Activation of 5′-3′ exoribonuclease Xrn1 by cofactor Dcs1 is essential for mitochondrial function in yeast

Abstract

The scavenger decapping enzyme Dcs1 has been shown to facilitate the activity of the cytoplasmic 5′-3′ exoribonuclease Xrn1 in eukaryotes. Dcs1 has also been shown to be required for growth in glycerol medium. We therefore wondered whether the capacity to activate RNA degradation could account for its requirement for growth on this carbon source. Indeed, a catalytic mutant of Xrn1 is also unable to grow in glycerol medium, and removal of the nuclear localization signal of Rat1, the nuclear homolog of Xrn1, restores glycerol growth. A cytoplasmic 5′-3′ exoribonuclease activity is therefore essential for yeast growth on glycerol, suggesting that Xrn1 activation by Dcs1 is physiologically important. In fact, Xrn1 is essentially inactive in the absence of Dcs1 in vivo. We analyzed the role of Dcs1 in the control of exoribonuclease activity in vitro and propose that Dcs1 is a specific cofactor of Xrn1. Dcs1 does not stimulate the activity of other 5′-3′ exoribonucleases, such as Rat1, in vitro. We demonstrate that Dcs1 improves the apparent affinity of Xrn1 for RNA and that Xrn1 and Dcs1 can form a complex in vitro. We examined the biological significance of this regulation by performing 2D protein gel analysis. We observed that a set of proteins showing decreased levels in a DCS deletion strain, some essential for respiration, are also systematically decreased in an XRN1 deletion mutant. Therefore, we propose that the activation of Xrn1 by Dcs1 is important for respiration.RNA turnover | ribonuclease | post-transcriptional control | porin T urnover of messenger RNA (mRNA) is a regulated process and a key step in the control of gene expression (1). In eukaryotic cells, most cytoplasmic mRNAs are degraded through two alternative pathways, each of which is initiated by the removal of the poly(A) tail by deadenylases. Subsequently, the cap (5′-m 7 GpppN) structure is removed by the decapping complex Dcp1/Dcp2, and the mRNA is degraded in the 5′ to 3′ direction by the major cytoplasmic enzyme Xrn1. Alternatively, deadenylated mRNAs can be degraded from their 3′-ends by the exosome, a multimeric complex possessing 3′ to 5′ exoribonuclease activity (2). The cap structure resulting from 3′-end decay is hydrolyzed by conserved scavenger decapping enzyme Dcs1 (3). Dcs1 is also necessary for the 5′ to 3′ exonucleolytic activity of Xrn1, a requirement that functionally links these two alternative degradation pathways (4). The 5′-3′ exoribonuclease Xrn1 is highly conserved in eukaryotes and has been extensively described for its role in the degradation of cytoplasmic mRNAs (5, 6). Xrn1 also participates in the degradation of nonfunctional mRNAs (7) and noncoding RNAs (8,9).In addition to causing direct defects in RNA turnover, it has been known for a long time that a deletion of XRN1 is detrimental to other cellular functions. XRN1 mutants exhibit pleiotropic phenotypes, including slow growth, loss of viability upon nitrogen starvation, meiotic arrest, defective sporulation, defects in microtubule-related processes, telomere sho...

DcpS is a transcript-specific modulator of RNA in mammalian cells

“…Our data further demonstrate a transcript-specific role of DcpS in RNA decay where the stability of DRNT1, but not DRNT2 was increased in DcpS KD cells indicating DcpS promotes selective decay of DRNT1. Furthermore, the comparable increase of DRNT1 stability in DcpS KD , Xrn1 KD , and double DcpS KD and Xrn1 KD cells suggests both proteins function within the same pathway and DcpS likely accentuates Xrn1 function similar to that reported in S. cerevisiae (Sinturel et al 2012) and C. elegans (Bosse et al 2013). Interestingly, unlike the scenario in S. cerevisiae and C. elegans where the decapping activity of the DcpS homolog was dispensable for its stimulation of Xrn1 decay, human DcpS requires a functional catalytic decapping activity.…”

“…The resulting cap structure is a substrate of the scavenger decapping enzyme, DcpS to release m 7 Gp and ppN products (Wang and Kiledjian 2001;Liu et al 2002). In yeast, the DcpS homolog Dcs1p, facilitates 5 ′ to 3 ′ exoribonucleolytic decay (Liu and Kiledjian 2005) as an obligate cofactor for the Xrn1 exoribonuclease by an unknown mechanism independent of its decapping catalytic activity (Sinturel et al 2012). Moreover, the Caenorhabditis elegans Dcs-1 was shown to physically interact with Xrn-1 and promote specific microRNA degradation also independent of its decapping activity (Bosse et al 2013).…”

Interaction between the poly(A)-binding protein Pab1 and the eukaryotic release factor eRF3 regulates translation termination but not mRNA decay in Saccharomyces cerevisiae

“…In wild-type cells, the half-life of TIF51a mRNA was measured to be ∼11 min (Supplemental Fig. S3), which was slightly shorter than the previously reported (Sinturel et al 2012). The absence of the eRF3-NM domain did not affect the half-life of this mRNA (12.8 min).…”

Synaptic control of mRNA translation by reversible assembly of XRN1 bodies

“…Indirect evidence additionally indicates that the SX-bodies also lack the decapping co-activators Ge-1/ Hedls/RCD8/EDC4 and Rck/p54/DDX6. All this suggests that the SX-bodies are unlikely to represent sites for the processing of decapped mRNAs, although additional decapping enzymes exist in several organisms that might bind and activate XRN1 (Li et al, 2011;Sinturel et al, 2012;Bossé et al, 2013). Similarly, only a small fraction of dendritic DCP1a foci contain XRN1 (this work and Cougot et al, 2008), indicating that 59-39 degradation is unlikely to occur in the DCP1a foci.…”