Multi-transcriptomics identifies targets of the endoribonuclease DNE1 and highlights its coordination with decapping.

Decapping is a crucial step in mRNA degradation in eucaryotes and requires the formation of a holoenzyme complex between the decapping enzyme DECAPPING 2 (DCP2) and the decapping enhancer DCP1. In Arabidopsis (Arabidopsis thaliana), DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1) is a direct protein partner of DCP1. The function of both DNE1 and decapping are necessary to maintain phyllotaxis, the regularity of organ emergence in the apex. In this study, we combined in vivo mRNA editing, RNA degradome sequencing, transcriptomics and small RNA-omics to identify targets of DNE1 and study how DNE1 and DCP2 cooperate in controlling mRNA fate. Our data reveal that DNE1 mainly contacts and cleaves mRNAs in the coding sequence and has sequence cleavage preferences. DNE1 targets are also degraded through decapping, and both RNA degradation pathways influence the production of mRNA-derived small interfering RNAs. Finally, we detected mRNA features enriched in DNE1 targets including RNA G-quadruplexes and translated upstream open reading frames. Combining these four complementary high-throughput sequencing strategies greatly expands the range of DNE1 targets and allowed us to build a conceptual framework describing the influence of DNE1 and decapping on mRNA fate. These data will be crucial to unveil the specificity of DNE1 action and understand its importance for developmental patterning.

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.

An extensive survey of phytoviral RNA 3' uridylation identifies extreme variations and virus-specific patterns.

Viral RNAs can be uridylated in eukaryotic hosts. However, our knowledge of uridylation patterns and roles remains rudimentary for phytoviruses. Here, we report global 3' terminal RNA uridylation profiles for representatives of the main families of positive single-stranded RNA phytoviruses. We detected uridylation in all 47 viral RNAs investigated here, revealing its prevalence. Yet, uridylation levels of viral RNAs varied from 0.2% to 90%. Unexpectedly, most poly(A) tails of grapevine fanleaf virus (GFLV) RNAs, including encapsidated tails, were strictly monouridylated, which corresponds to an unidentified type of viral genomic RNA extremity. This monouridylation appears beneficial for GFLV because it became dominant when plants were infected with nonuridylated GFLV transcripts. We found that GFLV RNA monouridylation is independent of the known terminal uridylyltransferases (TUTases) HEN1 SUPPRESSOR 1 (HESO1) and UTP:RNA URIDYLYLTRANSFERASE 1 (URT1) in Arabidopsis (Arabidopsis thaliana). By contrast, both TUTases can uridylate other viral RNAs like turnip crinkle virus (TCV) and turnip mosaic virus (TuMV) RNAs. Interestingly, TCV and TuMV degradation intermediates were differentially uridylated by HESO1 and URT1. Although the lack of both TUTases did not prevent viral infection, we detected degradation intermediates of TCV RNA at higher levels in an Arabidopsis heso1 urt1 mutant, suggesting that uridylation participates in clearing viral RNA. Collectively, our work unveils an extreme diversity of uridylation patterns across phytoviruses and constitutes a valuable resource to further decipher pro- and antiviral roles of uridylation.

A NYN domain protein directly interacts with DECAPPING1 and is required for phyllotactic pattern.

In eukaryotes, general mRNA decay requires the decapping complex. The activity of this complex depends on its catalytic subunit, DECAPPING2 (DCP2), and its interaction with decapping enhancers, including its main partner DECAPPING1 (DCP1). Here, we report that in Arabidopsis thaliana, DCP1 also interacts with a NYN domain endoribonuclease, hence named DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1). Interestingly, we found DNE1 predominantly associated with DCP1, but not with DCP2, and reciprocally, suggesting the existence of two distinct protein complexes. We also showed that the catalytic residues of DNE1 are required to repress the expression of mRNAs in planta upon transient expression. The overexpression of DNE1 in transgenic lines led to growth defects and a similar gene deregulation signature than inactivation of the decapping complex. Finally, the combination of dne1 and dcp2 mutations revealed a functional redundancy between DNE1 and DCP2 in controlling phyllotactic pattern formation. Our work identifies DNE1, a hitherto unknown DCP1 protein partner highly conserved in the plant kingdom and identifies its importance for developmental robustness.

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.

The UPF1 interactome reveals interaction networks between RNA degradation and translation repression factors in Arabidopsis.

The RNA helicase UP-FRAMESHIFT (UPF1) is a key factor of nonsense-mediated decay (NMD), a mRNA decay pathway involved in RNA quality control and in the fine-tuning of gene expression. UPF1 recruits UPF2 and UPF3 to constitute the NMD core complex, which is conserved across eukaryotes. No other components of UPF1-containing ribonucleoproteins (RNPs) are known in plants, despite its key role in regulating gene expression. Here, we report the identification of a large set of proteins that co-purify with the Arabidopsis UPF1, either in an RNA-dependent or RNA-independent manner. We found that like UPF1, several of its co-purifying proteins have a dual localization in the cytosol and in P-bodies, which are dynamic structures formed by the condensation of translationally repressed mRNPs. Interestingly, more than half of the proteins of the UPF1 interactome also co-purify with DCP5, a conserved translation repressor also involved in P-body formation. We identified a terminal nucleotidyltransferase, ribonucleases and several RNA helicases among the most significantly enriched proteins co-purifying with both UPF1 and DCP5. Among these, RNA helicases are the homologs of DDX6/Dhh1, known as translation repressors in humans and yeast, respectively. Overall, this study reports a large set of proteins associated with the Arabidopsis UPF1 and DCP5, two components of P-bodies, and reveals an extensive interaction network between RNA degradation and translation repression factors. Using this resource, we identified five hitherto unknown components of P-bodies in plants, pointing out the value of this dataset for the identification of proteins potentially involved in translation repression and/or RNA degradation.

Uridylation Earmarks mRNAs for Degradation and More.

Groundbreaking discoveries have uncovered the widespread post-transcriptional modifications of all classes of RNA. These studies have led to the emerging notion of an 'epitranscriptome' as a new layer of gene regulation. Diverse modifications control RNA fate, including the 3' addition of untemplated nucleotides or 3' tailing. The most exciting recent discoveries in 3' tailing are related to uridylation. Uridylation targets various noncoding RNAs, from small RNAs and their precursors to rRNAs, and U tails mostly regulate processing or degradation. Interestingly, uridylation is also a pervasive modification of mRNAs. In this review, we discuss how the addition of few uridines to the 3' end of mRNAs influences mRNA decay. We also consider recent findings that reveal other consequences of uridylation on mRNA fate.

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.

Cell-type-specific control of secondary cell wall formation by Musashi-type translational regulators in Arabidopsis.

Deciphering the mechanism of secondary cell wall/SCW formation in plants is key to understanding their development and the molecular basis of biomass recalcitrance. Although transcriptional regulation is essential for SCW formation, little is known about the implication of post-transcriptional mechanisms in this process. Here we report that two bonafide RNA-binding proteins homologous to the animal translational regulator Musashi, MSIL2 and MSIL4, function redundantly to control SCW formation in Arabidopsis. MSIL2/4 interactomes are similar and enriched in proteins involved in mRNA binding and translational regulation. MSIL2/4 mutations alter SCW formation in the fibers, leading to a reduction in lignin deposition, and an increase of 4-O-glucuronoxylan methylation. In accordance, quantitative proteomics of stems reveal an overaccumulation of glucuronoxylan biosynthetic machinery, including GXM3, in the msil2/4 mutant stem. We showed that MSIL4 immunoprecipitates GXM mRNAs, suggesting a novel aspect of SCW regulation, linking post-transcriptional control to the regulation of SCW biosynthesis genes.

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 TUTase URT1 connects decapping activators and prevents the accumulation of excessively deadenylated mRNAs to avoid siRNA biogenesis.

Uridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators. URT1 directly interacts with DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6, and this interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. Nanopore direct RNA sequencing reveals a global role of URT1 in shaping poly(A) tail length, notably by preventing the accumulation of excessively deadenylated mRNAs. Based on in vitro and in planta data, we propose a model that explains how URT1 could reduce the accumulation of oligo(A)-tailed mRNAs both by favoring their degradation and because 3' terminal uridines intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb Arabidopsis growth and development.

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.

High-Resolution Mapping of 3' Extremities of RNA Exosome Substrates by 3' RACE-Seq.

The main 3'-5' exoribonucleolytic activity of eukaryotic cells is provided by the RNA exosome. The exosome is constituted by a core complex of nine subunits (Exo9), which coordinates the recruitment and the activities of distinct types of cofactors. The RNA exosome cofactors confer distributive and processive 3'-5' exoribonucleolytic, endoribonucleolytic, and RNA helicase activities. In addition, several RNA binding proteins and terminal nucleotidyltransferases also participate in the recognition of exosome RNA substrates.To fully understand the biological roles of the exosome, the respective functions of its cofactors must be deciphered. This entails the high-resolution analysis of 3' extremities of degradation or processing intermediates in different mutant backgrounds or growth conditions. Here, we describe a detailed 3' RACE-seq procedure for targeted mapping of exosome substrate 3' ends. This procedure combines a 3' RACE protocol with Illumina sequencing to enable the high-resolution mapping of 3' extremities and the identification of untemplated nucleotides for selected RNA targets.

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.