Catalytic activities, molecular connections, and biological functions of plant RNA exosome complexes.

RNA exosome complexes provide the main 3'-5'-exoribonuclease activities in eukaryotic cells and contribute to the maturation and degradation of virtually all types of RNA. RNA exosomes consist of a conserved core complex that associates with exoribonucleases and with multimeric cofactors that recruit the enzyme to its RNA targets. Despite an overall high level of structural and functional conservation, the enzymatic activities and compositions of exosome complexes and their cofactor modules differ among eukaryotes. This review highlights unique features of plant exosome complexes, such as the phosphorolytic activity of the core complex, and discusses the exosome cofactors that operate in plants and are dedicated to the maturation of ribosomal RNA, the elimination of spurious, misprocessed, and superfluous transcripts, or the removal of mRNAs cleaved by the RNA-induced silencing complex and other mRNAs prone to undergo silencing.

Decoding the consecutive lysosomal degradation of 3-O-sulfate containing heparan sulfate by Arylsulfatase G (ARSG).

The lysosomal degradation of heparan sulfate is mediated by the concerted action of nine different enzymes. Within this degradation pathway, Arylsulfatase G (ARSG) is critical for removing 3-O-sulfate from glucosamine, and mutations in ARSG are causative for Usher syndrome type IV. We developed a specific ARSG enzyme assay using sulfated monosaccharide substrates, which reflect derivatives of its natural substrates. These sulfated compounds were incubated with ARSG, and resulting products were analyzed by reversed-phase HPLC after chemical addition of the fluorescent dyes 2-aminoacridone or 2-aminobenzoic acid, respectively. We applied the assay to further characterize ARSG regarding its hydrolytic specificity against 3-O-sulfated monosaccharides containing additional sulfate-groups and N-acetylation. The application of recombinant ARSG and cells overexpressing ARSG as well as isolated lysosomes from wild-type and Arsg knockout mice validated the utility of our assay. We further exploited the assay to determine the sequential action of the different sulfatases involved in the lysosomal catabolism of 3-O-sulfated glucosamine residues of heparan sulfate. Our results confirm and extend the characterization of the substrate specificity of ARSG and help to determine the sequential order of the lysosomal catabolic breakdown of (3-O-)sulfated heparan sulfate.

SERRATE interacts with the nuclear exosome targeting (NEXT) complex to degrade primary miRNA precursors in Arabidopsis.

SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the nuclear exosome targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.

Agreement and Repeatability of Noncycloplegic and Cycloplegic Wavefront-based Autorefraction in Children.

SIGNIFICANCE: Increasing prevalence of refractive error requires assessment of ametropia as a screening tool in children. If cycloplegia is not an option, knowledge about the increase in uncertainty for wavefront-based autorefraction is needed. The cycloplegic agent as the principal variant presents cross-reference and allows for extraction of the influence of accommodation. PURPOSE: The purpose of this study was to determine the repeatability, agreement, and propensity to accommodate of cycloplegic (ARc) and noncycloplegic (ARnc) wavefront-based autorefraction (ZEISS i.Profiler plus; Carl Zeiss Vision, Aalen, Germany) in children aged 2 to 15 years. METHODS: In a clinical setting, three consecutive measurements were feasible for 145 eyes (OD) under both conditions. Data are described by spherical equivalent (M), horizontal or vertical astigmatic component (J0), and oblique astigmatic component (J45). In the case of M, the most positive value of the three measurements was chosen, whereas the mean was applied for astigmatic components. RESULTS: Regarding agreement, differences for ARc minus ARnc were statistically significant: for M, 0.55 (0.55 D; mean [SD]; P < .001), that is, more hyperopic in cycloplegia; for J0, -0.03 (0.11 D; P = .002); and for J45, -0.03 D (SD, 0.09 D; P < .001). Regarding repeatability, astigmatic components showed excellent repeatability: SD < 0.11 D (ARnc) and SD < 0.09 D (ARc). The repeatability of M was SD = 0.57 D with a 95% interval of 1.49 D (ARnc). Under cycloplegia, this decreased to SD = 0.17 D (ARc) with a 95% interval of 0.50 D. The mean propensity to accommodate was 0.44 D from repeated measurements; in cycloplegia, this was reduced to 0.19 D. CONCLUSIONS: Wavefront-based refraction measurement results are highly repeatable and precise for astigmatic components. Noncycloplegic measurements of M show a systematic bias of 0.55 D. Cycloplegia reduces the propensity to accommodate by a factor of 2.4; for noncycloplegic repeated measurements, accommodation is controlled to a total interval of 1.49 D (95%). Without cycloplegia, results improve drastically when measurements are repeated.

The Zinc-Finger Protein SOP1 Is Required for a Subset of the Nuclear Exosome Functions in Arabidopsis.

Correct gene expression requires tight RNA quality control both at transcriptional and post-transcriptional levels. Using a splicing-defective allele of PASTICCINO2 (PAS2), a gene essential for plant development, we isolated suppressor mutations modifying pas2-1 mRNA profiles and restoring wild-type growth. Three suppressor of pas2 (sop) mutations modified the degradation of mis-spliced pas2-1 mRNA species, allowing the synthesis of a functional protein. Cloning of the suppressor mutations identified the core subunit of the exosome SOP2/RRP4, the exosome nucleoplasmic cofactor SOP3/HEN2 and a novel zinc-finger protein SOP1 that colocalizes with HEN2 in nucleoplasmic foci. The three SOP proteins counteract post-transcriptional (trans)gene silencing (PTGS), which suggests that they all act in RNA quality control. In addition, sop1 mutants accumulate some, but not all of the misprocessed mRNAs and other types of RNAs that are observed in exosome mutants. Taken together, our data show that SOP1 is a new component of nuclear RNA surveillance that is required for the degradation of a specific subset of nuclear exosome targets.

SKI2 mediates degradation of RISC 5'-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis.

Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20-24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5', but not 3'-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5' to the cleavage site, but several examples of 3'-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5'-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5'-cleavage fragments.

The RNA helicases AtMTR4 and HEN2 target specific subsets of nuclear transcripts for degradation by the nuclear exosome in Arabidopsis thaliana.

The RNA exosome is the major 3'-5' RNA degradation machine of eukaryotic cells and participates in processing, surveillance and turnover of both nuclear and cytoplasmic RNA. In both yeast and human, all nuclear functions of the exosome require the RNA helicase MTR4. We show that the Arabidopsis core exosome can associate with two related RNA helicases, AtMTR4 and HEN2. Reciprocal co-immunoprecipitation shows that each of the RNA helicases co-purifies with the exosome core complex and with distinct sets of specific proteins. While AtMTR4 is a predominantly nucleolar protein, HEN2 is located in the nucleoplasm and appears to be excluded from nucleoli. We have previously shown that the major role of AtMTR4 is the degradation of rRNA precursors and rRNA maturation by-products. Here, we demonstrate that HEN2 is involved in the degradation of a large number of polyadenylated nuclear exosome substrates such as snoRNA and miRNA precursors, incompletely spliced mRNAs, and spurious transcripts produced from pseudogenes and intergenic regions. Only a weak accumulation of these exosome substrate targets is observed in mtr4 mutants, suggesting that MTR4 can contribute, but plays rather a minor role for the degradation of non-ribosomal RNAs and cryptic transcripts in Arabidopsis. Consistently, transgene post-transcriptional gene silencing (PTGS) is marginally affected in mtr4 mutants, but increased in hen2 mutants, suggesting that it is mostly the nucleoplasmic exosome that degrades aberrant transgene RNAs to limit their entry in the PTGS pathway. Interestingly, HEN2 is conserved throughout green algae, mosses and land plants but absent from metazoans and other eukaryotic lineages. Our data indicate that, in contrast to human and yeast, plants have two functionally specialized RNA helicases that assist the exosome in the degradation of specific nucleolar and nucleoplasmic RNA populations, respectively.

Distinct 18S rRNA precursors are targets of the exosome complex, the exoribonuclease RRP6L2 and the terminal nucleotidyltransferase TRL in Arabidopsis thaliana.

The biosynthesis of ribosomal RNA and its incorporation into functional ribosomes is an essential and intricate process that includes production of mature ribosomal RNA from large precursors. Here, we analyse the contribution of the plant exosome and its co-factors to processing and degradation of 18S pre-RNAs in Arabidopsis thaliana. Our data show that, unlike in yeast and humans, an RRP6 homologue, the nucleolar exoribonuclease RRP6L2, and the exosome complex, together with RRP44, function in two distinct steps of pre-18S rRNA processing or degradation in Arabidopsis. In addition, we identify TRL (TRF4/5-like) as the terminal nucleotidyltransferase that is mainly responsible for oligoadenylation of rRNA precursors in Arabidopsis. We show that TRL is required for efficient elimination of the excised 5' external transcribed spacer and of 18S maturation intermediates that escaped 5' processing. Our data also suggest involvement of additional nucleotidyltransferases, including terminal uridylyltransferase(s), in modifying rRNA processing intermediates in plants.

Uridylation prevents 3' trimming of oligoadenylated mRNAs.

Degradation of mRNAs is usually initiated by deadenylation, the shortening of long poly(A) tails to oligo(A) tails of 12-15 As. Deadenylation leads to decapping and to subsequent 5' to 3' degradation by XRN proteins, or alternatively 3' to 5' degradation by the exosome. Decapping can also be induced by uridylation as shown for the non-polyadenylated histone mRNAs in humans and for several mRNAs in Schizosaccharomyces pombe and Aspergillus nidulans. Here we report a novel role for uridylation in preventing 3' trimming of oligoadenylated mRNAs in Arabidopsis. We show that oligo(A)-tailed mRNAs are uridylated by the cytosolic UTP:RNA uridylyltransferase URT1 and that URT1 has no major impact on mRNA degradation rates. However, in absence of uridylation, oligo(A) tails are trimmed, indicating that uridylation protects oligoadenylated mRNAs from 3' ribonucleolytic attacks. This conclusion is further supported by an increase in 3' truncated transcripts detected in urt1 mutants. We propose that preventing 3' trimming of oligo(A)-tailed mRNAs by uridylation participates in establishing the 5' to 3' directionality of mRNA degradation. Importantly, uridylation prevents 3' shortening of mRNAs associated with polysomes, suggesting that a key biological function of uridylation is to confer 5' to 3' polarity in case of co-translational mRNA decay.

MTR4, a putative RNA helicase and exosome co-factor, is required for proper rRNA biogenesis and development in Arabidopsis thaliana.

The exosome is a conserved protein complex that is responsible for essential 3'→5' RNA degradation in both the nucleus and the cytosol. It is composed of a nine-subunit core complex to which co-factors confer both RNA substrate recognition and ribonucleolytic activities. Very few exosome co-factors have been identified in plants. Here, we have characterized a putative RNA helicase, AtMTR4, that is involved in the degradation of several nucleolar exosome substrates in Arabidopsis thaliana. We show that AtMTR4, rather than its closely related protein HEN2, is required for proper rRNA biogenesis in Arabidopsis. AtMTR4 is mostly localized in the nucleolus, a subcellular compartmentalization that is shared with another exosome co-factor, RRP6L2. AtMTR4 and RRP6L2 cooperate in several steps of rRNA maturation and surveillance, such as processing the 5.8S rRNA and removal of rRNA maturation by-products. Interestingly, degradation of the Arabidopsis 5' external transcribed spacer (5' ETS) requires cooperation of both the 5'→3' and 3'→5' exoribonucleolytic pathways. Accumulating AtMTR4 targets give rise to illegitimate small RNAs; however, these do not affect rRNA metabolism or contribute to the phenotype of mtr4 mutants. Plants lacking AtMTR4 are viable but show several developmental defects, including aberrant vein patterning and pointed first leaves. The mtr4 phenotype resembles that of several ribosomal protein and nucleolin mutants, and may be explained by delayed ribosome biogenesis, as we observed a reduced rate of rRNA accumulation in mtr4 mutants. Taken together, these data link AtMTR4 with rRNA biogenesis and development in Arabidopsis.

The exosome and 3'-5' RNA degradation in plants.

One of the most versatile RNA degradation machines in eukaryotes is the 3'-5' RNA exosome. It consists of nine conserved subunits forming the core complex, which associates with active ribonucleases, RNA binding proteins, helicases and additional co-factors. While yeast and human exosome core complexes are catalytically inactive, the plant core complex has probably retained a phosphorolytic activity. Intriguingly, the down-regulation of individual subunits of the plant core complex in Arabidopsis mutants led to distinct developmental defects, suggesting an unequal contribution of the core subunits to the in vivo activities of the plant exosome complex. In addition, some of the plant core subunits as well as some associated factors are encoded by duplicated genes, which may have both overlapping and specific functions. Together, these results suggest an unique and complex organisation of exosome-mediated RNA degradation processes in plants. This chapter reviews our current knowledge of plant exosomes and discusses the impact of 3'-5' RNA degradation on the posttranscriptional control of plant genome expression.

The exosome and 3'-5' RNA degradation in plants.

One of the most versatile RNA degradation machines in eukaryotes is the 3'-5' RNA exosome. It consists of nine conserved subunits forming the core complex, which associates with active ribonucleases, RNA binding proteins, helicases and additional co-factors. While yeast and human exosome core complexes are catalytically inactive, the plant core complex has probably retained a phosphorolytic activity. Intriguingly, the down-regulation of individual subunits of the plant core complex in Arabidopsis mutants led to distinct developmental defects, suggesting an unequal contribution of the core subunits to the in vivo activities of the plant exosome complex. In addition, some of the plant core subunits as well as some associated factors are encoded by duplicated genes, which may have both overlapping and specific functions. Together, these results suggest an unique and complex organisation of exosome-mediated RNA degradation processes in plants. This chapter reviews our current knowledge of plant exosomes and discusses the impact of 3'-5' RNA degradation on the posttranscriptional control of plant genome expression.

Coping with cryptic and defective transcripts in plant mitochondria.

Plant mitochondria are particularly prone to the production of both defective and cryptic transcripts as a result of the complex organisation and mode of expression of their genome. Cryptic transcripts are generated from intergenic regions due to a relaxed control of transcription. Certain intergenic regions are transcribed at higher rates than genuine genes and therefore, cryptic transcripts are abundantly produced in plant mitochondria. In addition, primary transcripts from genuine genes must go through complex post-transcriptional processes such as C to U editing and cis or trans splicing of group II introns. These post-transcriptional processes are rather inefficient and as a result, defective transcripts are constantly produced in plant mitochondria. In this review, we will describe the nature of cryptic and defective transcripts as well as their fate in plant mitochondria. Although RNA surveillance is crucial to establishing the final transcriptome by degrading cryptic transcripts, plant mitochondria are able to tolerate a surprising high level of defective transcripts.

Relaxed transcription in Arabidopsis mitochondria is counterbalanced by RNA stability control mediated by polyadenylation and polynucleotide phosphorylase.

Plant mitochondrial genomes are extraordinarily large and complex compared to their animal counterparts, due to the presence of large noncoding regions. Multiple promoters are common for plant mitochondrial genes, and transcription exhibits little or no modulation. Mature functional RNAs are produced through various posttranscriptional processes, and control of RNA stability has a major impact on RNA abundance. This control involves polyadenylation which targets RNA for degradation by polynucleotide phosphorylase (PNPase). Here, we have analyzed polyadenylated RNA fragments from Arabidopsis plants down-regulated for PNPase (PNP- plants). Because of their polyadenylated status and the accumulation of the corresponding RNA in PNP- versus wild-type plants, these sequences represent mitochondrial RNA degradation tags. Analysis of these tags revealed that PNPase is involved in degrading rRNA and tRNA maturation by-products but also RNA transcribed from regions that are in some cases highly expressed although lacking known functional genes. Some of these transcripts, such as RNA containing chimeric open reading frames created by recombination or antisense RNA transcribed on the opposite strand of a known gene, may present potential detrimental effects to mitochondrial function. Taken together, our data show that the relaxed transcription in Arabidopsis mitochondria is counterbalanced by RNA stability control mediated by polyadenylation and PNPase.

RST1 and RIPR connect the cytosolic RNA exosome to the Ski complex in Arabidopsis.

The RNA exosome is a key 3'-5' exoribonuclease with an evolutionarily conserved structure and function. Its cytosolic functions require the co-factors SKI7 and the Ski complex. Here we demonstrate by co-purification experiments that the ARM-repeat protein RESURRECTION1 (RST1) and RST1 INTERACTING PROTEIN (RIPR) connect the cytosolic Arabidopsis RNA exosome to the Ski complex. rst1 and ripr mutants accumulate RNA quality control siRNAs (rqc-siRNAs) produced by the post-transcriptional gene silencing (PTGS) machinery when mRNA degradation is compromised. The small RNA populations observed in rst1 and ripr mutants are also detected in mutants lacking the RRP45B/CER7 core exosome subunit. Thus, molecular and genetic evidence supports a physical and functional link between RST1, RIPR and the RNA exosome. Our data reveal the existence of additional cytosolic exosome co-factors besides the known Ski subunits. RST1 is not restricted to plants, as homologues with a similar domain architecture but unknown function exist in animals, including humans.

RNR1, a 3'-5' exoribonuclease belonging to the RNR superfamily, catalyzes 3' maturation of chloroplast ribosomal RNAs in Arabidopsis thaliana.

Arabidopsis thaliana chloroplasts contain at least two 3' to 5' exoribonucleases, polynucleotide phosphorylase (PNPase) and an RNase R homolog (RNR1). PNPase has been implicated in both mRNA and 23S rRNA 3' processing. However, the observed maturation defects do not affect chloroplast translation, suggesting that the overall role of PNPase in maturation of chloroplast rRNA is not essential. Here, we show that this role can be largely ascribed to RNR1, for which homozygous mutants germinate only on sucrose-containing media, and have white cotyledons and pale green rosette leaves. Accumulation of chloroplast-encoded mRNAs and tRNAs is unaffected in such mutants, suggesting that RNR1 activity is either unnecessary or redundant for their processing and turnover. However, accumulation of several chloroplast rRNA species is severely affected. High-resolution RNA gel blot analysis, and mapping of 5' and 3' ends, revealed that RNR1 is involved in the maturation of 23S, 16S and 5S rRNAs. The 3' extensions of the accumulating 5S rRNA precursors can be efficiently removed in vitro by purified RNR1, consistent with this view. Our data suggest that decreased accumulation of mature chloroplast ribosomal RNAs leads to a reduction in the number of translating ribosomes, ultimately compromising chloroplast protein abundance and thus plant growth and development.

RNA degradation by the plant RNA exosome involves both phosphorolytic and hydrolytic activities.

The RNA exosome provides eukaryotic cells with an essential 3'-5' exoribonucleolytic activity, which processes or eliminates many classes of RNAs. Its nine-subunit core (Exo9) is structurally related to prokaryotic phosphorolytic exoribonucleases. Yet, yeast and animal Exo9s have lost the primordial phosphorolytic capacity and rely instead on associated hydrolytic ribonucleases for catalytic activity. Here, we demonstrate that Arabidopsis Exo9 has retained a distributive phosphorolytic activity, which contributes to rRNA maturation processes, the hallmark of exosome function. High-density mapping of 3' extremities of rRNA maturation intermediates reveals the intricate interplay between three exoribonucleolytic activities coordinated by the plant exosome. Interestingly, the analysis of RRP41 protein diversity across eukaryotes suggests that Exo9's intrinsic activity operates throughout the green lineage, and possibly in some earlier-branching non-plant eukaryotes. Our results reveal a remarkable evolutionary variation of this essential RNA degradation machine in eukaryotes.