Exoribonuclease Xrn1 by Cofactor Dcs1 Is Essential for Mitochondrial Function in Yeast

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 25, 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. 23) to measure Xrn1 activity in the presence of dsc1 mutant as an xrn1 mutant when cells are shifted from glucose m7GDP, m7GMP, m7G, and m7GpppN. None of these potential to glycerol media (Fig. S3A), and that the protein Xrn1 is similarly effectors affected Xrn1 activity at concentrations above those expressed in the wild-type, dcs1Δ, dcs2Δ,ordcs1Δ/dcs2Δ strains expected in vivo (24), whereas a known inhibitor of Xrn1, 3′- (Fig. S3B). Therefore, Xrn1 is essentially inactive for TIF51A phospho-adenosine-5′-phosphate (pAp), significantly reduced its mRNA degradation in the absence of Dcs1 in vivo, and Dcs1 is activity (Fig. S2). We thus conclude that Xrn1 is not a target for not a regulator of XRN1 gene expression. the potential substrates or end-products of Dcs1 activity. Direct and Specific Activation of Xrn1 by Dcs1. To determine Catalytic Activity of Dcs1 Is Not Necessary for Xrn1 Activity in Vivo. whether the activation of Xrn1 by Dcs1 was direct, we assayed The correlation between growth on glycerol and Xrn1 activation Xrn1 in the presence of Dcs1 in vitro. RNA degradation assays by Dcs1 is inconsistent with a previous observation that a cata- were performed by using either RT-FeDEx assays (23) (Fig. 3) ′ lytic mutant of Dcs1, Dcs1H-N (SI Materials and Methods), could or a 5 -labeled 30-nt substrate (Fig. S4). Both approaches showed grow on glycerol but was unable to activate Xrn1 (4). that the activity of Xrn1 is increased by the presence of Dcs1. We therefore revisited the original observation by measuring Furthermore, both wild-type Dcs1 and the catalytic mutant, mRNA half-life to determine the impact of Dcs1H-N on Xrn1- Dcs1H-N, were able to enhance Xrn1 activity in vitro. Control dependent mRNA decay in vivo. Northern blots were performed experiments showed that neither form of Dcs could degrade RNA to detect TIF51A mRNA, a known substrate of Xrn1 (4), at times alone, excluding the possibility that these preparations contained after addition of thiolutin to stop transcription (Fig. 2A). Quan- a contaminating RNase activity (Fig. 3). Moreover, all Dcs factors tifications are shown in Fig. 2B and showed that the absence of the tested, such as DcpS, the Dcs1 human ortholog, or Dcs2, activated catalytic activity of Dcs1 has no real impact on TIF51A mRNA Xrn1 in vitro (Table 1). The activation ability of Dcs factors is stability in vivo. Only the DCS1 deletion strain and double de- apparently specific to Xrn1, because Dcs factors were unable to letion strain, in which a catalytically inactive paralog of Dcs1, stimulate exoribonuclease activity of other 5′-3′ RNases, such as Dcs2 is also deleted, showed increased stabilization of TIF51A Rat1, RNase J1, or the 3′-5′ exoribonuclease RNase PH (Table 1). mRNA compared with the WT, dcs2Δ,orDCS1H-N strains. A Because Rat1 is active independently of Dcs1 in vitro (Table clear correlation therefore exists between the ability to grow on 1), we asked whether Rat1ΔNLS could compensate for the

Sinturel et al. PNAS | May 22, 2012 | vol. 109 | no. 21 | 8265 Downloaded by guest on September 25, 2021 Table 2. Cytoplasmic mRNA degradation by Rat1ΔNLS does not require the presence of Dcs1 Strains Plasmids TIF51A mRNA half-life, min

Wild-type + Xrn1 17 ± 0.5

+ Rat1ΔNLS 15 ± 1 dcs1Δ + Xrn1 >60

+ Rat1ΔNLS 27 ± 1.7

DCS1, and even more so DCS2, gene expression is induced when the carbon source is limited (24). To examine this phenomenon Fig. 3. Direct and specific activation of Xrn1 by Dcs1 or Dcs1H-N in vitro. In ′ ′ fl using 2D protein gel analysis, we decided to measure the impact vitro assays of 5 to 3 -exoribonuclease activity by RT-FeDEX (23). The uo- XRN1 DCS rophore (FAM) linked to RNA is hybridized to a DNA oligonucleotide con- of an deletion and a double deletion on the accu- taining a quencher (TAMRA). The generation of FAM fluorescence signal mulation of specific proteins when cells are shifted from glucose was monitored in real time in the presence of 50 nM Xrn1 exoribonuclease. to glycerol medium for 3 h. We used the dcs1Δdcs2Δ strain be- RT-FeDEx analysis shows the increased activity of Xrn1 in the presence of cause Dcs2 was also found to be a potential activator of Xrn1 both wild-type 100 nM purified Dcs1 protein or catalytic mutant 100 nM in vitro (Table 1). We analyzed proteins extracted from wild-type purified Dcs1 protein (H268N), whereas no contaminating exoribonuclease and both xrn1Δ and dcs1Δ/dcs2Δ cells. Protein spots (230) were activity of Dcs1 (wild-type or mutant) can be detected. quantified that were common to three independent experiments. All spots showing at least a twofold decrease in mutant versus fi absence of cytoplasmic Xrn1 for the degradation of TIF51A wild-type strain were identi ed by mass spectrometry (16 spots in xrn1 dcs mRNA in the absence of Dcs1 in vivo. In Table 2, we show that the mutant and 11 in the double deletion mutant). These fi Rat1ΔNLS can destabilize the TIF51A mRNA (twofold) in the proteins could be classi ed into three categories, those down- xrn1 absence of Dcs1, albeit not as efficiently as Xrn1 in the presence regulated in both mutants and those down-regulated in or in dcs of Dcs1 (fourfold). mutants only (Fig. 5). We found that many of the proteins in the first category are involved in mitochondrial function (Fig. 5A) Specific Interaction Between Dcs1 and Xrn1. The experiments de- and are essential for respiration. Because this 2D protein gel scribed above suggest a specific interaction between Dcs1 and approach is limited to a portion of the proteome, under specific Xrn1. We therefore performed Far Western blot experiments to growth conditions, it is not possible to say whether these are the demonstrate this interaction in vitro (Fig. S5A). Different quan- primary targets of a defective 5′-3′ RNA degradation pathway. tities of Xrn1 were spotted on membranes, incubated in the However, this degradation defect directly or indirectly produces a presence of Dcs1 protein, and then revealed with anti-Dcs1 anti- down-regulation of mitochondrial factors that is sufficient to ex- body. A clear positive signal was seen with Xrn1 and the Dcs1- plain the absence of growth on a nonfermentable carbon source positive control, whereas no signal was seen with either BSA or such as glycerol. RNase J1. To determine at what level Dcs1 affected Xrn1 activity, The mitochondrial porin, Por1 (25), which is essential for mi- we also performed RNA gel-shift assays in the absence of Mg2+ to tochondrial function, is one of the most affected proteins in both inhibit the exoribonuclease activity of Xrn1 (Fig. 4). These exper- mutant strains. We confirmed the results of the 2D protein-gel iments revealed that the presence of Dcs1 improves the apparent analysis by performing Western and Northern blot analysis for affinity of Xrn1 for its RNA substrate, whereas it has no effect on POR1 (Fig. 5 B and C). The expression of POR1 clearly depends the binding of the Rat1/Rai1 complex to the same RNA. As ex- on both XRN1 and DCS1 (see also Fig. S5 that shows the specific pected based on its primary function, Dcs1 did not bind the RNA role of DCS1 in POR1 mRNA expression). In conclusion, the substrate of Xrn1, a 5′ monophosphorylated RNA. We were un- activation of Xrn1 by its cofactor Dcs1 is critical for the expression able to visualize the formation of a stable RNA/Xrn1/Dcs1 com- of genes necessary for mitochondrial function and respiration. plex (Fig. 4), but on the hypothesis that the complex between Dcs1 and Xrn1 is transitory, we performed formaldehyde cross-link Discussion experiments to try to trap the complex. Under denaturing gel In this paper, we show that a cytoplasmic 5′-3′ exoribonucleolytic conditions, a supershift in the mobility of Dcs1 was visible spe- activity is necessary for growth on glycerol and other nonferment- cifically in the presence of Xrn1 (Fig. S5B). These experiments are able carbon sources in yeast. This activity is usually provided by consistent with Dcs1 forming a transitory complex with Xrn1. Xrn1, the major cytoplasmic exoribonuclease in S. cerevisiae,but we were able to demonstrate that Rat1 lacking a nuclear local- Importance of Xrn1 Activation by Dcs1 for Mitochondrial Function. ization signal can compensate for the growth defect in an XRN1 The observations that Dcs1 is a cofactor of Xrn1 and that Xrn1 deletion strain. This observation reveals that degradation must exoribonuclease activity is important for growth on glycerol sug- be optimal to ensure respiration on glycerol. It is remarkable gests that activation of Xrn1 by Dcs1 is physiologically important. that the cell’s3′-5′ exoribonuclease activities are not able to compensate for the absence of Xrn1 when cells are grown on glycerol. Table 1. Dcs family proteins and activation of exoribonucleases The absence of Dcs1 or Xrn1 led to a strong defect in POR1 in vitro expression (Fig. 5 A–C and Fig. S6). Although defects in the ex- Protein added in RNase assays Xrn1 Rat1/Rai1 RNase J1 RNase PH pression of other mRNAs may also play a role, the defect in the POR1 ———expression of the gene alone, which is essential gene for Dcs1-his tag (E. coli)+ respiration, is sufficient to explain the observation that 5′-3′ DcpS-flag (human) + ——— cytoplasmic exoribonuclease activity is required for growth on Dcs1-his tag catalytic + ——N/D glycerol (Fig. 1). We also considered the possibility that the ab- mutant (E. coli) sence of Xrn1 or Dcs1 could have caused a loss of mitochondrial Dcs2-his tag (E. coli)+N/D— N/D DNA over several generations. However, replacing the deleted +, activation; —, Dcs-independent exoribonuclease activity; N/D, not done. XRN1 locus by a wild-type copy of XRN1 allowed the new strain

8266 | www.pnas.org/cgi/doi/10.1073/pnas.1120090109 Sinturel et al. Downloaded by guest on September 25, 2021 Fig. 4. Dcs1 improves apparent affinity of Xrn1 for RNA in vitro. (A and B) Electrophoretic mobility shift assay of FAM-linked RNA by Xrn1. FAM-linked RNA (1.5 μM) was incubated with increased amounts of purified Xrn1protein (in a buffer without Mg2+, in the absence (A) or in the presence of 1μM of purified Dcs1 (B), and electrophoretically separated on a 4% nondenaturing polyacrylamide gel. (C) Comparison of the quantification of Xrn1-associated RNA as a percentage of the RNA input, in the absence or presence of purified Dcs1 (A and B). (D and E) Electrophoretic mobility shift assay of FAM-linked RNA by Xrn1 or Rat1. FAM-linked RNA (1.5 μM) was incubated with increased amounts of purified Dcs1 protein in a buffer without Mg2+, in the presence of 0.5 μM Xrn1 (D)or1.3μM of Rat1/Rai1 (B) exoribonucleases, and electrophoretically separated on a 4% nondenaturing polyacrylamide gel. (F) Comparison of the quantification of Xrn1-associated RNA (D) with the quantification of Rat1/Rai1-associated RNA (E) in the presence of purified Dcs1, as a percentage of the RNA input.

to grow on glycerol again, ruling out a permanent loss of mito- tween Dcs1 and Xrn1 showed that Dcs1 interacts with Xrn1 and chondrial DNA in xnr1Δ strains (Fig. S7A). Similar observations that this interaction probably activates RNA degradation by in- have been made for a DCS1 deletion strain (4, 19). creasing the affinity of Xrn1 for its substrate, perhaps through Our results showing that the catalytic activity of Dcs1 is not im- a conformational change after a transitory interaction. The role ′ ′ fi portant to stimulate 5 -3 mRNA degradation are contradictory of Dcs1 is speci c to Xrn1 because no such activation was ob- MICROBIOLOGY with a prior study (4). To measure mRNA half-life in this study, we served for the other RNases tested in vitro, such as Rat1 (Table used thiolutin to block RNA synthesis at 30 °C, whereas in the 1). We were able to demonstrate that, in contrast to Xrn1, a cy- previous study, transcription was stopped by shifting temperature toplasmic Rat1ΔNLS still shows detectable RNA decay activity from 25 °C to 37 °C in a strain harboring a thermosensitive RNA in the absence of Dcs1 in vivo (Table 2). polymerase mutant rbp1-1 (4). To determine whether the Dcs1H-N Our data show that one important role of Dcs1 is to maintain catalytic mutant was temperature sensitive, we therefore per- Xrn1 activity and, because 5′-3′ exoribonuclease activity is criti- formed mRNA half-life measurements at 30 °C and 37 °C in the cal for growth on glycerol, this interaction clearly has physio- presence of thiolutin and observed that the Dcs1H-N catalytic logical importance. The 2D protein gel analysis revealed, how- mutant loses its ability to activate TIF51A mRNA degradation at ever, that the absence of Xrn1 and Dcs1 has distinct effects on 37 °C, and not at 30 °C (Fig. S8A). The Dcs1H-N mutant also shows the expression of some genes. Dps1 and Cys3 for example, which a growth defect on glycerol at 37 °C, and not at 30 °C, in agreement have both been shown to be involved in respiration (26, 27), are with previous observations (Fig. S8B). The apparent contradiction affected differently by Dcs1 and Xrn1 (Fig. 5A). Thus, Xrn1 and with the previous study is therefore likely explained by a thermo- Dcs1 appear to be involved in other pathways related to the sensitivity of the catalytic mutant. mitochondrial function independently of their functions as an In vitro experiments with purified proteins showed that Dcs1 exoribonuclease and an exoribonuclease cofactor. The activa- acts directly on Xrn1 (Fig. 2). A similar observation was made for tion of Xrn1 by Dcs1 is therefore necessary, but not sufficient, to Dcs1H-N, Dcs2 (Table 1). Although Dcs2 also stimulated Xrn1 maintain an optimal cellular respiration. activity in vitro, it is not implicated in the 5′-3′ RNA degradation The ability of Rat1ΔNLS to compensate for Xrn1 shows that pathway, and it is not required for respiration in vivo (Fig. 1). 5′-3′ exoribonuclease activity is important for mitochondrial DCS2 is poorly expressed in glucose media but is induced under function. Because Rat1ΔNLS 5′-3′ exoribonuclease activity is less conditions of carbon source limitation such as glycerol (24), which affected by the absence of Dcs1 in vivo, we wondered whether could explain the lack of phenotype in a DCS2 deletion strain in a DCS1 deletion strain could grow on glycerol with Rat1ΔNLS the presence of glucose. We asked whether Dcs2 could play a role replacing Xrn1. The answer was no (Fig. S7B), and one reason is in the kinetics of TIF51A mRNA degradation, when cells were probably, as mentioned above, that Dcs1 affects the expression of shifted from glucose to glycerol. However, only the dcs1Δ muta- other genes important for respiration independently of the 5′-3′ tion affects the degradation rate of the TIF51A mRNA, even in RNA degradation pathway (such as Dps1; Fig. 5A). glycerol growth conditions (Fig. S3A). A particular cellular lo- One could imagine that the accumulation of a particular class calization of Dcs2 could also explain why Dcs2 does not impact on of RNAs is potentially inhibitory for the expression of key genes TIF51A mRNA decay like Dcs1; Dcs2 has been reported to be of the respiration pathway. If these inhibitory RNAs exist, our localized in processing bodies under stress conditions (24). data suggest that they are likely to belong to a class of specific RNA gel shift experiments, cross-link experiments using form- targets of the 5′-3′ degradation pathway. Recently, a novel class aldehyde, and Far Western blot analysis of the interaction be- of RNAs mainly degraded by Xrn1 was discovered that includes

Sinturel et al. PNAS | May 22, 2012 | vol. 109 | no. 21 | 8267 Downloaded by guest on September 25, 2021 The interaction between Dcs1 and Xrn1 activates Xrn1. Dcs1 is a conserved pyrophosphatase of the histidine triad (HIT) family that catalyses the cleavage of m7GpppN resulting from the 3′-5′ mRNA degradation process (32). Cytoplasmic Xrn1 and nuclear Rat1 belong to a large family of conserved 5′-3′ exoribonucleases (33). Interestingly, Rat1 also forms a complex with a pyrophosphatase, Rai1, that was recently shown to cleave unmethylated capped RNA (34). Rai1 helps the exoribonuclease activity by stabilizing Rat1, and the catalytic activity of Rai1 is also dis- pensable for the activation of Rat1. The complex formation between a pyrophosphatase and a 5′-3′ exoribonuclease might have a particular importance in evolution. Materials and Methods mRNA decay measurements (Northern blot analysis) were done by following standard procedures with details given in SI Materials and Methods. Tran- scription was blocked by adding thiolutin (15 μg/mL) to the cultures (gen- erous gift from Pﬁzer). Scans were performed with a Typhoon (Amersham Bioscience), and quantiﬁcations by using ImageQuant software. All half-life values reported are an average of at least three experiments.

RNA Degradation by Fluorescence Analysis. The ﬂuorescence assay was performed by RT-FeDeX assays as described (23). See SI Materials and Methods for reaction buffers.

Measuring Protein–RNA Affinity by EMSA. We used the FAM-modified RNA (23) at a constant concentration (1 μM) and varied the protein concentration. The Fig. 5. Different classes of down-regulated proteins in glycerol in the protein and RNA were incubated together on ice in the Xrn1/Rat1-Rai1 buffer 2+ · context of a defective 5′-3′ exoribonuclease activity. (A) Down-regulated without Mg (30 mM Tris HCl at pH 8, 50 mM NH4Cl, 0.5 mM DTT, and 20 mg/ (>twofold) proteins analyzed by 2D gel electrophoresis in the wild-type, mL Acetylated Bovine Serum). The samples were separated on 4% (wt/vol) xrn1Δ, and dcs1Δ dcs2Δ strains. Total proteins were isolated from cultures nondenaturing polyacrylamide (19:1) gels in Tris borate/EDTA buffer at grown in rich media containing glucose and shifted for 3 h into media room temperature. Gel images were scanned with a Typhoon (Amersham containing glycerol as the carbon source. The average value and SDs for each Bioscience). time point were obtained from at least three independent experiments. Genes involved in mitochondrial function are underlined, and essential Western Blot Analysis. Crude extracts were prepared from cultures by vor- genes for respiration are marked with an asterisk (http://www.yeastgenome. texing with glass beads. Samples were resolved by SDS/PAGE and transferred org). (B) Western blot of Por1 protein in wild-type, dcs1Δ dcs2Δ and xrn1Δ onto nitrocellulose membranes. Membranes were probed with either a strains. Prp4 was used as a loading control. (C) POR1 mRNA expression in 1:5,000 dilution of polyclonal anti-Prp4 antibodies with a 1:2,000 dilution of wild-type, dcs1Δdcs2Δ, and xrn1Δ strains. POR1 mRNA levels were analyzed monoclonal anti-Porin antibodies (Invitrogen). The blotted membranes were by Northern blot analysis. The ScR1 RNA served as a loading control. (B and developed with a 1:20,000 dilution of peroxidase-conjugated anti-rabbit and C) Total proteins or total RNA were isolated from cultures grown in media anti-mouse IgG (Sigma), respectively, and an enhanced chemiluminescence containing either glucose (Left) or shifted for 3 h into medium containing substrate (ECL; Amersham Bioscience). glycerol (Right) as the sole carbon source. 2D Gel Electrophoresis. Three independent cultures were grown to expo- nential phase in YPD and shifted to glycerol for 3 h at 30 °C. Soluble proteins potential regulatory antisense noncoding RNAs (ncRNAs) (16). were prepared according to ref. 35. First-dimension electrophoresis was car- Accumulation of these ncRNAs, called XUTs, in the absence of ried out on a nonlinear immobilized pH 3–11 gradient IPG strip (Bio-Rad) and 5′-3′ exoribonuclease activity could explain the down-regulation the second dimension by 12% SDS/PAGE. The six samples were analyzed in fi of the expression of specific genes. This model also relies on parallel. Densitometric quanti cation of the six blue-colloidal-stained 2D gels was performed by using the PDQuest 2D Analysis software. Protein spots previously published work in which it was demonstrated that the were excised from gels for trypsin in-gel digestion and MALDI-TOF analysis. expression of the PHO84 and TY1 genes can be down-regulated by the accumulation of antisense transcripts that are substrates Plasmids, Yeast Strains, and Growth Condition. All details can be found in SI of the exoribonucleases Rrp6 and Xrn1, respectively (28–30). Materials and Methods and Tables S1 and S2. We therefore checked to see whether any of the genes corre- sponding to the down-regulated proteins in our 2D gel analysis Purification of Enzymes. Details can be found in SI Materials and Methods. also contained potential XUTs (16), but did not find any. The xrn1 mutant has been analyzed by microarrays and, more Extraction of RNA. Preparation of total RNA was performed according stan- recently, in a high-throughput sequencing approach (16, 31), but dard procedures (36). Additional details are in SI Materials and Methods. we do not observe a major correlation with our 2D gel analysis. These earlier studies were conducted on total RNA, poly- ACKNOWLEDGMENTS. We thank Arlen W. Johnson for plasmids pAJ228, adenylated RNAs, or total RNA depleted of ribosomal RNAs. pAJ152, and pRDK307; Roy Parker for strains yRP840 and yRP844; and Josette xrn1 Banroques for providing polyclonal anti-Prp4 antibodies. This work was RNAs isolated from mutants are known to be enriched for supported by funds from the Centre National de la Recherche Scientifique, decapped mRNAs, making it impossible to accurately quantify Université Pierre et Marie Curie, Université Paris VII-Denis Diderot, Associa- functional messenger RNAs, possibly explaining why we do not tion pour la Recherche sur le Cancer Grant A07/2/4831, National Institutes of observe a significant correlation between mRNAs and protein Health Grant GM67005, and a grant from the Agence Nationale pour la xrn1 Recherche (ANR REGULncRNA). F.S. was a recipient of a fellowship from the levels. Moreover, no transcriptome data are available on Ministère pour la Recherche et la Technologie and from la Fondation pour la mutant cells grown in glycerol medium. Recherche Médicale.

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