1 mediated microRNA turnover. In both cases, this function is independent of the decapping activity. Whether DcpS and its decapping activity can affect mRNA steady state or stability in mammalian cells remains unknown. We sought to determine DcpS target genes in mammalian cells using a cell-permeable DcpS inhibitor compound, RG3039 initially developed for therapeutic treatment of spinal muscular atrophy. Global mRNA levels were examined following DcpS decapping inhibition with RG3039. The steady-state levels of 222 RNAs were altered upon RG3039 treatment. Of a subset selected for validation, two transcripts that appear to be long noncoding RNAs HS370762 and BC011766, were dependent on DcpS and its scavenger decapping catalytic activity and referred to as DcpS-responsive noncoding transcripts (DRNT) 1 and 2, respectively. Interestingly, only the increase in DRNT1 transcript was accompanied with an increase of its RNA stability and this increase was dependent on both DcpS and Xrn1. Importantly, unlike in yeast where the DcpS homolog is an obligate cofactor for Xrn1, stability of additional Xrn1 dependent RNAs were not altered by a reduction in DcpS levels. Collectively, our data demonstrate that DcpS in conjunction with Xrn1 has the potential to regulate RNA stability in a transcript-selective manner in mammalian cells. Keywords: RNA decay; DcpS; XRN1; lncRNA INTRODUCTION enzyme, DcpS to release m7Gp and ppN products (Wang and Kiledjian 2001; Liu et al. 2002). In yeast, the DcpS homolog In eukaryotic cells, mRNA degradation plays an important ′ ′ Dcs1p, facilitates 5 to 3 exoribonucleolytic decay (Liu and role in the control of gene expression and therefore is highly Kiledjian 2005) as an obligate cofactor for the Xrn1 exoribo- regulated. Following an initial deadenylation step to remove nuclease by an unknown mechanism independent of its the poly(A) tail, the remaining mRNA can undergo exonu- ′ ′ ′ ′ decapping catalytic activity (Sinturel et al. 2012). Moreover, cleolytic decay from either 3 or the 5 end. In the 5 to 3 decay ′ the Caenorhabditis elegans Dcs-1 was shown to physically in- pathway, the 5 cap is initially removed by Dcp2 or Nudt16 ′ teract with Xrn-1 and promote specific microRNA degrada- decapping enzymes, resulting in a 5 end monophosphory- tion also independent of its decapping activity (Bosse et al. lated RNA (Lykke-Andersen 2002; van Dijk et al. 2002; 2013). Wang et al. 2002; Song et al. 2010). The uncapped product un- ′ ′ DcpS hydrolyzes the triphosphate linkage of the cap struc- dergoes 5 to 3 exoribonuclease decay by, Xrn1 (Hsu and ture utilizing an evolutionarily conserved Histidine Triad Stevens 1993). Xrn1 is highly conserved in eukaryotes and (HIT) motif (Liu et al. 2002) with the central histidine serv- functions in the decay of cytoplasmic mRNAs (Muhlrad et ing as the nucleophile for the hydrolase activity (Lima et al. al. 1995), in the quality control of aberrant mRNAs (Conti 1997). Insights into the molecular mechanism of DcpS and Izaurralde 2005) and noncoding RNAs (Chernyakov ′ ′ decapping were provided by structural analysis, revealing a et al. 2008; van Dijk et al. 2011). In the 3 to 5 pathway, the homo-dimer complex where each monomer possesses a dis- multisubunit exosome complex degrades the deadenylated ′ tinct binding and active site (Gu et al. 2004). The homo- RNA from the 3 end, leaving the residual m7GpppN cap dimer displays an asymmetric structure in the ligand-bound structure (Anderson and Parker 1998; Liu et al. 2006). The re- sulting cap structure is a substrate of the scavenger decapping © 2015 Zhou et al. This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http:// Corresponding author: [email protected] rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available Article published online ahead of print. Article and publication date are at under a Creative Commons License (Attribution-NonCommercial 4.0 Inter- http://www.rnajournal.org/cgi/doi/10.1261/rna.051573.115. national), as described at http://creativecommons.org/licenses/by-nc/4.0/. 1306 RNA 21:1306–1312; Published by Cold Spring Harbor Laboratory Press for the RNA Society Downloaded from rnajournal.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press DcpS in RNA decay form and a dynamic state, where each monomer can alternate creased. Approximately 75% of the RNAs corresponded to from an open to closed state (Liu et al. 2008). Hydrolysis of mRNAs, while ∼25% mapped to uncharacterized transcripts cap structure only occurs in the closed conformation (Gu consistent with noncoding RNA. A subset of 12 RNAs were et al. 2004). randomly chosen for further validation by direct quantitative A potent DcpS decapping inhibitor was identified in efforts reverse-transcription (qRT)-PCR. The steady-state levels of to isolate small molecules that increase expression of the sur- six were found to reproducibly and significantly change great- vival of motor neuron 2 (SMN2) gene in spinal muscular at- er than twofold upon RG3039 treatment. The levels of four rophy (SMA) patients. Cocrystal structures of a C5 substituted RNAs, HS370762, BC011766, ATOH7, and RAB26, were ele- quinazoline compound, D156844, revealed it binds to the vated, while PAQR8 and MAOB were reduced compared with DcpS cap binding active site and inhibits its decapping untreated cells (Fig. 1A). Similar changes for the six RNAs (Singh et al. 2008; Thurmond et al. 2008). Further optimiza- were also detected in HEK293T cells treated with 100 nM tion generated the RG3039 derivative, which is also a potent RG3039 (Fig. 1B) demonstrating the generality of the results DcpS decapping inhibitor (Gogliotti et al. 2013). Although in different cell lines. All subsequent analyses were carried the quinazoline compounds were capable of improving mo- out in HEK293T cells. tor neuron function and extending survival of SMA model mice (Butchbach et al. 2010; Gogliotti et al. 2013; Van Meerbeke et al. 2013), statistically significant increases in RNA steady-state levels are influenced by DcpS SMN2 mRNA or SMN protein were not evident. In this study, To determine whether the changes in the levels of the six we took advantage of the DcpS decapping inhibition property RNAs were mediated through DcpS, we utilized a of RG3039 to determine the impact of DcpS decapping HEK293T cell line constitutively expressing a DcpS-directed on mRNA expression. Interestingly, only 222 RNAs were de- shRNA (DcpSKD) which reduced DcpS protein levels by 90% tected to be altered upon RG3039 treatment and of a subset of (Fig. 2A; Shen et al. 2008). Consistent with the outcome of genes validated, four were found to be responsive to RG3039 RG3039 treated cells, steady-state levels of both HS370762 independent of DcpS and two were mediated through DcpS. Importantly, both the DcpS-responsive transcripts are puta- A 7 tive noncoding RNAs, with the stability of one RNA modulat- *** 6 ed by both DcpS and Xrn1. Our data indicate that DcpS has *** 5 *** Control the capacity to be a transcript-restricted modulator of RNA RG3039 4 *** stability in mammalian cells and the RG3039 quinazoline 3 compound is more pleotropic than initially anticipated and 2 1 can influence gene expression in both an apparent DcpS de- *** mRNA Levels (SH-SY5Y) mRNA *** pendent and independent manner. 0 RESULTS B 8 7 *** Steady-state levels of RNAs are altered in cells Control 6 RG3039 treated with RG3039 5 *** *** ′ 4 Despite its role in the last step of 3 end mRNA decay pathway, 3 DcpS also contributes to mRNA or miRNA turnover in 2 *** 1 S. cerevisiae and C. elegans, respectively. To determine whether Levels (HEK293T) mRNA *** *** the human DcpS decapping activity influences the expression 0 of mRNAs in mammalian cells, we took advantage of a small molecule inhibitor of DcpS decapping, RG3039, to monitor changes in global mRNA levels. RG3039 is a cell-permeable FIGURE 1. Validation of a subset of RG3039 target RNAs. (A) Levels of 12 randomly chosen RNAs from the 222 RNAs that deviated by at least quinazoline derivative that is a potent inhibitor of DcpS twofold in SH-SY5Y neuroblastoma cells treated for 48 h with 100 nM decapping (Singh et al. 2008; Gogliotti et al. 2013) and was RG3039 were tested by quantitative reverse-transcription (qRT)-PCR. used to inhibit cellular DcpS activity and monitor potential RNA levels are presented relative to actin mRNA and derived from at changes in RNA steady-state levels. RNA isolated from human least three independent experiments. Six of the RNAs were reproducibly altered greater than twofold and constitute RG3039 responsive RNAs. SH-SY5Y retinoblastoma cells treated for 2 d with 100 nM (B) Validated RG3039 target RNAs from SH-SY5Y cells were further RG3039 were analyzed on an Affymetrix U133 Plus 2.0 hu- confirmed in HEK293T cells to determine the consistency of RG3039 man microarray. RNA profiles from treated and untreated in different cell backgrounds. RNA levels are presented relative to the cells revealed levels of 222 RNA were altered more than two- GAPDH mRNA and derived from at least three independent experi- ments. All six RNAs were similarly altered in the HEK293T cells as fold between the two cell parameters with P values <0.05 they were in SH-SY5Y cells.
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