The roles of 3′-exoribonucleases and the exosome in trypanosome mRNA degradation

The degradation of eukaryotic mRNAs can be initiated by deadenylation, decapping, or endonuclease cleavage. This is followed by 5′–3′ degradation by homologs of Xrn1, and/or 3′–5′ degradation by the exosome. We previously reported that, in African trypanosome Trypanosoma brucei, most mRNAs are deadenylated prior to degradation, and that depletion of the major 5′–3′ exoribonuclease XRNA preferentially stabilizes unstable mRNAs. We now show that depletion of either CAF1 or CNOT10, two components of the principal deadenylation complex, strongly inhibits degradation of most mRNAs. RNAi targeting another deadenylase, PAN2, or RRP45, a core component of the exosome, preferentially stabilized mRNAs with intermediate half-lives. RRP45 depletion resulted in a 5′ bias of mRNA sequences, suggesting action by a distributive 3′–5′ exoribonuclease. Results suggested that the exosome is involved in the processing of trypanosome snoRNAs. There was no correlation between effects on half-lives and on mRNA abundance.     

Yeast RNA exosome activity is necessary for maintaining cell wall stability through proper protein glycosylation

Nuclear RNA exosome is the main 3′→5′ RNA degradation and processing complex in eukaryotic cells and its dysregulation therefore impacts gene expression and viability. In this work we show that RNA exosome activity is necessary for maintaining cell wall stability in yeast Saccharomyces cerevisiae. While the essential RNA exosome catalytic subunit Dis3 provides exoribonuclease catalytic activity, the second catalytic subunit Rrp6 has a noncatalytic role in this process. RNA exosome cofactors Rrp47 and Air1/2 are also involved. RNA exosome mutants undergo osmoremedial cell lysis at high temperature or at physiological temperature upon treatment with cell wall stressors. Finally, we show that a defect in protein glycosylation is a major reason for cell wall instability of RNA exosome mutants. Genes encoding enzymes that act in the early steps of the protein glycosylation pathway are down-regulated at high temperature in cells lacking Rrp6 protein or Dis3 exoribonuclease activity and overexpression of the essential enzyme Psa1, that catalyzes synthesis of the mannosylation precursor, suppresses temperature sensitivity and aberrant morphology of these cells. Furthermore, this defect is connected to a temperature-dependent increase in accumulation of noncoding RNAs transcribed from loci of relevant glycosylation-related genes.     

RNA cleavage products generated by antisense oligonucleotides and siRNAs are processed by the RNA surveillance machinery

DNA-based antisense oligonucleotides (ASOs) elicit cleavage of the targeted RNA by the endoribonuclease RNase H1, whereas siRNAs mediate cleavage through the RNAi pathway. To determine the fates of the cleaved RNA in cells, we lowered the levels of the factors involved in RNA surveillance prior to treating cells with ASOs or siRNA and analyzed cleavage products by RACE. The cytoplasmic 5′ to 3′ exoribonuclease XRN1 was responsible for the degradation of the downstream cleavage products generated by ASOs or siRNA targeting mRNAs. In contrast, downstream cleavage products generated by ASOs targeting nuclear long non-coding RNA Malat 1 and pre-mRNA were degraded by nuclear XRN2. The downstream cleavage products did not appear to be degraded in the 3′ to 5′ direction as the majority of these products contained intact poly(A) tails and were bound by the poly(A) binding protein. The upstream cleavage products of Malat1 were degraded in the 3′ to 5′ direction by the exosome complex containing the nuclear exoribonuclease Dis3. The exosome complex containing Dis3 or cytoplasmic Dis3L1 degraded mRNA upstream cleavage products, which were not bound by the 5′-cap binding complex and, consequently, were susceptible to degradation in the 5′ to 3′ direction by the XRN exoribonucleases.     

RRP41L,a Putative Core Subunit of the Exosome,Plays an Important Role in Seed Germination and Early Seedling Growth in Arabidopsis

In prokaryotic and eukaryotic cells, the 3′-5′-exonucleolytic decay and processing of RNAs are essential for RNA metabolism. However, the understanding of the mechanism of 3′-5′-exonucleolytic decay in plants is very limited. Here, we report the characterization of an Arabidopsis (Arabidopsis thaliana) transfer DNA insertional mutant that shows severe growth defects in early seedling growth, including delayed germination and cotyledon expansion, thinner yellow/pale-green leaves, and a slower growth rate. High-efficiency thermal asymmetric interlaced polymerase chain reaction analysis showed that the insertional locus was in the sixth exon of AT4G27490, encoding a predicted 3′-5′-exonuclease, that contained a conserved RNase phosphorolytic domain with high similarity to RRP41, designated RRP41L. Interestingly, we detected highly accumulated messenger RNAs (mRNAs) that encode seed storage protein and abscisic acid (ABA) biosynthesis and signaling pathway-related protein during the early growth stage in rrp41l mutants. The mRNA decay kinetics analysis for seed storage proteins, 9-cis-epoxycarotenoid dioxygenases, and ABA INSENSITIVEs revealed that RRP41L catalyzed the decay of these mRNAs in the cytoplasm. Consistent with these results, the rrp41l mutant was more sensitive to ABA in germination and root growth than wild-type plants, whereas overexpression lines of RRP41L were more resistant to ABA in germination and root growth than wild-type plants. RRP41L was localized to both the cytoplasm and nucleus, and RRP41L was preferentially expressed in seedlings. Altogether, our results showed that RRP41L plays an important role in seed germination and early seedling growth by mediating specific cytoplasmic mRNA decay in Arabidopsis.RNA decay is an essential step in gene expression regulation that influences many aspects of development and growth. In eukaryotes, mRNA decay is normally initiated by the removal of the poly(A) tail (Couttet et al., 1997; Parker and Song, 2004) and then enters one of two decay pathways: (1) the decapping complex cleaves the 5′ cap, after which the 5′-3′-exoribonuclease, such as XRN1 in animals and yeast (Saccharomyces cerevisiae) and XRN4 in plants, hydrolyzes the mRNA from the 5′ end (Hsu and Stevens, 1993; Kastenmayer and Green, 2000; Garneau et al., 2007; Rymarquis et al., 2011), and (2) the mRNA decays from the 3′ end by the 3′-5′-exonuclease.In eukaryotic cells, the 3′-5′-exonuclease can act alone to process the substrate in some cases, but the vast majority of 3′-5′-exonuclease activity is attributed to the exosome, which is an evolutionarily conserved macromolecular complex that mediates numerous reactions of 3′-5′ RNA processing/degradation and is essential for viability (Mitchell et al., 1997; Estévez et al., 2003). The structure of the exosome has been determined in archaea and eukaryotes, with the core forming a ring-shaped structure (Büttner et al., 2005; Lorentzen et al., 2005; Liu et al., 2006). In eukaryotes, the salient feature of the ring is defined by three distinct heterodimers of six RNase phosphorolytic (PH) domain-type proteins, MTR3-RRP42, RRP41-RRP45, and RRP43-RRP46 (Lehner and Sanderson, 2004; Hernández et al., 2006; Liu et al., 2006). However, the six-protein ring is not stable on its own in vitro and requires three subunits that contain S1 and KH domains (RRP4 links RRP41 and RRP42, RRP40 links RRP45 and RRP46, and CSL4 contacts MTR3 and RRP43) to form a stable core complex (Liu et al., 2006). In yeast, the loss of any individual subunit of the nine-component conserved core is lethal, resulting in similar ribosomal RNA (rRNA) processing defect profiles (Allmang et al., 1999a, 1999b). Moreover, x-ray crystallographic analysis of the human exosome revealed that all of its core subunits are required for its integrity (Liu et al., 2006). Using tandem affinity purification tagging in Arabidopsis (Arabidopsis thaliana) transgenic lines that expressed tagged versions of RRP4 and RRP41, Chekanova et al. (2007) first purified and characterized the exosome complex and revealed that the plant exosome complex contains six RNase PH domain-containing proteins and three S1 and/or KH domain proteins. Although the composition and structure of the plant exosome is similar to other eukaryotes, the function of individual subunits of the exosome appears to be different in Arabidopsis. Down-regulation of distinct subunits of the core complex results in different defects in plant development and RNA-processing profiles. For example, csl4 null mutant plants did not manifest any obvious phenotype, and the null mutation affected only a subset of exosome targets (Chekanova et al., 2007). Therefore, the CSL4 subunit appears to be nonessential for exosome function in Arabidopsis. However, the CSL4 subunit is essential for viability in yeast (Baker et al., 1998; Allmang et al., 1999b). In contrast, the rrp4 mutant shows seed arrest during early stages of embryonic development. RRP41 was shown to be essential for the development of female gametophytes, and homozygous rrp41 is lethal (Chekanova et al., 2007). Additionally, RRP45 is encoded by duplicate genes: RRP45A and RRP45B. Arabidopsis single mutants that lack either RRP45A or RRP45B have no phenotype or only a mild one, respectively, whereas simultaneous down-regulation of both proteins is lethal (Hooker et al., 2007). These data indicate that subunits of the Arabidopsis exosome core complex have specialized roles in plant growth and development and make unequal contributions to the activity of the exosome in vivo. However, the functions of other predicted core subunits of the exosome, with the exception of those mentioned above, are still unclear in Arabidopsis.Here, we report the characterization of an Arabidopsis transfer DNA (T-DNA) insertional mutant that displays severe defects in early seedling growth. High-efficiency thermal asymmetric interlaced (hiTAIL)-PCR analysis revealed that the insertional locus was in the sixth exon of AT4G27490, encoding a predicted 3′-5′-exonuclease that contained a conserved RNase PH domain. A previous study presumed that AT4G27490 was one subunit of the core exosome in Arabidopsis, a homolog of yeast Mtr3 (Chekanova et al., 2007), but another study suggested that it was a homolog of yeast Rrp41 (Zimmer et al., 2008). Here, we refer to AT4G27490 as RRP41L. Interestingly, we detected highly accumulated mRNAs that encode seed storage protein (SSP) and abscisic acid (ABA) biosynthesis and signaling pathway-related protein during the early growth stage in the rrp41l mutant. The mRNA decay kinetics analysis for SSPs, 9-cis-epoxycarotenoid dioxygenases (NCEDs), and ABA INSENSITIVEs (ABIs) revealed that RRP41L catalyzed the decay of these mRNAs in the cytoplasm. Consistent with these results, the rrp41l mutant was more sensitive in seed germination and root growth than wild-type plants, whereas the overexpression (OE) lines of RRP41L were more resistant to ABA in seed germination and root growth than wild-type plants. RRP41L is localized to both the cytoplasm and nucleus, and RRP41L is preferentially expressed in seedlings. Collectively, our results showed that RRP41L plays an important role in seed germination and early seedling growth by mediating specific cytoplasmic mRNA decay in Arabidopsis.     

Artificial MicroRNAs highly accessible to targets confer efficient virus resistance in plants

Short-hairpin RNAs based on microRNA (miRNA) precursors to express the artificial miRNAs (amiRNAs) can specifically induce gene silencing and confer virus resistance in plants. The efficacy of RNA silencing depends not only on the nature of amiRNAs but also on the local structures of the target mRNAs. However, the lack of tools to accurately and reliably predict secondary structures within long RNAs makes it very hard to predict the secondary structures of a viral genome RNA in the natural infection conditions in vivo. In this study, we used an experimental approach to dissect how the endogenous silencing machinery acts on the 3′ untranslated region (UTR) of the Cucumber mosaic virus (CMV) genome. Transiently expressed 3′UTR RNAs were degraded by site-specific cleavage. By comparing the natural cleavage hotspots within the 3′UTR of the CMV-infected wild-type Arabidopsis to those of the triple dcl2/3/4 mutant, we acquired true small RNA programmed RNA-induced silencing complex (siRISC)-mediated cleavage sites to design valid amiRNAs. We showed that the tRNA-like structure within the 3′UTR impeded target site access and restricted amiRNA-RISC-mediated cleavage of the target viral RNA. Moreover, target recognition in the less-structured area also influenced siRISC catalysis, thereby conferring different degrees of resistance to CMV infection. Transgenic plants expressing the designed amiRNAs that target the putative RISC accessible target sites conferred high resistance to the CMV challenge from both CMV subgroup strains. Our work suggests that the experimental approach is credible for studying the course of RISC target recognition to engineer effective gene silencing and virus resistance in plants by amiRNAs.     

MPP6 is an exosome-associated RNA-binding protein involved in 5.8S rRNA maturation

The exosome is a complex of 3′→5′ exoribonucleases which is involved in many RNA metabolic processes. To regulate these functions distinct proteins are believed to recruit the exosome to specific substrate RNAs. Here, we demonstrate that M-phase phosphoprotein 6 (MPP6), a protein reported previously to co-purify with the TAP-tagged human exosome, accumulates in the nucleoli of HEp-2 cells and associates with a subset of nuclear exosomes as evidenced by co-immunoprecipitation and biochemical fractionation experiments. In agreement with its nucleolar accumulation, siRNA-mediated knock-down experiments revealed that MPP6 is involved in the generation of the 3′ end of the 5.8S rRNA. The accumulation of the same processing intermediates after reducing the levels of either MPP6 or exosome components strongly suggests that MPP6 is required for the recruitment of the exosome to the pre-rRNA. Interestingly, MPP6 appeared to display RNA-binding activity in vitro with a preference for pyrimidine-rich sequences, and to bind to the ITS2 element of pre-rRNAs. Our data indicate that MPP6 is a nucleolus-specific exosome co-factor required for its role in the maturation of 5.8S rRNA.     

Crystal Structure of the S. solfataricus Archaeal Exosome Reveals Conformational Flexibility in the RNA-Binding Ring

Background

The exosome complex is an essential RNA 3′-end processing and degradation machinery. In archaeal organisms, the exosome consists of a catalytic ring and an RNA-binding ring, both of which were previously reported to assume three-fold symmetry.

Methodology/Principal Findings

Here we report an asymmetric 2.9 Å Sulfolobus solfataricus archaeal exosome structure in which the three-fold symmetry is broken due to combined rigid body and thermal motions mainly within the RNA-binding ring. Since increased conformational flexibility was also observed in the RNA-binding ring of the related bacterial PNPase, we speculate that this may reflect an evolutionarily conserved mechanism to accommodate diverse RNA substrates for degradation.

Conclusion/Significance

This study clearly shows the dynamic structures within the RNA-binding domains, which provides additional insights on mechanism of asymmetric RNA binding and processing.     

RNA degradation by the exosome is promoted by a nuclear polyadenylation complex

The exosome complex of 3'-5' exonucleases participates in RNA maturation and quality control and can rapidly degrade RNA-protein complexes in vivo. However, the purified exosome showed weak in vitro activity, indicating that rapid RNA degradation requires activating cofactors. This work identifies a nuclear polyadenylation complex containing a known exosome cofactor, the RNA helicase Mtr4p; a poly(A) polymerase, Trf4p; and a zinc knuckle protein, Air2p. In vitro, the Trf4p/Air2p/Mtr4p polyadenylation complex (TRAMP) showed distributive RNA polyadenylation activity. The presence of the exosome suppressed poly(A) tail addition, while TRAMP stimulated exosome degradation through structured RNA substrates. In vivo analyses showed that TRAMP is required for polyadenylation and degradation of rRNA and snoRNA precursors that are characterized exosome substrates. Poly(A) tails stimulate RNA degradation in bacteria, suggesting that this is their ancestral function. We speculate that this function was maintained in eukaryotic nuclei, while cytoplasmic mRNA poly(A) tails acquired different roles in translation.     

Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition

Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3′-to-5′ in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNA-binding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3′ ends and strain-specific alternative 3′ editing within 3′ UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs.     

Arabidopsis thaliana LSM proteins function in mRNA splicing and degradation

Sm-like (Lsm) proteins have been identified in all organisms and are related to RNA metabolism. Here, we report that Arabidopsis nuclear AtLSM8 protein, as well as AtLSM5, which localizes to both the cytoplasm and nucleus, function in pre-mRNA splicing, while AtLSM5 and the exclusively cytoplasmic AtLSM1 contribute to 5′–3′ mRNA decay. In lsm8 and sad1/lsm5 mutants, U6 small nuclear RNA (snRNA) was reduced and unspliced mRNA precursors accumulated, whereas mRNA stability was mainly affected in plants lacking AtLSM1 and AtLSM5. Some of the mRNAs affected in lsm1a lsm1b and sad1/lsm5 plants were also substrates of the cytoplasmic 5′–3′ exonuclease AtXRN4 and of the decapping enzyme AtDCP2. Surprisingly, a subset of substrates was also stabilized in the mutant lacking AtLSM8, which supports the notion that plant mRNAs are actively degraded in the nucleus. Localization of LSM components, purification of LSM-interacting proteins as well as functional analyses strongly suggest that at least two LSM complexes with conserved activities in RNA metabolism, AtLSM1-7 and AtLSM2-8, exist also in plants.     

Arabidopsis thaliana exosome subunit AtRrp4p is a hydrolytic 3′→5′ exonuclease containing S1 and KH RNA-binding domains

The exosome, an evolutionarily conserved complex of multiple 3′→5′ exoribonucleases, is responsible for a variety of RNA processing and degradation events in eukaryotes. In this report Arabidopsis thaliana AtRrp4p is shown to be an active 3′→5′ exonuclease that requires a free 3′-hydroxyl and degrades RNA hydrolytically and distributively, releasing nucleoside 5′-monophosphate products. AtRrp4p behaves as an ~500 kDa species during sedimentation through a 10–30% glycerol gradient, co-migrating with AtRrp41p, another exosome subunit, and it interacts in vitro with AtRrp41p, suggesting that it is also present in the plant cell as a subunit of the exosome. We found that, in addition to a previously reported S1-type RNA-binding domain, members of the Rrp4p family of proteins contain a KH-type RNA-binding domain in the C-terminal half and show that either domain alone can bind RNA. However, only the full-length protein is capable of degrading RNA and interacting with AtRrp41p.