Activation of 5′-3′ exoribonuclease Xrn1 by cofactor Dcs1 is essential for mitochondrial function in yeast Flore Sinturela,b, Dominique Bréchemier-Baeyb, Megerditch Kiledjianc, Ciarán Condonb, and Lionel Bénarda,b,1 aLaboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie Physico-Chimique, Centre National de la Recherche Scientifique, Formation de Recherche en Evolution (FRE) 3354, Université Pierre et Marie Curie, 75005 Paris, France; bLaboratoire de l’Expression Génétique Microbienne, Institut de Biologie Physico-Chimique, Centre National de la Recherche Scientifique, Unité Propre de Recherche 9073, Université de Paris 7-Denis-Diderot, 75005 Paris, France; and cDepartment of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854 Edited* by Reed B. Wickner, National Institutes of Health, Bethesda, MD, and approved April 6, 2012 (received for review December 6, 2011) The scavenger decapping enzyme Dcs1 has been shown to facilitate Saccharomyces cerevisiae (16). Known regulatory RNAs belong to the activity of the cytoplasmic 5′-3′ exoribonuclease Xrn1 in eukar- this class, and their stabilization in the absence of Xrn1 could yotes. Dcs1 has also been shown to be required for growth in glyc- explain the aberrant expression of some genes. erol medium. We therefore wondered whether the capacity to Few studies have investigated how the activity of exoribonu- activate RNA degradation could account for its requirement for cleases is modulated in connection to cell physiology (17, 18). growth on this carbon source. Indeed, a catalytic mutant of Xrn1 Here, we focus on Dcs1, a factor that potentially controls the is also unable to grow in glycerol medium, and removal of the activity of the exoribonuclease Xrn1 (4). We have a particular nuclear localization signal of Rat1, the nuclear homolog of Xrn1, interest in the fact that Dcs1 is also required for growth in restores glycerol growth. A cytoplasmic 5′-3′ exoribonuclease ac- glycerol medium because we show that Xrn1 is also necessary for tivity is therefore essential for yeast growth on glycerol, suggest- growth on this carbon source. More precisely, we demonstrate ing that Xrn1 activation by Dcs1 is physiologically important. In that a cytoplasmic 5′-3′ exoribonuclease activity is required un- fact, Xrn1 is essentially inactive in the absence of Dcs1 in vivo. der these conditions, suggesting that a potential connection We analyzed the role of Dcs1 in the control of exoribonuclease exists between the ability to grow on glycerol and the capacity to activity in vitro and propose that Dcs1 is a specific cofactor of degrade RNA or to activate RNA degradation in the presence of Xrn1. Dcs1 does not stimulate the activity of other 5′-3′ exoribonu- Dcs1. We demonstrate that Dcs1 is a specific cofactor of Xrn1. cleases, such as Rat1, in vitro. We demonstrate that Dcs1 improves We decided to examine the physiological consequences of this the apparent affinity of Xrn1 for RNA and that Xrn1 and Dcs1 can regulation by 2D protein gel analysis. We studied the impact of form a complex in vitro. We examined the biological significance of shifting cells from glucose to glycerol on the accumulation of this regulation by performing 2D protein gel analysis. We observed specific proteins in the absence of Xrn1 or its activator Dcs1. A that a set of proteins showing decreased levels in a DCS deletion majority of the down-regulated proteins are essential for mito- strain, some essential for respiration, are also systematically de- chondrial function such as respiration, a prerequisite for growth creased in an XRN1 deletion mutant. Therefore, we propose that on nonfermentable carbon sources like glycerol. We thus show the activation of Xrn1 by Dcs1 is important for respiration. that 5′-3′ exoribonuclease activity is important for mitochondrial function and propose that one role of Dcs1 is the modulation of RNA turnover | ribonuclease | post-transcriptional control | porin Xrn1 activity. urnover of messenger RNA (mRNA) is a regulated process Results Tand a key step in the control of gene expression (1). In eu- 5′-3′ Exoribonuclease Activity Is Required for Growth on Glycerol. karyotic cells, most cytoplasmic mRNAs are degraded through Dcs1 has been shown to stimulate Xrn1 activity (4), and a DCS1 two alternative pathways, each of which is initiated by the re- deletion strain cannot grow on glycerol (19) (Fig. 1A). We asked moval of the poly(A) tail by deadenylases. Subsequently, the cap whether these two observations were connected by spotting serial (5′-m7GpppN) structure is removed by the decapping complex dilutions of the xrn1 mutant on glycerol plates. In fact, we observed Dcp1/Dcp2, and the mRNA is degraded in the 5′ to 3′ direction that both mutants show a growth defect on nonfermentable carbon by the major cytoplasmic enzyme Xrn1. Alternatively, dead- sources such as glycerol, ethanol, and lactate (Fig. S1). Complemen- enylated mRNAs can be degraded from their 3′-ends by the tation of this strain with a wild-type XRN1 gene restored growth on exosome, a multimeric complex possessing 3′ to 5′ exoribonu- glycerol, whereas complementation with a catalytic mutant did not clease activity (2). The cap structure resulting from 3′-end decay (Fig. 1B), showing that this defect is related to the enzyme’sexor- is hydrolyzed by conserved scavenger decapping enzyme Dcs1 ibonuclease activity. We were also able to rescue this growth defect (3). Dcs1 is also necessary for the 5′ to 3′ exonucleolytic activity by expressing the nuclear 5′-3′ exoribonuclease Rat1 in the cyto- of Xrn1, a requirement that functionally links these two alter- plasm in the absence of its nuclear localization signal, Rat1ΔNLS native degradation pathways (4). The 5′-3′ exoribonuclease Xrn1 (20) (Fig. 1B). Thus, a cytoplasmic 5′-3′ exoribonuclease activity is highly conserved in eukaryotes and has been extensively de- is important for growth on glycerol. An interesting connection scribed for its role in the degradation of cytoplasmic mRNAs (5, therefore exists between the ability to grow on this carbon source 6). Xrn1 also participates in the degradation of nonfunctional and the capacity to degrade RNA in the 5′-3′ direction or to activate mRNAs (7) and noncoding RNAs (8, 9). this degradation through the presence of Dcs1. In addition to causing direct defects in RNA turnover, it has been known for a long time that a deletion of XRN1 is detrimental XRN1 to other cellular functions. mutants exhibit pleiotropic Author contributions: F.S. and L.B. designed research; F.S., D.B.-B., and L.B. performed phenotypes, including slow growth, loss of viability upon nitrogen research; M.K. contributed new reagents/analytic tools; F.S., C.C., and L.B. analyzed data; starvation, meiotic arrest, defective sporulation, defects in micro- and F.S. and L.B. wrote the paper. tubule-related processes, telomere shortening, and chromosomal The authors declare no conflict of interest. stability (10–15). It has yet to be shown that these phenotypes are *This Direct Submission article had a prearranged editor. directly related to a deficiency in exoribonuclease activity, how- 1To whom correspondence should be addressed. E-mail: [email protected]. ever. More recently, a new class of noncoding RNAs, Xrn1-sen- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sitive unstable transcripts named XUTs, has been described in 1073/pnas.1120090109/-/DCSupplemental. 8264–8269 | PNAS | May 22, 2012 | vol. 109 | no. 21 www.pnas.org/cgi/doi/10.1073/pnas.1120090109 Downloaded by guest on September 23, 2021 Fig. 1. Correlation between cytoplasmic 5′-3′ exoribonuclease activity and growth on glycerol. Serial dilutions of the indicated strains were spotted onto plates containing either glucose (Left) or glycerol (Right) as the sole carbon source. (A) Absence of Dcs1 impedes growth on glycerol. (B) Cyto- plasmic 5′-3′ exoribonuclease activity is necessary for growth on glycerol. The xrn1Δ strain was transformed with different plasmids encoding wild-type (WT) XRN1,theXRN1 catalytic mutant (E176G substitution), or the mutated RAT1ΔNLS gene that restores cytoplasmic exoribonuclease activity by expressing Rat1 in the cytoplasm through removal of the nuclear localization signal (NLS). Growth on plates of xrn1Δ and dcs1Δ strains versus WT are Fig. 2. The catalytic activity of Dcs1 is not required for activation of TIF51A shown in Fig. S1. mRNA degradation. DCS1H-N denotes the catalytically inactive DCS1 mutant harboring a H268N substitution in its HIT motif (4). Degradation of TIF51A mRNA was monitored after transcriptional shutoff with thiolutin (15 μg/mL), Xrn1 Activity Is Not Inhibited by Products or Substrates of Dcs1 in and RNA was isolated at the indicated time points. (A) Northern blot showing degradation in dcs1Δ and/or dcs2Δ mutant backgrounds. The ScR1 RNA Vitro. Dcs1 cleaves the m7GpppN cap of messenger RNAs that are served as a loading control. (B) Half-life measurements of TIF51A transcripts digested by the exosome, and it was proposed that this catalytic were carried out by quantification of the mRNA remaining at each time point activity is necessary for Xrn1-dependent RNA decay (4). Dcs1 after correcting for loading differences using ScR1 RNA. The average value 7 MICROBIOLOGY produces m GMP and a 5′ diphosphate-nucleotide upon cleavage and SDs were obtained from at least three independent experiments. of m7GpppN and is also able to convert the m7GDP that results from the cleavage of capped RNA by the Dcp1/Dcp2 complex to m7GMP (21, 22). The previous model suggested that one or more glycerol and the capacity of Dcs1 to activate 5′-3′ RNA degra- of these compounds, which accumulate or are missing in the ab- dation by Xrn1. A possible explanation for the discrepancy with sence of the activity of Dcs1, are effectors of Xrn1 activity (4). We the previous observation is given in the discussion. We also ver- therefore performed in vitro RNA degradation assays (RT- ified that TIF51A mRNA has a similar half-life (>60 min) in a FeDEx; ref.