Ambro van Hoof

Messenger RNA regulation: to translate or to degrade

Quality control of gene expression operates post‐transcriptionally at various levels in eukaryotes. Once transcribed, mRNAs associate with a host of proteins throughout their lifetime. These mRNA–protein complexes (mRNPs) undergo a series of remodeling events that are influenced by and/or influence the translation and mRNA decay machinery. In this review we discuss how a decision to translate or to degrade a cytoplasmic mRNA is reached. Nonsense‐mediated mRNA decay (NMD) and microRNA (miRNA)‐mediated mRNA silencing are provided as examples. NMD is a surveillance mechanism that detects and eliminates aberrant mRNAs whose expression would result in truncated proteins that are often deleterious to the organism. miRNA‐mediated mRNA silencing is a mechanism that ensures a given protein is expressed at a proper level to permit normal cellular function. While NMD and miRNA‐mediated mRNA silencing use different decision‐making processes to determine the fate of their targets, both are greatly influenced by mRNP dynamics. In addition, both are linked to RNA processing bodies. Possible modes involving 3′ untranslated region and its associated factors, which appear to play key roles in both processes, are discussed.

Yeast Exosome Mutants Accumulate 3′-Extended Polyadenylated Forms of U4 Small Nuclear RNA and Small Nucleolar RNAs

ABSTRACT The exosome is a protein complex consisting of a variety of 3′-to-5′ exonucleases that functions both in 3′-to-5′ trimming of rRNA precursors and in 3′-to-5′ degradation of mRNA. To determine additional exosome functions, we examined the processing of a variety of RNAs, including tRNAs, small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), RNase P, RNase MRP, and SRP RNAs, and 5S rRNAs in mutants defective in either the core components of the exosome or in other proteins required for exosome function. These experiments led to three important conclusions. First, exosome mutants accumulate 3′-extended forms of the U4 snRNA and a wide variety of snoRNAs, including snoRNAs that are independently transcribed or intron derived. This finding suggests that the exosome functions in the 3′ end processing of these species. Second, in exosome mutants, transcripts for U4 snRNA and independently transcribed snoRNAs accumulate as 3′-extended polyadenylated species, suggesting that the exosome is required to process these 3′-extended transcripts. Third, processing of 5.8S rRNA, snRNA, and snoRNA by the exosome is affected by mutations of the nuclear proteins Rrp6p and Mtr4p, whereas mRNA degradation by the exosome required Ski2p and was not affected by mutations inRRP6 or MTR4. This finding suggests that the cytoplasmic and nuclear forms of the exosome represent two functionally different complexes involved in distinct 3′-to-5′ processing and degradation reactions.

The exosome contains domains with specific endoribonuclease, exoribonuclease and cytoplasmic mRNA decay activities

The eukaryotic exosome is a ten-subunit 3′ exoribonucleolytic complex responsible for many RNA-processing and RNA-degradation reactions. How the exosome accomplishes this is unknown. Rrp44 (also known as Dis3), a member of the RNase II family of enzymes, is the catalytic subunit of the exosome. We show that the PIN domain of Rrp44 has endoribonucleolytic activity. The PIN domain is preferentially active toward RNA with a 5′ phosphate, suggesting coordination of 5′ and 3′ processing. We also show that the endonuclease activity is important in vivo. Furthermore, the essential exosome subunit Csl4 does not contain any domains that are required for viability, but its zinc-ribbon domain is required for exosome-mediated mRNA decay. These results suggest that specific exosome domains contribute to specific functions, and that different RNAs probably interact with the exosome differently. The combination of an endoRNase and an exoRNase activity seems to be a widespread feature of RNA-degrading machines.

The exosome: A proteasome for RNA?

is presumably because defects in one exosome subunit The eukaryotic cell contains a wide variety of RNA specause a failure to assemble the exosome properly, and cies that are either processed from 39-extended precurthe exosome per se is required for viability. This conclusors or degraded in a 39-to-59 direction. How 39-to-59 sion is supported by the observations that inactivation processing is controlled for different transcripts and disof any core component generally gives similar defects tinguished from complete 39-to-59 degradation of an in exosome-dependent events (Mitchell et al., 1997; JaRNA molecule is unknown. Surprisingly, a single comcobs Anderson and Parker, 1998; Allmang et al., 1999a, plex of multiple 39-to-59 exonucleases identified by the 1999b; van Hoof et al., 2000). In addition, there does not Tollervey lab, termed the exosome, catalyzes many of appear to be a substantial free pool of exosome subunits these reactions. For example, the exosome trims 5.8S (Mitchell et al., 1997; Allmang et al., 1999a). This hypothrRNA from a 39-extended precursor and functions in the esis would be similar to what has been observed for the 39-to-59 degradation of mRNA. In this review, we discuss proteasome. Here catalytic subunits are essential for the organization and functions of the exosome. structural reasons, but individual active sites are not The presence of multiple exonucleases in the exoessential (reviewed in Baumeister et al., 1998). However, some complex is analogous to a number of proteases two observations raise the formal possibility that exoboth in prokaryotes and eukaryotes, such as the proteasome subunits may have distinct essential functions some, that assemble in large complexes. In addition, both independent of the entire complex. First, in contrast to the exosome, and the proteasome, require ATPases subunits of the proteasome, which require assembly for for their functions (see below, and reviewed in Gottesactivity, isolated exosome subunits show exonucleoman et al., 1997; Baumeister et al., 1998; DeMartino and lytic activity (see above). Second, homologous human Slaughter, 1999). These similarities suggest that there cDNAs can complement at least some of the phenotypes may be a fundamental advantage to the compartmentalof yeast strains carrying mutations in the RRP4, RRP44, ization of degradative enzymes by their assembly into or CSL4 genes. This suggests that either the exosome larger complexes. subunits are sufficiently conserved to allow assembly What Is the Exosome? between different species, or that exosome subunits Based on copurification, the yeast exosome is a protein may be able to function individually. To resolve these complex that consists of a core of at least ten proteins issues, the analysis of mutant alleles of exosome sub(Rrp4p, Rrp40p to Rrp46p, Mtr3p, and Csl4p; Table 1; units that separate exonucleolytic activity from assemAllmang et al., 1999a). The stoichiometry of the different bly will be needed. subunits is unknown, but the sedimentation of the exoSimilar to the proteasome, the exosome is present in some in glycerol gradients (300–400 kDa; Mitchell et al., both the nucleus and the cytoplasm. This conclusion is 1997) is consistent with a single copy of each subunit. based on immunolocalization of core exosome subunits Strikingly, all ten subunits have been proposed to be and biochemical fractionation (Kinoshita et al., 1991; active 39-to-59 exoribonucleases (Allmang et al., 1999a). Mitchell et al., 1997; Allmang et al., 1999a; Zanchin and Six of the exosome subunits (Rrp41p, Rrp42p, Rrp43p, Goldfarb, 1999). However, the nuclear exosome has an Rrp45p, Rrp46p, and Mtr3p) appear to be 39-to-59 phosadditional subunit, Rrp6p, which is yet another active phorolytic enzymes, since they are related to the 39-to-59 39-to-59 exoribonuclease (Allmang et al., 1999a; Burkard exoribonucleases RNase PH and PNPase from Escheand Butler, 2000). Rrp6p is the only exosome subunit richia coli (Mian, 1997). These E. coli enzymes function that is not essential for viability, although rrp6D strains in the decay of mRNA and the processing of other RNAs. have strong defects in all the known nuclear exosome Unlike hydrolases they utilize phosphate as an attacking functions (see below). group during RNA digestion and produce nucleotide Several lines of evidence indicate that the exosome 59 diphosphates (NDPs). In support of these proposed is conserved in eukaryotes. For example, the majority activities, recombinant Rrp41p is a phosphate-stimuof the exosome subunits identified in yeast have strong lated exonuclease that produces NDPs (Mitchell et al., homologs in other eukaryotes. Moreover, two observa1997). In contrast, Rrp44p is related to the 39 hydrolases tions indicate that these homologs do form an exosome RNase II and RNase R from E. coli (Mian, 1997) and complex in other eukaryotes. First, the human homologs recombinant Rrp44p has 39-to-59 exonuclease activity of Rrp6p (PM-Scl100), Rrp4p, and Rrp45p (PM-Scl75) that releases nucleotide 59 monophosphates (NMPs; are found in the PM-Scl particle, which appears to be Mitchell et al., 1997). Recombinant Rrp4p, purified from the human exosome (Allmang et al., 1999a). Second, E. coli also has 39 exoribonuclease activity that releases the Schizosaccharomyces pombe homolog of Rrp44p,

Function of the ski4p (Csl4p) and Ski7p proteins in 3'-to-5' degradation of mRNA.

ABSTRACT One of two general pathways of mRNA decay in the yeastSaccharomyces cerevisiae occurs by deadenylation followed by 3′-to-5′ degradation of the mRNA body. Previous results have shown that this degradation requires components of the exosome and the Ski2p, Ski3p, and Ski8p proteins, which were originally identified due to their superkiller phenotype. In this work, we demonstrate that deletion of the SKI7 gene, which encodes a putative GTPase, also causes a defect in 3′-to-5′ degradation of mRNA. Deletion ofSKI7, like deletion of SKI2, SKI3, or SKI8, does not affect various RNA-processing reactions of the exosome. In addition, we show that a mutation in theSKI4 gene also causes a defect in 3′-to-5′ mRNA degradation. We show that the SKI4 gene is identical to theCSL4 gene, which encodes a core component of the exosome. Interestingly, the ski4-1 allele contains a point mutation resulting in a mutation in the putative RNA binding domain of the Csl4p protein. This point mutation strongly affects mRNA degradation without affecting exosome function in rRNA or snRNA processing, 5′ externally transcribed spacer (ETS) degradation, or viability. In contrast, thecsl4-1 allele of the same gene affects rRNA processing but not 3′-to-5′ mRNA degradation. We identify csl4-1 as resulting from a partial-loss-of-function mutation in the promoter of the CSL4 gene. These data indicate that the distinct functions of the exosome can be separated genetically and suggest that the RNA binding domain of Csl4p may have a specific function in mRNA degradation.

Three conserved members of the RNase D family have unique and overlapping functions in the processing of 5S, 5.8S, U4, U5, RNase MRP and RNase P RNAs in yeast

The biogenesis of a number of RNA species in eukaryotic cells requires 3′ processing. To determine the enzymes responsible for these trimming events, we created yeast strains lacking specific 3′ to 5′ exonucleases. In this work, we describe the analysis of three members of the RNase D family of exonucleases (Rex1p, Rex2p and Rex3p). This work led to three important conclusions. First, each of these exonucleases is required for the processing of distinct RNAs. Specifically, Rex1p, Rex2p and Rex3p are required for 5S rRNA, U4 snRNA and MRP RNA trimming, respectively. Secondly, some 3′ exonucleases are redundant with other exonucleases. Specifically, Rex1p and Rex2p function redundantly in 5.8S rRNA maturation, Rex1p, Rex2p and Rex3p are redundant for the processing of U5 snRNA and RNase P RNA, and Rex1p and the exonuclease Rrp6p have an unknown redundant essential function. Thirdly, the demonstration that the Rex proteins can affect reactions that have been attributed previously to the exosome complex indicates that an apparently simple processing step can be surprisingly complex with multiple exonucleases working sequentially in the same pathway.

Conserved Functions of Yeast Genes Support the Duplication, Degeneration and Complementation Model for Gene Duplication

Gene duplication is often cited as a potential mechanism for the evolution of new traits, but this hypothesis has not been thoroughly tested experimentally. A classical model of gene duplication states that after gene duplication one copy of the gene preserves the ancestral function, while the other copy is free to evolve a new function. In an alternative duplication, divergence, and complementation model, duplicated genes are preserved because each copy of the gene loses some, but not all, of its functions through degenerating mutations. This results in the degenerating mutations in one gene being complemented by the other and vice versa. These two models make very different predictions about the function of the preduplication orthologs in closely related species. These predictions have been tested here for several duplicated yeast genes that appeared to be the leading candidates to fit the classical model. Surprisingly, the results show that duplicated genes are maintained because each copy carries out a subset of the conserved functions that were already present in the preduplication gene. Therefore, the results are not consistent with the classical model, but instead fit the duplication, divergence, and complementation model.

The crystal structure of Mtr4 reveals a novel arch domain required for rRNA processing

The essential RNA helicase, Mtr4, performs a critical role in RNA processing and degradation as an activator of the nuclear exosome. The molecular basis for this vital function is not understood and detailed analysis is significantly limited by the lack of structural data. In this study, we present the crystal structure of Mtr4. The structure reveals a new arch‐like domain that is specific to Mtr4 and Ski2 (the cytosolic homologue of Mtr4). In vivo and in vitro analyses demonstrate that the Mtr4 arch domain is required for proper 5.8S rRNA processing, and suggest that the arch functions independently of canonical helicase activity. In addition, extensive conservation along the face of the putative RNA exit site highlights a potential interface with the exosome. These studies provide a molecular framework for understanding fundamental aspects of helicase function in exosome activation, and more broadly define the molecular architecture of Ski2‐like helicases.

A riboswitch-containing sRNA controls gene expression by sequestration of a response regulator

A dual-action RNA switch for expression Riboswitches are short segments of RNA that bind small molecules and switch between two different conformations, thereby regulating gene expression (see the Perspective by Chen and Gottesman). DebRoy et al. and Mellin et al. find a new class of riboswitches—in two different species of bacteria—that are both part of and regulate the production of a noncoding RNA. Each riboswitch ensures that a particular metabolic pathway is only activated in the presence of an essential small-molecule cofactor. In the absence of the cofactor, the full-length non-coding RNA is made and binds a regulator protein, preventing the regulator protein from inappropriately activating the metabolic pathway. Science, this issue p. 937 and p. 940; see also p. 876 A riboswitch is both part of, and regulates the activity of, a small noncoding regulatory RNA. [Also see Perspective by Chen and Gottesman] The ethanolamine utilization (eut) locus of Enterococcus faecalis, containing at least 19 genes distributed over four polycistronic messenger RNAs, appears to be regulated by a single adenosyl cobalamine (AdoCbl)–responsive riboswitch. We report that the AdoCbl-binding riboswitch is part of a small, trans-acting RNA, EutX, which additionally contains a dual-hairpin substrate for the RNA binding–response regulator, EutV. In the absence of AdoCbl, EutX uses this structure to sequester EutV. EutV is known to regulate the eut messenger RNAs by binding dual-hairpin structures that overlap terminators and thus prevent transcription termination. In the presence of AdoCbl, EutV cannot bind to EutX and, instead, causes transcriptional read through of multiple eut genes. This work introduces riboswitch-mediated control of protein sequestration as a posttranscriptional mechanism to coordinately regulate gene expression.

A Genomic Screen in Yeast Reveals Novel Aspects of Nonstop mRNA Metabolism

Nonstop mRNA decay, a specific mRNA surveillance pathway, rapidly degrades transcripts that lack in-frame stop codons. The cytoplasmic exosome, a complex of 3′–5′ exoribonucleases involved in RNA degradation and processing events, degrades nonstop transcripts. To further understand how nonstop mRNAs are recognized and degraded, we performed a genomewide screen for nonessential genes that are required for nonstop mRNA decay. We identified 16 genes that affect the expression of two different nonstop reporters. Most of these genes affected the stability of a nonstop mRNA reporter. Additionally, three mutations that affected nonstop gene expression without stabilizing nonstop mRNA levels implicated the proteasome. This finding not only suggested that the proteasome may degrade proteins encoded by nonstop mRNAs, but also supported previous observations that rapid decay of nonstop mRNAs cannot fully explain the lack of the encoded proteins. Further, we show that the proteasome and Ski7p affected expression of nonstop reporter genes independently of each other. In addition, our results implicate inositol 1,3,4,5,6-pentakisphosphate as an inhibitor of nonstop mRNA decay.