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Cells respond to environmental stress in multiple ways. In the germ line, heat shock and nutritive stress trigger the assembly of large ribonucleoprotein (RNP) granules via liquid-liquid phase separation (LLPS). The RNP granules are hypothesized to maintain the quality of oocytes during stress. The goal of this study was to investigate the cellular response to glucose in the germ line and determine if it is an osmotic stress response. We found that exposure to 500 mM glucose induces the assembly of RNP granules in the germ line within 1 h. Interestingly, the RNP granules are maintained for up to 3 h; however, they dissociate after longer periods of stress. The RNP granules include processing body and stress granule proteins, suggesting shared functions. Based on several lines of evidence, the germ line response to glucose largely appears to be an osmotic stress response, thus identifying osmotic stress as a trigger of LLPS. Although RNP granules are not maintained beyond 3 h of osmotic stress, the quality of oocytes does not appear to decrease after longer periods of stress, suggesting a secondary adaptation in the germ line. We used an indirect marker of glycerol and observed high levels after 5 and 20 h of glucose exposure. Moreover, in gpdh-1;gpdh-2 germ lines, glycerol levels are reduced concomitant with RNP granules being maintained for an extended period. We speculate that increased glycerol levels may function as a secondary osmoregulatory adaptive response in the germ line, following a primary response of RNP granule assembly.

Regulation of gene expression at the level of cytoplasmic polyadenylation is important for many biological phenomena including cell cycle progression, mitochondrial respiration, and learning and memory. GLD4 is one of the non-canonical poly(A) polymerases that regulates cytoplasmic polyadenylation-induced translation, but its target mRNAs and role in cellular physiology is not well known. To assess the full panoply of mRNAs whose polyadenylation is controlled by GLD4, we performed an unbiased whole genome-wide screen using poy(U) chromatography and thermal elution. We identified hundreds of mRNAs regulated by GLD4, several of which are involved in carbohydrate metabolism including GLUT1, a major glucose transporter. Depletion of GLD4 not only reduced GLUT1 poly(A) tail length, but also GLUT1 protein. GLD4-mediated translational control of GLUT1 mRNA is dependent of an RNA binding protein, CPEB1, and its binding elements in the 3΄ UTR. Through regulating GLUT1 level, GLD4 affects glucose uptake into cells and lactate levels. Moreover, GLD4 depletion impairs glucose deprivation-induced GLUT1 up-regulation. In addition, we found that GLD4 affects glucose-dependent cellular phenotypes such as migration and invasion in glioblastoma cells. Our observations delineate a novel post-transcriptional regulatory network involving carbohydrate metabolism and glucose homeostasis mediated by GLD4.

Glutamine synthetase (GS) is an important enzyme for nitrogen assimilation, and GS2, encoded by GLN2, is the only plastid-type GS in Arabidopsis thaliana. A co-expression analysis suggested that the expression level of the gene encoding a uridylyltransferase-like protein, ACR11, is strongly correlated with GLN2 expression levels. Here we showed that the recombinant ACR11 protein increased GS2 activity in vitro by reducing the Km values of its substrate glutamine. A T-DNA insertion mutant of ACR11 exhibited a reduced GS activity under low nitrate conditions and reduced glutamine levels. Biochemical analyses revealed that ACR11 and GS2 interacted both in vitro and in vivo. These data demonstrate that ACR11 is an activator of GS2, giving it a mechanistic role in the nitrogen assimilation of A. thaliana.

We have developed fluorescent 2′,5′ branched RNAs (bRNA) that permit real time monitoring of RNA lariat (intron) debranching enzyme (Dbr1) kinetics. These compounds contain fluorescein (FAM) on the 5′ arm of the bRNA that is quenched by a dabcyl moiety on the 2′ arm. Dbr1-mediated hydrolysis of the 2′,5′ linkage induces a large increase in fluorescence, providing a convenient assay for Dbr1 hydrolysis. We show that unlabeled bRNAs with non-native 2′,5′-phosphodiester linkages, such as phosphoramidate or phosphorothioate, can inhibit Dbr1-mediated debranching with IC50 values in the low nanomolar range. In addition to measuring kinetic parameters of the debranching enzyme, these probes can be used for high throughput screening (HTS) of chemical libraries with the aim of identifying Dbr1 inhibitors, compounds that may be useful in treating neurodegenerative diseases and retroviral infections.

Intron lariats are circular, branched RNAs (bRNAs) produced during pre-mRNA splicing. Their unusual chemical and topological properties arise from branch-point nucleotides harboring vicinal 2′,5′- and 3′,5′-phosphodiester linkages. The 2′,5′-bonds must be hydrolyzed by the RNA debranching enzyme Dbr1 before spliced introns can be degraded or processed into small nucleolar RNA and microRNA derived from intronic RNA. Here, we measure the activity of Dbr1 from Entamoeba histolytica by using a synthetic, dark-quenched bRNA substrate that fluoresces upon hydrolysis. Purified enzyme contains nearly stoichiometric equivalents of Fe and Zn per polypeptide and demonstrates turnover rates of ∼3 s −1 . Similar rates are observed when apo-Dbr1 is reconstituted with Fe(II)+Zn(II) under aerobic conditions. Under anaerobic conditions, a rate of ∼4.0 s −1 is observed when apoenzyme is reconstituted with Fe(II). In contrast, apo-Dbr1 reconstituted with Mn(II) or Fe(II) under aerobic conditions is inactive. Diffraction data from crystals of purified enzyme using X-rays tuned to the Fe absorption edge show Fe partitions primarily to the β-pocket and Zn to the α-pocket. Structures of the catalytic mutant H91A in complex with 7-mer and 16-mer synthetic bRNAs reveal bona fide RNA branchpoints in the Dbr1 active site. A bridging hydroxide is in optimal position for nucleophilic attack of the scissile phosphate. The results clarify uncertainties regarding structure/function relationships in Dbr1 enzymes, and the fluorogenic probe permits high-throughput screening for inhibitors that may hold promise as treatments for retroviral infections and neurodegenerative disease.

RNA uridylation is a significant transcriptome-shaping factor in protists, fungi, metazoans, and plants. The 3′ U-additions are catalyzed by terminal uridyltransferases (TUTases), a diverse group of enzymes that along with non-canonical poly(A) polymerases form a distinct group in the superfamily of DNA polymerase β-like nucleotidyl transferases. Within and across studied organisms and subcellular compartments, TUTases differ in nucleotide triphosphate selectivity, interacting partners, and RNA targets. A general premise linking RNA uridylation to 3′–5′ degradation received support from several studies of small RNAs and mRNA turnover. However, recent work on kinetoplastid protists typified by Trypanosoma brucei provides evidence that RNA uridylation may play a more nuanced role in generating functional small RNAs. In this pathogen's mitochondrion, most mRNAs are internally edited by U-insertions and deletions, and subjected to 3′ adenylation/uridylation; guide RNAs (gRNAs) required for editing are U-tailed. The prominent role of uridylation in mitochondrial RNA metabolism stimulated identification of the first TUTase, RNA editing TUTase 1 (RET1). Here we discuss functional studies of mitochondrial uridylation in trypanosomes that have revealed an unorthodox pathway of small RNA biogenesis. The current model accentuates physical coupling of RET1 and 3′–5′ RNase II/RNB-type exonuclease DSS1 within a stable complex termed the mitochondrial 3′ processome (MPsome). In the confines of this complex, RET1 initially uridylates a long precursor to activate its 3′–5′ degradation by DSS1, and then uridylates trimmed guide RNA to disengage the processing complex from the mature molecule. We also discuss a potential role of antisense transcription in the MPsome pausing at a fixed distance from gRNA's 5′ end. This step likely defines the mature 3′ end by enabling kinetic competition between TUTase and exonuclease activities.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.08.105. The nonsense-mediated decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological mRNAs that encode full-length proteins. In human cells, NMD-targeted mRNAs are degraded by endonucleolytic cleavage and exonucleolytic degradation from both 5-' and 3'-ends. This is done by a process not yet completely understood that recruits decapping and 5'-to-3' exonuclease activities, as well as deadenylating and 3'-to-5' exonuclease exosome activities. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome, but in humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. However, little is known about their role in NMD. Here, we show that some NMD-targets are DIS3L2 substrates in human cells. In addition, we observed that DIS3L2 acts over full-length transcripts, through a process that also involves UPF1. Moreover, DIS3L2-mediated decay is dependent on the activity of the terminal uridylyl transferases Zcchc6/11 (TUT7/4). Together, our findings establish a role for DIS3L2 and uridylation in NMD. Highlights: DIS3L2 functions in the decay of natural NMD-targets in a transcript-specific manner; DIS3L2 acts over full-length NMD-targets, through a process that also involves UPF1; DIS3L2 function in NMD is dependent on the terminal uridylyl transferases Zcchc6/11. This work was partially supported by Fundaçao para a Ci ~ encia e ^ a Tecnologia (FCT) (PTFC/BIM-MEC/3749/2014 to LR and UID/ MULTI/04046/2013 to BioISI). PJdC, HAS and JFG-M are recipients of a fellowship from BioSys PhD programme (SFRH/BD/52495/2014, SFRH/BD/52492/2014, and PD/BD/142898/2018, respectively) and JM is a posdoc fellow (SFRH/BPD/98360/2013) from FCT. Work at ITQB-NOVA was financially supported by: Project LISBOA-01-0145-FEDER-007660 funded by the European Regional Development Fund (FEDER) through COMPETE2020 - Programa Operacional Competitividade e Internacionalizaçao (POCI) and by FCT funds: ~ PTDC/BIA-MIC/1399/2014 to CMA and PTDC/BIM-MEC/3749/2014 to SCV. SCV was financed by program IF of FCT (IF/00217/2015). MS was financed by an FCT contract according to DL57/2016 [SFRH/ BPD/109464/2015]. We thank Dr. V. Narry Kim from Seoul National University, who kindly provided us with the pCK-FLAG-TUT4 and pCK-FLAG-TUT7 vectors. info:eu-repo/semantics/publishedVersion

TENT4 enzymes generate 'mixed tails' of diverse nucleotides at 3' ends of RNAs via nontemplated nucleotide addition to protect messenger RNAs from deadenylation. Here we discover extensive mixed tailing in transcripts of hepatitis B virus (HBV) and human cytomegalovirus (HCMV), generated via a similar mechanism exploiting the TENT4-ZCCHC14 complex. TAIL-seq on HBV and HCMV RNAs revealed that TENT4A and TENT4B are responsible for mixed tailing and protection of viral poly(A) tails. We find that the HBV post-transcriptional regulatory element (PRE), specifically the CNGGN-type pentaloop, is critical for TENT4-dependent regulation. HCMV uses a similar pentaloop, an interesting example of convergent evolution. This pentaloop is recognized by the sterile alpha motif domain-containing ZCCHC14 protein, which in turn recruits TENT4. Overall, our study reveals the mechanism of action of PRE, which has been widely used to enhance gene expression, and identifies the TENT4-ZCCHC14 complex as a potential target for antiviral therapeutics.

The 3′-termini of tRNA are the point of amino acid linkage and thus crucial for their function in delivering amino acids to the ribosome and other enzymes. Therefore, to provide tRNA functionality, cells have to ensure the integrity of the 3′-terminal CCA-tail, which is generated during maturation by the 3′-trailer processing machinery and maintained by the CCA-adding enzyme. We developed a new tRNA sequencing method that is specifically tailored to assess the 3′-termini of E. coli tRNA. Intriguingly, we found a significant fraction of tRNAs with damaged CCA-tails under exponential growth conditions and, surprisingly, this fraction decreased upon transition into stationary phase. Interestingly, tRNAs bearing guanine as a discriminator base are generally unaffected by CCA-tail damage. In addition, we showed tRNA species-specific 3′-trailer processing patterns and reproduced in vitro findings on preferences of the maturation enzyme RNase T in vivo.

Summary: Tolerance to severe tumor microenvironments, including hypoxia and nutrient starvation, is a common feature of aggressive cancer cells and can be targeted. However, metabolic alterations that support cancer cells upon nutrient starvation are not well understood. Here, by comprehensive metabolome analyses, we show that glutamine deprivation leads to phosphoethanolamine (PEtn) accumulation in cancer cells via the downregulation of PEtn cytidylyltransferase (PCYT2), a rate-limiting enzyme of phosphatidylethanolamine biosynthesis. PEtn accumulation correlated with tumor growth under nutrient starvation. PCYT2 suppression was partially mediated by downregulation of the transcription factor ELF3. Furthermore, PCYT2 overexpression reduced PEtn levels and tumor growth. In addition, PEtn accumulation and PCYT2 downregulation in human breast tumors correlated with poor prognosis. Thus, we show that glutamine deprivation leads to tumor progression by regulating PE biosynthesis via the ELF3-PCYT2 axis. Furthermore, manipulating glutamine-responsive genes could be a therapeutic approach to limit cancer progression. : Osawa et al. find that accumulation of phosphoethanolamine (PEtn) protects cancer cells under glutamine starvation through the downregulation of PCYT2. Glutamine regulates PE biosynthesis through PCYT2, resulting in pro-tumorigenic metabolite PEtn accumulation. PEtn stimulates the tolerance of cancer cells to starvation, and lowered PCYT2 expression correlates with decreased survival in patients. Keywords: cancer metabolism, tumor microenvironments, hypoxia, nutrient starvation, glutamine deprivation, amino acids, phosphoethanolamine, PCYT2, PE biosynthesis

Reduced levels of TERC, the telomerase RNA component, cause dyskeratosis congenita (DC) in patients harboring mutations in TERC, PARN, NOP10, NHP2, NAF1, or DKC1. Inhibition of the noncanonical poly(A) polymerase PAPD5, or the exosome RNA degradation complex, partially restores TERC levels in immortalized DKC1 mutant cells, but it remains unknown if modulation of posttranscriptional processing of TERC could improve hematopoietic output in DC. We used human embryonic stem cells (hESCs) with a common dyskerin mutation (DKC1_A353V), which have defective telomere maintenance and reduced definitive hematopoietic potential, to understand the effects of reducing EXOSC3 activity, or silencing PAPD5-mediated oligoadenylation, on hematopoietic progenitor specification and function in DC. Reduction of EXOSC3 or PAPD5 levels in DKC1 mutant hESCs led to functional improvements in TERC levels and telomerase activity, with concomitant telomere elongation and reduced levels of DNA damage signaling. Interestingly, the silencing of PAPD5, but not EXOSC3, significantly restored definitive hematopoietic potential in DKC1 mutant cells. Mechanistically, we show that PAPD5 inhibition is sustained in differentiated CD34+ cells, with a concomitant increase in mature, functional, forms of TERC, indicating that regulation of PAPD5 is a potential strategy to reverse hematologic dysfunction in DC patients.

Uridylation-dependent RNA decay is a widespread eukaryotic pathway modulating RNA homeostasis. Terminal uridylyltransferases (Tutases) add untemplated uridyl residues to RNA 3′-ends, marking them for degradation by the U-specific exonuclease Dis3L2. In Schizosaccharomyces pombe, Cid1 uridylates a variety of RNAs. In this study, we investigate the prevalence and impact of uridylation-dependent RNA decay in S. pombe by transcriptionally profiling cid1 and dis3L2 deletion strains. We found that the exonuclease Dis3L2 represents a bottleneck in uridylation-dependent mRNA decay, whereas Cid1 plays a redundant role that can be complemented by other Tutases. Deletion of dis3L2 elicits a cellular stress response, upregulating transcription of genes involved in protein folding and degradation. Misfolded proteins accumulate in both deletion strains, yet only trigger a strong stress response in dis3L2 deficient cells. While a deletion of cid1 increases sensitivity to protein misfolding stress, a dis3L2 deletion showed no increased sensitivity or was even protective. We furthermore show that uridylyl- and adenylyltransferases cooperate to generate a 5′-NxAUUAAAA-3′ RNA motif on dak2 mRNA. Our studies elucidate the role of uridylation-dependent RNA decay as part of a global mRNA surveillance, and we found that perturbation of this pathway leads to the accumulation of misfolded proteins and elicits cellular stress responses.

A key aspect of development in all metazoans is remodeling at the cellular level. During the development of gametes, remodeling occurs throughout the germ line. When Caenorhabditis elegans hermaphrodites become depleted of sperm after 4 days of adulthood, significant cellular remodeling occurs within the meiotically-arrested oocytes, including the formation of ribonucleoprotein granules. Since major remodeling of the endoplasmic reticulum (ER) occurs in early embryos, we investigated the extent of ER remodeling in meiotically-arrested oocytes. We found, using a combination of fluorescence reporters and transmission electron microscopy, that the ER in arrested oocytes accumulates in patches and sheets that are enriched at the cortex. Our findings suggest this remodeling is not due to simple displacement by large amounts of yolk that accumulate in arrested oocytes, and instead may be genetically regulated. We further identified the Ddx6 RNA helicase, CGH-1, as a key regulator of ER in the germ line. In cgh-1(tn691) oocytes, we detected cortical ER patches as well as aberrant granules of the RNA-binding proteins, PAB-1, MEX-3, and CGH-1. Taken together, our results suggest the possibility that the spatial organization of RNA binding proteins may regulate the translation of mRNAs associated with the ER that in turn, controls the organization of the ER in the adult germ line.

Summary Many microRNAs (miRNAs) exist alongside abundant miRNA isoforms (isomiRs), most of which arise from post-maturation sequence modifications such as 3′ uridylation. However, the ways in which these sequence modifications affect miRNA function remain poorly understood. Here, using human miR-27a in cell lines as a model, we discovered that a nonfunctional target site unable to base-pair extensively with the miRNA seed sequence can regain function when an upstream adenosine is able to base-pair with a post-transcriptionally added uridine in the miR-27a tail. This tail-U-mediated repression (TUMR) is abolished in cells lacking the uridylation enzymes TUT4 and TUT7, indicating that uridylation alters miRNA function by modulating target recognition. We identified a set of non-canonical targets in human cells that are specifically regulated by uridylated miR-27a. We provide evidence that TUMR expands the targets of other endogenous miRNAs. Our study reveals a function of uridylated isomiRs in regulating non-canonical miRNA targets.

In yeast, the Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP) complex acts as a cofactor for the nuclear exosome to promote degradation of various RNAs. However, the corresponding machinery in mammals is less characterized. We analyzed the interactions of the human TRAMP-like proteins, PAPD5, ZCCHC7, and MTR4, with the nuclear exosome. PAPD5 and ZCCHC7 exhibited mutual interactions in presence of the exosome catalytic subunit RRP6, whereas MTR4 was dispensable for their assembly. Furthermore, the human TRAMP-like proteins were involved in the RRP6-catalyzed turnover of pre-rRNA 5'ETS fragments. These results suggest the significant role for RRP6 in the assembly of TRAMP-like proteins during nucleolar RNA surveillance.

The telomerase RNA component (TERC) is a critical determinant of cellular self-renewal. Poly(A)-specific ribonuclease (PARN) is required for posttranscriptional maturation of TERC. PARN mutations lead to incomplete 3' end processing and increased destruction of nascent TERC RNA transcripts, resulting in telomerase deficiency and telomere diseases. Here, we determined that overexpression of TERC increased telomere length in PARN-deficient cells and hypothesized that decreasing posttranscriptional 3' oligo-adenylation of TERC would counteract the deleterious effects of PARN mutations. Inhibition of the noncanonical poly(A) polymerase PAP-associated domain-containing 5 (PAPD5) increased TERC levels in PARN-mutant patient cells. PAPD5 inhibition was also associated with increases in TERC stability, telomerase activity, and telomere elongation. Our results demonstrate that manipulating posttranscriptional regulatory pathways may be a potential strategy to reverse the molecular hallmarks of telomere disease.

Complexes of RNA and RNA binding proteins form large-scale supramolecular structures under many cellular contexts. In Caenorhabditis elegans, small germ granules are present in the germ line that share characteristics with liquid droplets that undergo phase transitions. In meiotically-arrested oocytes of middle-aged hermaphrodites, the germ granules appear to aggregate or condense into large assemblies of RNA-binding proteins and maternal mRNAs. Prior characterization of the assembly of large-scale RNP structures via candidate approaches has identified a small number of regulators of phase transitions in the C. elegans germ line; however, the assembly, function, and regulation of these large RNP assemblies remain incompletely understood. To identify genes that promote remodeling and assembly of large RNP granules in meiotically-arrested oocytes, we performed a targeted, functional RNAi screen and identified over 300 genes that regulate the assembly of the RNA-binding protein MEX-3 into large granules. Among the most common GO classes are several categories related to RNA biology, as well as novel categories such as cell cortex, ER, and chromosome segregation. We found that arrested oocytes that fail to localize MEX-3 into cortical granules display reduced oocyte quality, consistent with the idea that the larger RNP assemblies promote oocyte quality when fertilization is delayed. Interestingly, a relatively small number of genes overlap with the regulators of germ granule assembly during normal development, or with the regulators of solid RNP granules in cgh-1 oocytes, suggesting fundamental differences in the regulation of RNP granule phase transitions during meiotic arrest.

tRNAs undergo multiple conformational changes during the translation cycle that are required for tRNA translocation and proper communication between the ribosome and translation factors. Recent structural data on how destabilized tRNAs utilize the CCA-adding enzyme to proofread themselves put a spotlight on tRNA flexibility beyond the translation cycle. In analogy to tRNA surveillance, this review finds that other processes also exploit versatile tRNA folding to achieve, amongst others, specific aminoacylation, translational regulation by riboswitches or a block of bacterial translation. tRNA flexibility is thereby not restricted to the hinges utilized during translation. In contrast, the flexibility of tRNA is distributed all over its L-shape and is actively exploited by the tRNA-interacting partners to discriminate one tRNA from another. Since the majority of tRNA modifications also modulate tRNA flexibility it seems that cells devote enormous resources to tightly sense and regulate tRNA structure. This is likely required for error-free protein synthesis.

Mutations in the human telomerase RNA component (hTR), the telomerase ribonucleoprotein component dyskerin (DKC1) and the poly(A) RNase (PARN) can lead to reduced levels of hTR and to dyskeratosis congenita (DC). However, the enzymes and mechanisms responsible for hTR degradation are unknown. We demonstrate that defects in dyskerin binding lead to hTR degradation by PAPD5-mediated oligoadenylation, which promotes 3'-to-5' degradation by EXOSC10, as well as decapping and 5'-to-3' decay by the cytoplasmic DCP2 and XRN1 enzymes. PARN increased hTR levels by deadenylating hTR, thereby limiting its degradation by EXOSC10. Telomerase activity and proper hTR localization in dyskerin- or PARN-deficient cells were rescued by knockdown of DCP2 and/or EXOSC10. Prevention of hTR RNA decay also led to a rescue of localization of DC-associated hTR mutants. These results suggest that inhibition of RNA decay pathways might be a useful therapy for some telomere pathologies.

Pre-mRNA splicing is a central step in the shaping of the eukaryotic transcriptome and in the regulation of gene expression. Yet, due to a focus on fully processed mRNA, common approaches for defining pre-mRNA splicing genome-wide are suboptimal—especially with respect to defining the branch point sequence, a key cis-element that initiates the chemistry of splicing. Here, we report a complementary intron-centered approach designed to more efficiently, simply, and directly define splicing events genome-wide. Specifically, we developed a method distinguished by deep sequencing of lariat intron termini (LIT-seq). In a test of LIT-seq using the budding yeast Saccharomyces cerevisiae, we not only successfully captured the majority of annotated, expressed splicing events but also uncovered 45 novel splicing events, establishing the sensitivity of LIT-seq. Moreover, our libraries were highly enriched with reads that reported on splice sites; by a simple and direct inspection of sequencing reads, we empirically defined both 5′ splice sites and branch sites, as well as their consensus sequences, with nucleotide resolution. Additionally, our study revealed that the 3′ termini of lariat introns are subject to nontemplated addition of adenosines, characteristic of signals sensed by 3′ to 5′ RNA turnover machinery. Collectively, this work defines a novel, genome-wide approach for analyzing splicing with unprecedented depth, specificity, and resolution.

Choline plays a critical role in systemic lipid metabolism and hepatic function. Here we conducted a series of experiments to investigate the effect of choline supplementation on metabolically altered Pcyt2(+/-) mice. In Pcyt2(+/-) mice, the membrane phosphatidylethanolamine (PE) turnover is reduced and the formation of fatty acids (FA) and triglycerides (TAG) increased, resulting in hypertriglyceridemia, liver steatosis and obesity. One month of choline supplementation reduced the incorporation of FA into TAG and facilitated TAG degradation in Pcyt2(+/-) adipocytes, plasma and liver. Choline particularly stimulated adipocyte and liver TAG lipolysis by specific lipases (ATGL, LPL and HSL) and inhibited TAG formation by DGAT1 and DGAT2. Choline also activated the liver AMPK and mitochondrial FA oxidation gene PPARα and reduced the FA synthesis genes SREBP1, SCD1 and FAS. Liver (HPLC) and plasma (tandem mass spectroscopy and (1)H-NMR) metabolite profiling established that Pcyt2(+/-) mice have reduced membrane cholesterol/sphingomyelin ratio and the homocysteine/methionine cycle that were improved by choline supplementation. These data suggest that supplementary choline is beneficial for restoring FA and TAG homeostasis under conditions of obesity caused by impaired PE synthesis.

Cancer secretion can change the properties of adjacent cells, including peripheral blood mononuclear cells (PBMCs). We investigated whether such secretion influences messenger RNA expression in PBMCs of patients with head and neck squamous cell carcinoma (HNSCC). In the present study, co-culture model of normal PBMCs and HNSCC cell lines were established. The PBMCs were subsequently subjected to RNA sequencing for transcriptome analysis. Furthermore, expression data from the Gene Expression Omnibus repository, platform GPL4133, series GSE39400, were gathered to analyze, afterward identify zinc finger CysCysHisCys (CCHC)-type domain-containing protein 6 (ZCCHC6) as the main gene involved in HNSCC. This gene was then validated by a quantitative real-time polymerase chain reaction. The results showed that ZCCHC6 was expressed at significantly higher levels in the patients with HNSCC than in the healthy controls, and the sensitivity and specificity of these findings for diagnostic purposes were 100.00% and 70.83%, respectively. In summary, our findings demonstrated that the secretion of HNSCC cells could cause the alterations in messenger RNA expression by PBMCs. The ZCCHC6 expression level may apply in HNSCC screening.

Strand selection is a critical step in microRNA (miRNA) biogenesis. Although the dominant strand may change depending on cellular contexts, the molecular mechanism and physiological significance of such alternative strand selection (or "arm switching") remain elusive. Here we find miR-324 to be one of the strongly regulated miRNAs by arm switching and identify the terminal uridylyl transferases TUT4 and TUT7 to be the key regulators. Uridylation of pre-miR-324 by TUT4/7 re-positions DICER on the pre-miRNA and shifts the cleavage site. This alternative processing produces a duplex with a different terminus from which the 3' strand (3p) is selected instead of the 5' strand (5p). In glioblastoma, the TUT4/7 and 3p levels are upregulated, whereas the 5p level is reduced. Manipulation of the strand ratio is sufficient to impair glioblastoma cell proliferation. This study uncovers a role of uridylation as a molecular switch in alternative strand selection and implicates its therapeutic potential.

CTP: phosphoethanolamine cytidylyltransferase (Pcyt2) is the rate-limiting enzyme in mammalian phosphatidylethanolamine (PE) biosynthesis. Previously, we reported that increasedPcyt2 mRNA levels after serum starvation are suppressed by 25-hydroxycholesterol (HC) (25-HC), and that nuclear factor-Y (NF-Y) is involved in the inhibitory effects. Transcription of Hmgcr, which encodes 3-hydroxy-3-methylglutaryl-CoA reductase, is suppressed in the same manner. However, no typical sterol regulatory element (SRE) was detected in the Pcyt2 promoter. We were therefore interested in the effect of 25-HC on the modification of histones and thus treated cells with histone acetyltransferase inhibitor (anacardic acid) or histone deacetylase inhibitor (trichostatin A). The suppressive effect of 25-HC on Pcyt2 and Hmgcr mRNA transcription was ameliorated by trichostatin A. Anacardic acid, 25-HC and 24(S)-HC suppressed their transcription by inhibiting H3K27 acetylation in their promoters as evaluated by chromatin immunoprecipitation (ChIP) assays. 27-HC, 22(S)-HC and 22(R)-HC also suppressed their transcription, but 7α-HC, 7β-HC, the synthetic LXR agonist T0901317 and cholesterol did not. Furthermore, 25-HC inhibited p300 recruitment to the Pcyt2 and Hmgcr promoters, and suppressed H3K27 acetylation. 25-HC in the medium was easily conducted into cells. Based on these results, we concluded that 25-HC (and other side-chain oxysterols) in the medium was easily transferred into cells, suppressed H3K27 acetylation via p300 recruitment on the NF-Y complex in the Pcyt2 and Hmgcr promoters, and then suppressed transcription of these genes although LXR is not involved.

RNA viruses are a major threat to animals and plants. RNA interference (RNAi) and the interferon response provide innate antiviral defense against RNA viruses. Here, we performed a large-scale screen using Caenorhabditis elegans and its natural pathogen the Orsay virus (OrV), and we identified cde-1 as important for antiviral defense. CDE-1 is a homolog of the mammalian TUT4 and TUT7 terminal uridylyltransferases (collectively called TUT4(7)); its catalytic activity is required for its antiviral function. CDE-1 uridylates the 3' end of the OrV RNA genome and promotes its degradation in a manner independent of the RNAi pathway. Likewise, TUT4(7) enzymes uridylate influenza A virus (IAV) mRNAs in mammalian cells. Deletion of TUT4(7) leads to increased IAV mRNA and protein levels. Collectively, these data implicate 3'-terminal uridylation of viral RNAs as a conserved antiviral defense mechanism.

The RNA lariat debranching enzyme (Dbr1) has different functions in RNA metabolism, such as hydrolyzing the 2'-5' linkage in intron lariats, positively influencing Ty1 and HIV-1 retrotransposition, and modulating snRNP recycling during splicing reactions. It seems that Dbr1 is one of the major players in RNA turnover. It is remarkable that of all the studies carried out to date with Dbr1, to our knowledge, none of them have evaluated the expression profile of the endogenous Dbr1 gene. In this work, we describe, for the first time, that Entamoeba histolytica EhDbr1 mRNA has a very short half-life (less than 30 min) and encodes a very stable protein that is present until trophozoite cultures die. We also show that the EhDbr1 protein is present in the nuclear periphery on the cytoplasmic basal side, contrary to the localization of human Dbr1. Comparing these results with previous hypotheses and with results from different organisms suggests that Dbr1 gene expression is finely tuned and conserved across eukaryotes. Experiments describing the aspects of Dbr1 gene expression and Dbr1 mRNA turnover as well as other functions of the protein need to be performed. Particularly, a special emphasis is needed on the protozoan parasite E. histolytica, the causative agent of amoebiasis, since even though it is a unicellular organism, it is an intron-rich eukaryote whose intron lariats seem to be open to avoid intron lariat accumulation and to process them in non-coding RNAs that might be involved in its virulence.

RNA–protein interactions permeate biology. Transcription, translation, and splicing all hinge on the recognition of structured RNA elements by RNA-binding proteins. Models of RNA–protein interactions are generally limited to short linear motifs and structures because of the vast sequence sampling required to access longer elements. Here, we develop an integrated approach that calculates global pairwise interaction scores from in vitro selection and high-throughput sequencing. We examine four RNA-binding proteins of phage, viral, and human origin. Our approach reveals regulatory motifs, discriminates between regulated and non-regulated RNAs within their native genomic context, and correctly predicts the consequence of mutational events on binding activity. We design binding elements that improve binding activity in cells and infer mutational pathways that reveal permissive versus disruptive evolutionary trajectories between regulated motifs. These coupling landscapes are broadly applicable for the discovery and characterization of protein–RNA recognition at single nucleotide resolution., RNA–protein interactions often depend on the recognition of extended RNA elements but the identification of these motifs is challenging. Here, the authors present a global integrated approach to analyze RNA–protein binding landscapes, mapping extended RNA interaction motifs for four RNA-binding proteins.

PARN loss-of-function mutations cause a severe form of the hereditary disease dyskeratosis congenita (DC). PARN deficiency affects the stability of non-coding RNAs such as human telomerase RNA (hTR), but these effects do not explain the severe disease in patients. We demonstrate that PARN deficiency affects the levels of numerous miRNAs in human cells. PARN regulates miRNA levels by stabilizing either mature or precursor miRNAs by removing oligo(A) tails added by the poly(A) polymerase PAPD5, which if remaining recruit the exonuclease DIS3L or DIS3L2 to degrade the miRNA. PARN knockdown destabilizes multiple miRNAs that repress p53 translation, which leads to an increase in p53 accumulation in a Dicer-dependent manner, thus explaining why PARN-defective patients show p53 accumulation. This work also reveals that DIS3L and DIS3L2 are critical 3' to 5' exonucleases that regulate miRNA stability, with the addition and removal of 3' end extensions controlling miRNA levels in the cell.

Objective Cytokine expression is tightly regulated posttranscriptionally, but high levels of interleukin-6 (IL-6) in patients with osteoarthritis (OA) indicate that regulatory mechanisms are disrupted in this disorder. The enzyme ZCCHC6 (zinc-finger CCHC domain-containing protein 6; TUT-7) has been implicated in posttranscriptional regulation of inflammatory cytokine expression, but its role in OA pathogenesis is unknown. The present study was undertaken to investigate whether ZCCHC6 directs the expression of IL-6 and influences OA pathogenesis in vivo. Methods Human and mouse chondrocytes were stimulated with recombinant IL-1β. Expression of ZCCHC6 in human chondrocytes was knocked down using small interfering RNAs. IL-6 transcript stability was determined by actinomycin D chase, and 3'-uridylation of microRNAs was determined by deep sequencing. Zcchc6-/- mice were produced by gene targeting. OA was surgically induced in the knee joints of mice, and disease severity was scored using a semiquantitative grading system. Results ZCCHC6 was markedly up-regulated in damaged cartilage from human OA patients and from wild-type mice with surgically induced OA. Overexpression of ZCCHC6 induced the expression of IL-6, and its knockdown reduced IL-6 transcript stability and IL-1β-induced IL-6 expression in chondrocytes. Reintroduction of Zcchc6 in Zcchc6-/- mouse chondrocytes rescued the IL-1β-induced IL-6 expression. Knockdown of ZCCHC6 reduced the population of micro-RNA 26b (miR-26b) with 3'-uridylation by 60%. Zcchc6-/- mice with surgically induced OA produced low levels of IL-6 and exhibited reduced cartilage damage and synovitis in the joints. Conclusion These findings indicate that ZCCHC6 enhances IL-6 expression in chondrocytes through transcript stabilization and by uridylating miR-26b, which abrogates repression of IL-6. Inhibition of IL-6 expression and significantly reduced OA severity in Zcchc6-/- mice identify ZCCHC6 as a novel therapeutic target to inhibit disease pathogenesis.

Terminal uridylyl transferase (TUTase) is one type of enzyme that modifies RNA molecules by facilitating the post-transcriptional addition of uridyl ribonucleotides to their 3′ ends. Recent researches have reported that Drosophila TUTase, Tailor, exhibits an intrinsic preference for RNA substrates ending in 3′G, distinguishing it from any other known TUTases. Through this unique feature, Tailor plays a crucial role as the repressor in the biogenesis pathway of splicing-derived mirtron pre-miRNAs. Here we describe crystal structures of core catalytic domain of Tailor and its complexes with RNA stretches 5′-AGU-3′ and 5′-AGUU-3′. We demonstrate that R327 and N347 are two key residues contributing cooperatively to Tailor's preference for 3′G, and R327 may play an extra role in facilitating the extension of polyuridylation chain. We also demonstrate that conformational stability of the exit of RNA-binding groove also contributes significantly to Tailor's activity. Overall, our work reveals useful insights to explain why Drosophila Tailor can preferentially select RNA substrates ending in 3′G and provides important values for further understanding the biological significances of biogenesis pathway of mirtron in flies.

tRNA maturation and quality control are crucial for proper functioning of these transcripts in translation. In several organisms, defective tRNAs were shown to be tagged by poly(A) or CCACCA tails and subsequently degraded by 3′-exonucleases. In a deep-sequencing analysis of tRNA 3′-ends, we detected the CCACCA tag also in Escherichia coli. However, this tag closely resembles several 3′-trailers of tRNA precursors targeted for maturation and not for degradation. Here, we investigate the ability of two important exonucleases, RNase R and RNase T, to distinguish tRNA precursors with a native 3′-trailer from tRNAs with a CCACCA tag. Our results show that the degrading enzyme RNase R breaks down both tRNAs primed for degradation as well as precursor transcripts, indicating that it is a rather nonspecific RNase. RNase T, a main processing exonuclease involved in trimming of 3′-trailers, is very inefficient in converting the CCACCA-tagged tRNA into a mature transcript. Hence, while both RNases compete for trailer-containing tRNA precursors, the inability of RNase T to process CCACCA tails ensures that defective tRNAs cannot reenter the functional tRNA pool, representing a safeguard to avoid detrimental effects of tRNAs with erroneous integrity on protein synthesis. Furthermore, these data indicate that the RNase T-mediated end turnover of the CCA sequence represents a means to deliver a tRNA to a repeated quality control performed by the CCA-adding enzyme. Hence, originally described as a futile side reaction, the tRNA end turnover seems to fulfill an important function in the maintenance of the tRNA pool in the cell.

The ACT domain (aspartate kinase, chorismate mutase and TyrA), an allosteric effector binding domain, is commonly found in amino acid metabolic enzymes. In addition to ACT domain-containing enzymes, plants have a novel family of ACT domain repeat (ACR) proteins, which do not contain any recognizable catalytic domain. Arabidopsis has 12 ACR proteins, whose functions are largely unknown. To study the functions of Arabidopsis ACR11, we have characterized two independent T-DNA insertion mutants, acr11-2 and acr11-3. RNA gel-blot analysis revealed that the expression of wild-type ACR11 transcripts was not detectable in the acr11 mutants. Interestingly, a lesion-mimic phenotype occurs in some rosette leaves of the acr11 mutants. In addition, high levels of reactive oxygen species (ROS), salicylic acid (SA), and callose accumulate in the mutant leaves when grown under normal conditions. The expression of several SA marker genes and the key SA biosynthetic gene ISOCHORISMATE SYNTHASE1 is up-regulated in the acr11 mutants. Furthermore, the acr11 mutants are more resistant to the infection of bacterial pathogen Pseudomonas syringae pathovar tomato DC3000. These results suggest that ACR11 may be directly or indirectly involved in the regulation of ROS and SA accumulation, which in turn modulates SA-associated defense responses and disease resistance in Arabidopsis.

Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients’ fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.

PMC5785979; Cold adaptation is an evolutionary process that has dramatic impact on enzymatic activity. Increased flexibility of the protein structure represents the main evolutionary strategy for efficient catalysis and reaction rates in the cold, but is achieved at the expense of structural stability. This results in a significant activity-stability tradeoff, as it was observed for several metabolic enzymes. In polymerases, however, not only reaction rates, but also fidelity plays an important role, as these enzymes have to synthesize copies of DNA and RNA as exact as possible. Here, we investigate the effects of cold adaptation on the highly accurate CCA-adding enzyme, an RNA polymerase that uses an internal amino acid motif within the flexible catalytic core as a template to synthesize the CCA triplet at tRNA 3'-ends. As the relative orientation of these residues determines nucleotide selection, we characterized how cold adaptation impacts template reading and fidelity. In a comparative analysis of closely related psychro-, meso-, and thermophilic enzymes, the cold-adapted polymerase shows a remarkable error rate during CCA synthesis in vitro as well as in vivo. Accordingly, CCA-adding activity at low temperatures is not only achieved at the expense of structural stability, but also results in a reduced polymerization fidelity.

Previous studies in our laboratory showed that the RNA debranching enzyme (DBR1) is not required for early steps in HIV cDNA formation but is necessary for synthesis of intermediate and late cDNA products. To further characterize this effect, we evaluated the topology of the 5′ end of the HIV-1 RNA genome during early infection with and without inhibition of DBR1 synthesis. Cells were transfected with DBR1 short hairpin RNA (shRNA) followed 48 h later by infection with an HIV-1-derived vector containing an RNase H-deficient reverse transcriptase (RT). RNA was isolated at several times postinfection and treated with various RNA-modifying enzymes prior to rapid amplification of 5′ cDNA ends (5′ RACE) for HIV-1 RNA and quantitative reverse transcriptase PCR (qRT-PCR). In infected cells, DBR1 knockdown inhibited detection of free HIV-1 RNA 5′ ends at all time points. The difference in detection of free HIV-1 RNA 5′ ends in infected DBR1 knockdown versus control cells was eliminated by in vitro incubation of infected cell RNAs with yeast or human DBR1 enzyme prior to 5′ RACE and qRT-PCR. This was dependent on the 2′-5′ phosphatase activity of DBR1, since it did not occur when we used the catalytically inactive DBR1(N85A) mutant. Finally, HIV-1 RNA from infected DBR1 knockdown cells was resistant to RNase R that degrades linear RNAs but not RNAs in circular or lariat-like conformations. These results provide evidence for formation of a lariat-like structure involving the 5′ end of HIV-1 RNA during an early step in infection and the involvement of DBR1 in resolving it. IMPORTANCE Our findings support a new view of the early steps in HIV genome replication. We show that the HIV genomic RNA is rapidly decapped and forms a lariat-like structure after entering a cell. The lariat-like structure is subsequently resolved by the cellular enzyme DBR1, leaving a 5′ phosphate. This pathway is similar to the formation and resolution of pre-mRNA intron lariats and therefore suggests that similar mechanisms may be used by HIV. Our work therefore opens a new area of investigation in HIV replication and may ultimately uncover new targets for inhibiting HIV replication and for preventing the development of AIDS.

Phosphatidylethanolamine (PE) is the most abundant inner membrane phospholipid. PE synthesis from ethanolamine and diacylglycerol is regulated primarily by CTP:phosphoethanolamine cytidylyltransferase (Pcyt2). Pcyt2(+/-) mice have reduced PE synthesis and, as a consequence, perturbed glucose and fatty acid metabolism, which gradually leads to the development of hyperlipidemia, obesity, and insulin resistance. Glucose and fatty acid uptake and the corresponding transporters Glut4 and Cd36 are similarly impaired in male and female Pcyt2(+/-) hearts. These mice also have similarly reduced phosphatidylinositol 3-kinase (PI3K)/Akt1 signaling and increased reactive oxygen species (ROS) production in the heart. However, only Pcyt2(+/-) males develop hypertension and cardiac hypertrophy. Pcyt2(+/-) males have upregulated heart AceI expression, heart phospholipids enriched in arachidonic acid and other n-6 polyunsaturated fatty acids, and dramatically increased ROS production in the aorta. In contrast, Pcyt2(+/-) females have unmodified heart phospholipids but have reduced heart triglyceride levels and altered expression of the structural genes Acta (low) and Myh7 (high). These changes together protect Pcyt2(+/-) females from cardiac dysfunction under conditions of reduced glucose and fatty acid uptake and heart insulin resistance. Our data identify Pcyt2 and membrane PE biogenesis as important determinants of gender-specific differences in cardiac lipids and heart function.

Protozoan mitochondrial tRNAs (mt-tRNAs) are repaired by a process known as 5′-editing. Mt-tRNA sequencing revealed organism-specific patterns of editing G–U base pairs, wherein some species remove G–U base pairs during 5′-editing, while others retain G–U pairs in the edited tRNA. We tested whether 3′–5′ polymerases that catalyze the repair step of 5′-editing exhibit organism-specific preferences that explain the treatment of G–U base pairs. Biochemical and kinetic approaches revealed that a 3′–5′ polymerase from Acanthamoeba castellanii tolerates G–U wobble pairs in editing substrates much more readily than several other enzymes, consistent with its biological pattern of editing.

The RNA-binding protein Lin28 regulates the expression of the let-7 family of microRNAs (miRNAs) during early embryonic development. Lin28 recruits the 3' terminal uridylyl transferase (TUTase) Zcchc11 (TUT4) and/or Zcchc6 (TUT7) to precursor let-7 RNA (pre-let-7) to selectively block let-7 biogenesis. Uridylated pre-let-7 is targeted for decay by the downstream exonuclease Dis3l2 thereby preventing processing to mature let-7. Activation of this oncogenic pathway via up-regulation of Lin28 expression promotes cellular transformation, drives tumorigenesis in mouse models, and is frequently observed in a wide variety of cancer. Recent proof-of-principle experiments showed that Zcchc11 knockdown inhibits the tumorigenicity of Lin28-expressing human cancer cells and established this enzyme as a possible new therapeutic target for human malignancies. However, there are currently no known pharmacological agents capable of targeting this novel enzyme. In this study we developed and applied a sensitive biochemical assay that monitors Zcchc11 activity. Using this assay we performed an automated high-throughput screen of ∼ 15,000 chemicals to identify putative TUTase inhibitors. Several of these small molecules were validated as specific inhibitors of Zcchc11 activity. Our results demonstrate the feasibility of screening for TUTase inhibitors and present a relatively simple platform that can be exploited for future drug discovery efforts aimed at restoring let-7 expression in cancer.

We report on the clinical data and molecular cytogenetic findings in three unrelated patients presenting with intellectual disability and behavior abnormalities. An overlapping microduplication involving 3p26.2-26.3 was identified in these patients. All three aberrations were confirmed and proven to be parentally inherited. The sizes of the duplications were different, with a common minimal region of 423,754 bp containing two genes - TRNT1 and CRBN. Here, we hypothesize that the copy number gain of CRBN gene might be responsible for developmental delay/intellectual disability.

The spliceosome is a large molecular machine that serves to remove the intervening sequences that are present in most eukaryotic pre-mRNAs. At its core are five small nuclear ribonucleoprotein complexes, the U1, U2, U4, U5 and U6 snRNPs, which undergo dynamic rearrangements during splicing. Their reutilization for subsequent rounds of splicing requires reversion to their original configurations, but little is known about this process. Here, we show that ZK863.4/USIP-1 (U Six snRNA-Interacting Protein-1) is a ribonucleotidyl transferase that promotes accumulation of the Caenorhabditis elegans U6 snRNA. Endogenous USIP-1-U6 snRNA complexes lack the Lsm proteins that constitute the protein core of the U6 snRNP, but contain the U6 snRNP recycling factor SART3/B0035.12. Furthermore, co-immunoprecipitation experiments suggest that SART3 but not USIP-1 occurs also in a separate complex containing both the U4 and U6 snRNPs. Based on this evidence, genetic interaction between usip-1 and sart-3, and the apparent dissociation of Lsm proteins from the U6 snRNA during spliceosome activation, we propose that USIP-1 functions upstream of SART3 to promote U6 snRNA recycling.

DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5' end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA.

Addition of the trinucleotide cytosine/cytosine/adenine (CCA) to the 3' end of transfer RNAs (tRNAs) is essential for translation and is catalyzed by the enzyme TRNT1 (tRNA nucleotidyl transferase), which functions in both the cytoplasm and mitochondria. Exome sequencing revealed TRNT1 mutations in two unrelated subjects with different clinical features. The first presented with acute lactic acidosis at 3 weeks of age and developed severe developmental delay, hypotonia, microcephaly, seizures, progressive cortical atrophy, neurosensorial deafness, sideroblastic anemia and renal Fanconi syndrome, dying at 21 months. The second presented at 3.5 years with gait ataxia, dysarthria, gross motor regression, hypotonia, ptosis and ophthalmoplegia and had abnormal signals in brainstem and dentate nucleus. In subject 1, muscle biopsy showed combined oxidative phosphorylation (OXPHOS) defects, but there was no OXPHOS deficiency in fibroblasts from either subject, despite a 10-fold-reduction in TRNT1 protein levels in fibroblasts of the first subject. Furthermore, in normal controls, TRNT1 protein levels are 10-fold lower in muscle than in fibroblasts. High resolution northern blots of subject fibroblast RNA suggested incomplete CCA addition to the non-canonical mitochondrial tRNA(Ser(AGY)), but no obvious qualitative differences in other mitochondrial or cytoplasmic tRNAs. Complete knockdown of TRNT1 in patient fibroblasts rendered mitochondrial tRNA(Ser(AGY)) undetectable, and markedly reduced mitochondrial translation, except polypeptides lacking Ser(AGY) codons. These data suggest that the clinical phenotypes associated with TRNT1 mutations are largely due to impaired mitochondrial translation, resulting from defective CCA addition to mitochondrial tRNA(Ser(AGY)), and that the severity of this biochemical phenotype determines the severity and tissue distribution of clinical features.

Terminal uridylyl transferases (TUTs) are responsible for the post-transcriptional addition of uridyl residues to RNA 3' ends, leading in some cases to altered stability. The Schizosaccharomyces pombe TUT Cid1 is a model enzyme that has been characterized structurally at moderate resolution and provides insights into the larger and more complex mammalian TUTs, ZCCHC6 and ZCCHC11. Here, we report a higher resolution (1.74 Å) crystal structure of Cid1 that provides detailed evidence for uracil selection via the dynamic flipping of a single histidine residue. We also describe a novel closed conformation of the enzyme that may represent an intermediate stage in a proposed product ejection mechanism. The structural insights gained, combined with normal mode analysis and biochemical studies, demonstrate that the plasticity of Cid1, particularly about a hinge region (N164-N165), is essential for catalytic activity, and provide an explanation for its distributive uridylyl transferase activity. We propose a model clarifying observed differences between the in vitro apparently processive activity and in vivo distributive monouridylylation activity of Cid1. We suggest that modulating the flexibility of such enzymes-for example by the binding of protein co-factors-may allow them alternatively to add single or multiple uridyl residues to the 3' termini of RNA molecules.

Summary Uridylation of RNA species represents an emerging theme in post-transcriptional gene regulation. In the microRNA pathway, such modifications regulate small RNA biogenesis and stability in plants, worms, and mammals. Here, we report Tailor, an uridylyltransferase that is required for the majority of 3′ end modifications of microRNAs in Drosophila and predominantly targets precursor hairpins. Uridylation modulates the characteristic two-nucleotide 3′ overhang of microRNA hairpins, which regulates processing by Dicer-1 and destabilizes RNA hairpins. Tailor preferentially uridylates mirtron hairpins, thereby impeding the production of non-canonical microRNAs. Mirtron selectivity is explained by primary sequence specificity of Tailor, selecting substrates ending with a 3′ guanosine. In contrast to mirtrons, conserved Drosophila precursor microRNAs are significantly depleted in 3′ guanosine, thereby escaping regulatory uridylation. Our data support the hypothesis that evolutionary adaptation to Tailor-directed uridylation shapes the nucleotide composition of precursor microRNA 3′ ends. Hence, hairpin uridylation may serve as a barrier for the de novo creation of microRNAs in Drosophila., Graphical Abstract, Highlights • Tailor is a small RNA uridylyltransferase in Drosophila • Tailor uridylates pre-miRNAs and regulates miRNA maturation • Tailor prevents the maturation of non-canonical miRNAs, i.e., mirtrons • Tailor may act as a barrier for the de novo creation of miRNAs, Reimão-Pinto et al. report a small RNA-specific terminal uridylyltransferase in Drosophila, which acts on precursor miRNAs to regulate miRNA maturation and may serve as a barrier for the de novo creation of miRNAs in flies.

Modifiers of aberrant solid RNP granules suggest new insights into pathways that control dynamics of large-scale RNP bodies and mRNAs during C. elegans oogenesis., Ribonucleoproteins (RNPs) often coassemble into supramolecular bodies with regulated dynamics. The factors controlling RNP bodies and connections to RNA regulation are unclear. During Caenorhabditis elegans oogenesis, cytoplasmic RNPs can transition among diffuse, liquid, and solid states linked to mRNA regulation. Loss of CGH-1/Ddx6 RNA helicase generates solid granules that are sensitive to mRNA regulators. Here, we identified 66 modifiers of RNP solids induced by cgh-1 mutation. A majority of genes promote or suppress normal RNP body assembly, dynamics, or metabolism. Surprisingly, polyadenylation factors promote RNP coassembly in vivo, suggesting new functions of poly(A) tail regulation in RNP dynamics. Many genes carry polyglutatmine (polyQ) motifs or modulate polyQ aggregation, indicating possible connections with neurodegenerative disorders induced by CAG/polyQ expansion. Several RNP body regulators repress translation of mRNA subsets, suggesting that mRNAs are repressed by multiple mechanisms. Collectively, these findings suggest new pathways of RNP modification that control large-scale coassembly and mRNA activity during development.

SummaryUridylation occurs pervasively on mRNAs, yet its mechanism and significance remain unknown. By applying TAIL-seq, we identify TUT4 and TUT7 (TUT4/7), also known as ZCCHC11 and ZCCHC6, respectively, as mRNA uridylation enzymes. Uridylation readily occurs on deadenylated mRNAs in cells. Consistently, purified TUT4/7 selectively recognize and uridylate RNAs with short A-tails (less than ∼25 nt) in vitro. PABPC1 antagonizes uridylation of polyadenylated mRNAs, contributing to the specificity for short A-tails. In cells depleted of TUT4/7, the vast majority of mRNAs lose the oligo-U-tails, and their half-lives are extended. Suppression of mRNA decay factors leads to the accumulation of oligo-uridylated mRNAs. In line with this, microRNA induces uridylation of its targets, and TUT4/7 are required for enhanced decay of microRNA targets. Our study explains the mechanism underlying selective uridylation of deadenylated mRNAs and demonstrates a fundamental role of oligo-U-tail as a molecular mark for global mRNA decay.