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In Trypanosoma brucei, mitochondrial pre-mRNAs undergo 3′-5′ exonucleolytic processing, 3′ adenylation and uridylation, 5′ pyrophosphate removal, and, often, U-insertion/deletion editing. The 3′ modifications are modulated by pentatricopeptide repeat (PPR) Kinetoplast Polyadenylation Factors (KPAFs). We have shown that KPAF3 binding to the 3′ region stabilizes properly trimmed transcripts and stimulates their A-tailing by KPAP1 poly(A) polymerase. Conversely, poly(A) binding KPAF4 shields the nascent A-tail from uridylation and decay thereby protecting pre-mRNA upon KPAF3 displacement by editing. While editing concludes in the 5′ region, KPAF1/2 dimer induces A/U-tailing to activate translation. Remarkably, 5′ end recognition and pyrophosphate hydrolysis by the PPsome complex also contribute to mRNA stabilization. Here, we demonstrate that KPAF4 functions as a heterodimer with KPAF5, a protein lacking discernable motifs. We show that KPAF5 stabilizes KPAF4 to enable poly(A) tail recognition, which likely leads to mRNA stabilization during the editing process and impedes spontaneous translational activation of partially-edited transcripts. Thus, KPAF4/5 represents a poly(A) binding element of the mitochondrial polyadenylation complex. We present evidence that RNA editing substrate binding complex bridges the 5′ end-bound PPsome and 3′ end-bound polyadenylation complexes. This interaction may enable mRNA circularization, an apparently critical element of mitochondrial mRNA stability and quality control.

BNC1 is a transcription factor that is crucial for spermatogenesis and male fertility, although the underlying mechanism remains unclear. To study BNC1's specific role in spermatogenesis, we characterized a previously developed mouse model carrying a truncating mutation in Bnc1 (termed Bnc1

The RNA-binding protein Ataxin-2 binds to and stabilizes a number of mRNA sequences, including that of the transactive response DNA-binding protein of 43 kDa (TDP-43). Ataxin-2 is additionally involved in several processes requiring translation, such as germline formation, long-term habituation, and circadian rhythm formation. However, it has yet to be unambiguously demonstrated that Ataxin-2 is actually involved in activating the translation of its target mRNAs. Here we provide direct evidence from a polysome profile analysis showing that Ataxin-2 enhances translation of target mRNAs. Our recently established method for transcriptional pulse-chase analysis under conditions of suppressing deadenylation revealed that Ataxin-2 promotes post-transcriptional polyadenylation of the target mRNAs. Furthermore, Ataxin-2 binds to a poly(A)-binding protein PABPC1 and a noncanonical poly(A) polymerase PAPD4 via its intrinsically disordered region (amino acids 906–1095) to recruit PAPD4 to the targets. Post-transcriptional polyadenylation by Ataxin-2 explains not only how it activates translation but also how it stabilizes target mRNAs, including TDP-43 mRNA. Ataxin-2 is known to be a potent modifier of TDP-43 proteinopathies and to play a causative role in the neurodegenerative disease spinocerebellar ataxia type 2, so these findings suggest that Ataxin-2–induced cytoplasmic polyadenylation and activation of translation might impact neurodegeneration (i.e. TDP-43 proteinopathies), and this process could be a therapeutic target for Ataxin-2–related neurodegenerative disorders.

Family with sequence similarity (FAM46) proteins are newly identified metazoan-specific poly(A) polymerases (PAPs). Although predicted as Gld-2-like eukaryotic non-canonical PAPs, the detailed architecture of FAM46 proteins is still unclear. Exact biological functions for most of FAM46 proteins also remain largely unknown. Here, we report the first crystal structure of a FAM46 protein, FAM46B. FAM46B is composed of a prominently larger N-terminal catalytic domain as compared to known eukaryotic PAPs, and a C-terminal helical domain. FAM46B resembles prokaryotic PAP/CCA-adding enzymes in overall folding as well as certain inter-domain connections, which distinguishes FAM46B from other eukaryotic non-canonical PAPs. Biochemical analysis reveals that FAM46B is an active PAP, and prefers adenosine-rich substrate RNAs. FAM46B is uniquely and highly expressed in human pre-implantation embryos and pluripotent stem cells, but sharply down-regulated following differentiation. FAM46B is localized to both cell nucleus and cytosol, and is indispensable for the viability of human embryonic stem cells. Knock-out of FAM46B is lethal. Knock-down of FAM46B induces apoptosis and restricts protein synthesis. The identification of the bacterial-like FAM46B, as a pluripotent stem cell-specific PAP involved in the maintenance of translational efficiency, provides important clues for further functional studies of this PAP in the early embryonic development of high eukaryotes.

The de-regulation of microRNAs (miRNAs) is associated with multiple human diseases, yet cellular mechanisms governing miRNA abundance remain largely elusive. Human miR-122 is required for Hepatitis C proliferation, and low miR-122 abundance is associated with hepatic cancer. The adenylyltransferase Gld2 catalyses the post-transcriptional addition of a single adenine residue (A + 1) to the 3ʹ-end of miR-122, enhancing its stability. Gld2 activity is inhibited by binding to the Hepatitis C virus core protein during HepC infection, but no other mechanisms of Gld2 regulation are known. We found that Gld2 activity is regulated by site-specific phosphorylation in its disordered N-terminal domain. We identified two phosphorylation sites (S62, S110) where phosphomimetic substitutions increased Gld2 activity and one site (S116) that markedly reduced activity. Using mass spectrometry, we confirmed that HEK 293 cells readily phosphorylate the N-terminus of Gld2. We identified protein kinase A (PKA) and protein kinase B (Akt1) as the kinases that site-specifically phosphorylate Gld2 at S116, abolishing Gld2-mediated nucleotide addition. The data demonstrate a novel phosphorylation-dependent mechanism to regulate Gld2 activity, revealing tumour suppressor miRNAs as a previously unknown target of Akt1-dependent signalling.

Small regulatory RNAs (sRNAs) are an important class of bacterial post-transcriptional regulators that control numerous physiological processes, including stress responses. In Gram-negative bacteria including Escherichia coli, the RNA chaperone Hfq binds many sRNAs and facilitates pairing to target transcripts, resulting in changes in mRNA transcription, translation, or stability. Here, we report that poly(A) polymerase (PAP I), which promotes RNA degradation by exoribonucleases through the addition of poly(A) tails, has a crucial role in the regulation of gene expression by Hfq-dependent sRNAs. Specifically, we show that deletion of pcnB, encoding PAP I, paradoxically resulted in an increased turnover of certain Hfq-dependent sRNAs, including RyhB. RyhB instability in the pcnB deletion strain was suppressed by mutations in hfq or ryhB that disrupt pairing of RyhB with target RNAs, by mutations in the 3′ external transcribed spacer of the glyW-cysT-leuZ transcript (3′ETSLeuZ) involved in pairing with RyhB, or an internal deletion in rne, which encodes the endoribonuclease RNase E. Finally, the reduced stability of RyhB in the pcnB deletion strain resulted in impaired regulation of some of its target mRNAs, specifically sodB and sdhCDAB. Altogether our data support a model where PAP I plays a critical role in ensuring the efficient decay of the 3′ETSLeuZ. In the absence of PAP I, the 3′ETSLeuZ transcripts accumulate, bind Hfq, and pair with RyhB, resulting in its depletion via RNase E-mediated decay. This ultimately leads to a defect in RyhB function in a PAP I deficient strain.

Production of the translation apparatus of E. coli is carefully matched to the demand for protein synthesis posed by a given growth condition. For example, the fraction of RNA polymerases that transcribe rRNA and tRNA drops from 80% during rapid growth to 24% within minutes of a sudden amino acid starvation. We recently reported in Nucleic Acids Research that the tRNA pool is more dynamically regulated than previously thought. In addition to the regulation at the level of synthesis, we found that tRNAs are subject to demand-based regulation at the level of their degradation. In this point-of-view article we address the question of why this phenomenon has not previously been described. We also present data that expands on the mechanism of tRNA degradation, and we discuss the possible implications of tRNA instability for the ability of E. coli to cope with stresses that affect the translation process.

Mutant mice lacking a testis-specific cytoplasmic poly(A) polymerase, PAPOLB/TPAP, exhibit spermiogenesis arrest and male infertility. However, the mechanism by which PAPOLB regulates spermiogenesis remains unclear. In this study, we examined the relationships between PAPOLB and other spermiogenesis regulators present in the chromatoid body (CB). The loss of PAPOLB had no impact either on the abundance of CB components such as PIWIL1, TDRD6, YBX2, and piRNAs, or on retrotransposon expression. In addition, localization of CB proteins and CB architecture were both normal in PAPOLB-null mice. No interactions were observed between PAPOLB and PIWIL1 or YBX2. While PIWIL1 and YBX2 were associated with translationally inactive messenger ribonucleoproteins and translating polyribosomes, PAPOLB was present almost exclusively in the mRNA-free fractions of sucrose gradients. These results suggest that PAPOLB may regulate spermiogenesis through a pathway distinct from that mediated by CB-associated factors.

The parasite Trypanosoma brucei is the causative agent of African sleeping sickness and is known for its unique RNA processing mechanisms that are common to all the kinetoplastidea including Leishmania and Trypanosoma cruzi. Trypanosomes possess two canonical RNA poly (A) polymerases (PAPs) termed PAP1 and PAP2. PAP1 is encoded by one of the only two genes harboring cis-spliced introns in this organism, and its function is currently unknown. In trypanosomes, all mRNAs, and non-coding RNAs such as small nucleolar RNAs (snoRNAs) and long non-coding RNAs (lncRNAs), undergo trans-splicing and polyadenylation. Here, we show that the function of PAP1, which is located in the nucleus, is to polyadenylate non-coding RNAs, which undergo trans-splicing and polyadenylation. Major substrates of PAP1 are the snoRNAs and lncRNAs. Under the silencing of either PAP1 or PAP2, the level of snoRNAs is reduced. The dual polyadenylation of snoRNA intermediates is carried out by both PAP2 and PAP1 and requires the factors essential for the polyadenylation of mRNAs. The dual polyadenylation of the precursor snoRNAs by PAPs may function to recruit the machinery essential for snoRNA processing.

Polyadenylation of nascent RNA by poly(A) polymerase (PAP) is important for 3′ end maturation of almost all eukaryotic mRNAs. Most mammalian genes harbor multiple polyadenylation sites (PASs), leading to expression of alternative polyadenylation (APA) isoforms with distinct functions. How poly(A) polymerases may regulate PAS usage and hence gene expression is poorly understood. Here, we show that the nuclear canonical (PAPα and PAPγ) and non-canonical (Star-PAP) PAPs play diverse roles in PAS selection and gene expression. Deficiencies in the PAPs resulted in perturbations of gene expression, with Star-PAP impacting lowly expressed mRNAs and long-noncoding RNAs to the greatest extent. Importantly, different PASs of a gene are distinctly regulated by different PAPs, leading to widespread relative expression changes of APA isoforms. The location and surrounding sequence motifs of a PAS appear to differentiate its regulation by the PAPs. We show Star-PAP-specific PAS usage regulates the expression of the eukaryotic translation initiation factor EIF4A1, the tumor suppressor gene PTEN and the long non-coding RNA NEAT1. The Star-PAP-mediated APA of PTEN is essential for DNA damage-induced increase of PTEN protein levels. Together, our results reveal a PAS-guided and PAP-mediated paradigm for gene expression in response to cellular signaling cues.

RNAi is a conserved mechanism in which small RNAs induce silencing of complementary targets. How Argonaute-bound small RNAs are targeted for degradation is not well understood. We show that the adenyl-transferase Cid14, a member of the TRAMP complex, and the uridyl-transferase Cid16 add non-templated nucleotides to Argonaute-bound small RNAs in fission yeast. The tailing of Argonaute-bound small RNAs recruits the 3′–5′ exonuclease Rrp6 to degrade small RNAs. Failure in degradation of Argonaute-bound small RNAs results in accumulation of ‘noise' small RNAs on Argonaute and targeting of diverse euchromatic genes by RNAi. To protect themselves from uncontrolled RNAi, cid14Δ cells exploit the RNAi machinery and silence genes essential for RNAi itself, which is required for their viability. Our data indicate that surveillance of Argonaute-bound small RNAs by Cid14/Cid16 and the exosome protects the genome from uncontrolled RNAi and reveal a rapid RNAi-based adaptation to stress conditions., While RNA interference is a highly conserved mechanism of gene regulation, how Argonaute-bound small RNAs are targeted for degradation is not well understood. Here the authors show that Cid14 and Cid16 target Argonaute-bound small RNAs for degradation and protect the genome from uncontrolled RNAi activity.

Pantoea agglomerans YS19 is a preponderant endophytic bacterium isolated from rice. It is characterized by the formation of symplasmata, a type of multicellular aggregate structure, contributing to a strong stress resistance and specific adaptation of YS19 in endophyte-host associations. Indole is an important signal molecule in intra- or interspecies relationships, regulating a variety of bacterial behaviours such as cell aggregation and stress resistance; however, the regulatory mechanism remains an ongoing area of investigation. This study selected YS19 as a model strain to construct a mutant library, utilizing the mTn5 transposon mutagenesis method, thus obtaining a positive mutant with an indole-inhibited mutation gene. Via thermal asymmetric interlaced PCR, the mutational site was identified as the gene of pcnB, which encodes the poly(A) polymerase I to catalyse the polyadenylation of RNAs. The full length of the pcnB sequence was 1332 bp, and phylogenetic analysis revealed that pcnB is extremely conserved among strains of P. agglomerans. The expression of the gene was significantly inhibited (by 36.6 % as detected via quantitative PCR) by indole (0.5 mM). Many physiological behaviours of YS19 were affected by this mutation: the cell decay rate in the post-stationary growth phase was promoted, symplasmata formation and motility were inhibited in the late stationary growth phase and the colonization ability and growth-promoting effect of YS19 on the host plant were also inhibited. This study discusses the indole regulatory pathways from the point of RNA post-transcriptional modification, thus enriching our knowledge of polyadenylation and expanding current research ideas of indole regulation.

MicroRNA-122 (miR-122) is highly expressed in hepatocytes, where it plays an important role in regulating cholesterol and fatty acid metabolism, and it is also a host factor required for hepatitis C virus replication. miR-122 is selectively stabilized by 3′ adenylation mediated by the cytoplasmic poly(A) polymerase GLD-2 (also known as PAPD4 or TENT2). However, it is unclear how GLD-2 specifically stabilizes miR-122. Here, we show that QKI7 KH domain-containing RNA binding (QKI-7), one of three isoforms of the QKI proteins, which are members of the signal transduction and activation of RNA (STAR) family of RNA-binding proteins, is involved in miR-122 stabilization. QKI down-regulation specifically decreased the steady-state level of mature miR-122, but did not affect the pre-miR-122 level. We also found that QKI-7 uses its C-terminal region to interact with GLD-2 and its QUA2 domain to associate with the RNA-induced silencing complex protein Argonaute 2 (Ago2), indicating that the GLD-2–QKI-7 interaction recruits GLD-2 to Ago2. QKI-7 exhibited specific affinity to miR-122 and significantly promoted GLD-2–mediated 3′ adenylation of miR-122 in vitro. Taken together, our findings indicate that miR-122 binds Ago2–interacting QKI-7, which recruits GLD-2 for 3′ adenylation and stabilization of miR-122.

Family with sequence similarity 46 member C (FAM46C) is a non-canonical poly(A) polymerase that is associated with tumorigenesis. However, its role in prostate cancer development is not fully understood. Herein, we determined expression pattern of FAM46C in prostate cancer and further identified its effect on the tumorigenesis and chemosensitivity. FAM46C expression was decreased in prostate cancer tissues and cell lines compared with corresponding controls. FAM46C expression was significantly associated with the Gleason score, tumor size and overall survival. FAM46C knockdown in 22RV1 and DU145 cells significantly inhibited apoptosis and promoted cell proliferation and cell cycle progression as well as activation of AKT. FAM46C overexpression had an inverse effect in DU145 cells and inhibited tumor growth in vivo. FAM46C inhibited cell proliferation and cell cycle progression and induced apoptosis via the PTEN/AKT signaling pathway. FAM46C promoted PTEN expression through inhibiting PTEN ubiquitination. The prostate cancer cells and patient-derived xenograft (PDX) mice with high-FAM46C-expressing demonstrated an enhanced chemosensitivity to docetaxel. These findings suggest that FAM46C control cell proliferation, cell cycle and apoptosis through PTEN/AKT signaling pathway and is associated with chemosensitivity of prostate cancer. Modulation of their levels may offer a new approach for improving anti-tumor efficacy for chemotherapeutic agents in prostate cancer.

Pantoea agglomerans YS19 is a predominant diazotrophic endophyte with multiple growth-promoting effects on its host plant that was isolated from rice. Indole is confirmed to induce many changes of physiological and biochemical characteristics in bacteria. Although YS19 cannot produce indole, it can sense indole in the environment and be regulated by indole. Here, using gfp as a reporter gene, we constructed a series of recombinant plasmids containing the promoter region of the poly(A) polymerase I gene (pcnB) fused with gfp, and compared the green fluorescence intensity at different concentrations of exogenous indole by a flow cytometer. In this research, we confirmed that exogenous indole significantly inhibited the expression of pcnB by its promoter; the regulation sequence sensitive to indole in the promoter region of the pcnB gene (In-pcnB) was between −129 and −88 bp. In-pcnB is widely distributed and strictly conserved in the same genus. These results suggest novel roles of In-pcnB in P. agglomerans YS19, showing its special relation to the indole regulatory pathway.

Coronavirus nonstructural protein 8 (nsp8) has been suggested to have diverse activities, including noncanonical template-dependent polymerase activities. Here, we characterized a recombinant form of the human coronavirus 229E (HCoV-229E) nsp8 and found that the protein has metal ion-dependent RNA 3′-terminal adenylyltransferase (TATase) activity, while other nucleotides were not (or very inefficiently) transferred to the 3′ ends of single-stranded and (fully) double-stranded acceptor RNAs. Using partially double-stranded RNAs, very efficient TATase activity was observed if the opposite (template) strand contained a short 5′ oligo(U) sequence, while very little (if any) activity was detected for substrates with other homopolymeric or heteropolymeric sequences in the 5′ overhang. The oligo(U)-assisted/templated TATase activity on partial-duplex RNAs was confirmed for two other coronavirus nsp8 proteins, suggesting that the activity is conserved among coronaviruses. Replacement of a conserved Lys residue with Ala abolished the in vitro RNA-binding and TATase activities of nsp8 and caused a nonviable phenotype when the corresponding mutation was introduced into the HCoV-229E genome, confirming that these activities are mediated by nsp8 and critical for viral replication. In additional experiments, we obtained evidence that nsp8 has a pronounced specificity for adenylate and is unable to incorporate guanylate into RNA products, which strongly argues against the previously proposed template-dependent RNA polymerase activity of this protein. Given the presence of an oligo(U) stretch at the 5′ end of coronavirus minus-strand RNAs, it is tempting to speculate (but remains to be confirmed) that the nsp8-mediated TATase activity is involved in the 3′ polyadenylation of viral plus-strand RNAs. IMPORTANCE Previously, coronavirus nsp8 proteins were suggested to have template-dependent RNA polymerase activities resembling those of RNA primases or even canonical RNA-dependent RNA polymerases, while more recent studies have suggested an essential cofactor function of nsp8 (plus nsp7) for nsp12-mediated RNA-dependent RNA polymerase activity. In an effort to reconcile conflicting data from earlier studies, the study revisits coronavirus nsp8-associated activities using additional controls and proteins. The data obtained for three coronavirus nsp8 proteins provide evidence that the proteins share metal ion-dependent RNA 3′ polyadenylation activities that are greatly stimulated by a short oligo(U) stretch in the template strand. In contrast, nsp8 was found to be unable to select and incorporate appropriate (matching) nucleotides to produce cRNA products from heteropolymeric and other homooligomeric templates. While confirming the critical role of nsp8 in coronavirus replication, the study amends the list of activities mediated by coronavirus nsp8 proteins in the absence of other proteins.

Summary Cleavage and polyadenylation factor (CPF/CPSF) is a multi-protein complex essential for formation of eukaryotic mRNA 3ʹ ends. CPF cleaves pre-mRNAs at a specific site and adds a poly(A) tail. The cleavage reaction defines the 3ʹ end of the mature mRNA, and thus the activity of the endonuclease is highly regulated. Here, we show that reconstitution of specific pre-mRNA cleavage with recombinant yeast proteins requires incorporation of the Ysh1 endonuclease into an eight-subunit “CPFcore” complex. Cleavage also requires the accessory cleavage factors IA and IB, which bind substrate pre-mRNAs and CPF, likely facilitating assembly of an active complex. Using X-ray crystallography, electron microscopy, and mass spectrometry, we determine the structure of Ysh1 bound to Mpe1 and the arrangement of subunits within CPFcore. Together, our data suggest that the active mRNA 3ʹ end processing machinery is a dynamic assembly that is licensed to cleave only when all protein factors come together at the polyadenylation site., Graphical Abstract, Highlights • Crystallography and cryo-EM reveal a Ysh1-Mpe1 interface • Specific pre-mRNA 3ʹ end cleavage is reconstituted from recombinant proteins • Electron microscopy shows that CPFcore assembles around a central scaffold • A model for activation of the 3ʹ endonuclease on substrate RNAs is proposed, The 3ʹ ends of eukaryotic mRNAs are formed by endonucleolytic cleavage by Ysh1/CPSF73. Hill et al. define a minimal machinery for cleavage and polyadenylation in vitro using recombinant yeast proteins and short substrates. They elucidate the architecture of an ∼500 kDa eight-protein assembly, including the atomic details of a Ysh1-Mpe1 interface.

Summary: A hallmark of chronic hepatitis B (CHB) virus infection is the presence of high circulating levels of non-infectious small lipid HBV surface antigen (HBsAg) vesicles. Although rare, sustained HBsAg loss is the idealized endpoint of any CHB therapy. A small molecule, RG7834, has been previously reported to inhibit HBsAg expression by targeting terminal nucleotidyltransferase proteins 4A and 4B (TENT4A and TENT4B). In this study, we describe a genome-wide CRISPR screen to identify other potential host factors required for HBsAg expression and to gain further insights into the mechanism of RG7834. We report more than 60 genes involved in regulating HBsAg and identify additional factors involved in RG7834 activity, including a zinc finger CCHC-type containing 14 (ZCCHC14) protein. We show that ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing, providing a potential new therapeutic target to achieve functional cure in CHB patients. : Hyrina et al. employ a non-biased functional CRISPR screening approach to identify host factors regulating HBsAg expression as well as those targeted by RG7834, a HBsAg inhibitor. The screen highlighted over 60 genes and identified a mechanism by which ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing. Keywords: HBV, HBsAg, CRISPR, genome-wide screen, RG7834, ZCCHC14, TENT4B, RNA tailing

Drosophila Trf4-1 (DmTrf4-1) is a polyadenylation polymerase or terminal nucleotidyl transferase (PAP/TENT) that has been reported to add poly(A) tails to snRNAs in nucleus or mRNAs in cytoplasm. Here, we found that the loss of Trf4-1 resulted in the reduction of mRNAs and primary miRNAs (pri-miRNAs) in both Drosophila S2 cells and adult flies. Interestingly, the role of Trf4-1 in transcription is independent of its PAP/TENT activity. Moreover, using the chromatin immunoprecipitation assay, we uncovered that the loss of Trf4-1 led to abnormal RNA polymerase II accumulation and reduced H3K4me3 binding in promoter regions. Thus, our study indicates a positive role of Trf4-1 in the transcription of mRNAs and pri-miRNAs.

PARN, poly(A)-specific ribonuclease, regulates the turnover of mRNAs and the maturation and stabilization of the hTR RNA component of telomerase. Biallelic PARN mutations were associated with Høyeraal–Hreidarsson (HH) syndrome, a rare telomere biology disorder that, because of its severity, is likely not exclusively due to hTR down-regulation. Whether PARN deficiency was affecting the expression of telomere-related genes was still unclear. Using cells from two unrelated HH individuals carrying novel PARN mutations and a human PARN knock-out (KO) cell line with inducible PARN complementation, we found that PARN deficiency affects both telomere length and stability and down-regulates the expression of TRF1, TRF2, TPP1, RAP1, and POT1 shelterin transcripts. Down-regulation of dyskerin-encoding DKC1 mRNA was also observed and found to result from p53 activation in PARN-deficient cells. We further showed that PARN deficiency compromises ribosomal RNA biogenesis in patients' fibroblasts and cells from heterozygous Parn KO mice. Homozygous Parn KO however resulted in early embryonic lethality that was not overcome by p53 KO. Our results refine our knowledge on the pleiotropic cellular consequences of PARN deficiency. © 2019 The Authors. Published under the terms of the CC BY 4.0 license

Objective To study the expression of FAM46A in glioblastoma (GBM) and analyze its significance in predicting the prognosis of patients. Materials and methods mRNA expression and clinical data of patients with GBM were retrieved from ONCOMINE databases and The Cancer Genome Atlas (TCGA) database. Immunohistochemistry was performed in a tissue microarray including 110 GBM cases and 12 normal controls to determine the expression of FAM46A protein. Then, Kaplan-Meier curve and Cox regression model were used to investigate the relationship between FAM46A expression and clinical outcome. Coexpressed genes of FAM46A were analyzed by Linked Omics, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Results Upregulated expression of FAM46A was found in both TCGA and our cohort. High FAM46A expression was associated with the poor prognosis of patients with GBM and could be identified as an independent risk factor for overall survival (HR = 1.652, p = 0.022). Further bioinformatics analysis revealed that FAM46A might be involved in cell motility and endoplasmic reticulum proteostasis and stress to promote GBM progression. Conclusion Our findings suggest that increased expression of FAM46A in GBM is a novel biomarker for predicting poor outcome of patients and that targeting FAM46A may serve as a potential therapy for this disease.

Polyadenylation is thought to be involved in the degradation and quality control of bacterial RNAs but relatively few examples have been investigated. We used a combination of 5 '-tagRACE and RNA-seq to analyze the total RNA content from a wild-type strain and from a poly(A) polymerase deleted Mutant. A total of 178 transcripts were either up- or down-regulated in the mutant when compared to the wild-type strain. Poly(A) polymerase up-regulates the expression of all genes related to the FliA regulon and several previously unknown transcripts, including numerous transporters. Notable down-regulation of genes in the expression of antigen 43 and components of the type 1 fimbriae was detected. The major consequence of the absence of poly(A) polymerase was the accumulation of numerous sRNAs, antisense transcripts, REP sequences and RNA fragments resulting from the processing of entire transcripts. A new algorithm to analyze the position and composition of post-transcriptional modifications based on the sequence of unencoded 3 '-ends, was developed to identify polyadenylated molecules. Overall our results shed new light on the broad spectrum of action of polyadenylation on gene expression and demonstrate the importance of poly(A) dependent degradation to remove structured RNA fragments.

Eukaryotic mRNAs are subject to multiple types of tailing that critically influence mRNA stability and translatability. To investigate RNA tails at the genomic scale, we previously developed TAIL-seq, but its low sensitivity precluded its application to biological materials of minute quantity. In this study, we report a new version of TAIL-seq (mRNA TAIL-seq [mTAIL-seq]) with enhanced sequencing depth for mRNAs (by ∼1000-fold compared with the previous version). The improved method allows us to investigate the regulation of poly(A) tails in Drosophila oocytes and embryos. We found that maternal mRNAs are polyadenylated mainly during late oogenesis, prior to fertilization, and that further modulation occurs upon egg activation. Wispy, a noncanonical poly(A) polymerase, adenylates the vast majority of maternal mRNAs, with a few intriguing exceptions such as ribosomal protein transcripts. By comparing mTAIL-seq data with ribosome profiling data, we found a strong coupling between poly(A) tail length and translational efficiency during egg activation. Our data suggest that regulation of poly(A) tails in oocytes shapes the translatomic landscape of embryos, thereby directing the onset of animal development. By virtue of the high sensitivity, low cost, technical robustness, and broad accessibility, mTAIL-seq will be a potent tool to improve our understanding of mRNA tailing in diverse biological systems.

Across kingdoms, RNA interference (RNAi) has been shown to control gene expression at the transcriptional- or the post-transcriptional level. Here, we describe a mechanism which involves both aspects: truncated transgenes, which fail to produce intact mRNA, induce siRNA accumulation and silencing of homologous loci in trans in the ciliate Paramecium. We show that silencing is achieved by co-transcriptional silencing, associated with repressive histone marks at the endogenous gene. This is accompanied by secondary siRNA accumulation, strictly limited to the open reading frame of the remote locus. Our data shows that in this mechanism, heterochromatic marks depend on a variety of RNAi components. These include RDR3 and PTIWI14 as well as a second set of components, which are also involved in post-transcriptional silencing: RDR2, PTIWI13, DCR1 and CID2. Our data indicates differential processing of nascent un-spliced and long, spliced transcripts thus suggesting a hitherto-unrecognized functional interaction between post-transcriptional and co-transcriptional RNAi. Both sets of RNAi components are required for efficient trans-acting RNAi at the chromatin level and our data indicates similar mechanisms contributing to genome wide regulation of gene expression by epigenetic mechanisms.

Emerging evidence has demonstrated that regulating the length of the poly(A) tail on an mRNA is an efficient means of controlling gene expression at the post-transcriptional level. In early development, transcription is silenced and gene expression is primarily regulated by cytoplasmic polyadenylation. In somatic cells, considerable progress has been made toward understanding the mechanisms of negative regulation by deadenylation. However, positive regulation through elongation of the poly(A) tail has not been widely studied due to the difficulty in distinguishing whether any observed increase in length is due to the synthesis of new mRNA, reduced deadenylation or cytoplasmic polyadenylation. Here, we overcame this barrier by developing a method for transcriptional pulse-chase analysis under conditions where deadenylases are suppressed. This strategy was used to show that a member of the Star family of RNA binding proteins, QKI, promotes polyadenylation when tethered to a reporter mRNA. Although multiple RNA binding proteins have been implicated in cytoplasmic polyadenylation during early development, previously only CPEB was known to function in this capacity in somatic cells. Importantly, we show that only the cytoplasmic isoform QKI-7 promotes poly(A) tail extension, and that it does so by recruiting the non-canonical poly(A) polymerase PAPD4 through its unique carboxyl-terminal region. We further show that QKI-7 specifically promotes polyadenylation and translation of three natural target mRNAs (hnRNPA1, p27(kip1)and β-catenin) in a manner that is dependent on the QKI response element. An anti-mitogenic signal that induces cell cycle arrest at G1 phase elicits polyadenylation and translation of p27(kip1)mRNA via QKI and PAPD4. Taken together, our findings provide significant new insight into a general mechanism for positive regulation of gene expression by post-transcriptional polyadenylation in somatic cells.

Post-transcriptional 3′ end addition of nucleotides is important in a variety of RNA decay pathways. We have examined the 3′ end addition of nucleotides during the decay of the Hsp70 mRNA and a corresponding reporter RNA in Drosophila S2 cells by conventional sequencing of cDNAs obtained after mRNA circularization and by deep sequencing of dedicated libraries enriched for 3′ decay intermediates along the length of the mRNA. Approximately 5%–10% of 3′ decay intermediates carried nonencoded oligo(A) tails with a mean length of 2–3 nucleotides. RNAi experiments showed that the oligoadenylated RNA fragments were intermediates of exosomal decay and the noncanonical poly(A) polymerase Trf4-1 was mainly responsible for A addition. A hot spot of A addition corresponded to an intermediate of 3′ decay that accumulated upon inhibition of decapping, and knockdown of Trf4-1 increased the abundance of this intermediate, suggesting that oligoadenylation facilitates 3′ decay. Oligoadenylated 3′ decay intermediates were found in the cytoplasmic fraction in association with ribosomes, and fluorescence microscopy revealed a cytoplasmic localization of Trf4-1. Thus, oligoadenylation enhances exosomal mRNA degradation in the cytoplasm.

The testis-specific cytoplasmic poly(A) polymerase PAPOLB/TPAP is essential for spermatogenesis. Although this enzyme is responsible for poly(A) tail extension of a subset of mRNAs in round spermatids, the stability and translational efficiency of these mRNAs are unaffected by the absence of PAPOLB. To clarify the functional importance of this enzyme’s adenylation activity, we produced PAPOLB-null mice expressing a polyadenylation-defective PAPOLB mutant (PAPOLBD114A), in which the catalytic Asp at residue 114 was mutated to Ala. Introducing PAPOLBD114A failed to rescue PAPOLB-null phenotypes, such as reduced expression of haploid-specific mRNAs, spermiogenesis arrest, and male infertility. These results suggest that PAPOLB regulates spermatogenesis through its adenylation activity.

Engineering a microbial strain for production sometimes entails metabolic modifications that impair essential physiological processes for growth or production. Restoring these functions may require amending a variety of non-obvious physiological networks, and thus, rational design strategies may not be practical. Here we demonstrate that growth and production may be restored by evolution that repairs impaired metabolic function. Furthermore, we use genomics, metabolomics and proteomics to identify several underlying mutations and metabolic perturbations that allow metabolism to repair. Previously, high titers of butanol production were achieved by Escherichia coli using a growth-coupled, modified Clostridial CoA-dependent pathway after all native fermentative pathways were deleted. However, production was only observed in rich media. Native metabolic function of the host was unable to support growth and production in minimal media. We use directed cell evolution to repair this phenotype and observed improved growth, titers and butanol yields. We found a mutation in pcnB which resulted in decreased plasmid copy numbers and pathway enzymes to balance resource utilization. Increased protein abundance was measured for biosynthetic pathways, glycolytic enzymes have increased activity, and adenosyl energy charge was increased. We also found mutations in the ArcAB two-component system and integration host factor (IHF) that tune redox metabolism to alter byproduct formation. These results demonstrate that directed strain evolution can enable systematic adaptations to repair metabolic function and enhance microbial production. Furthermore, these results demonstrate the versatile repair capabilities of cell metabolism and highlight important aspects of cell physiology that are required for production in minimal media.

A major event in germline development is the transition from stem/progenitor cells to entry into meiosis and gametogenesis. This transition requires downregulation of mitotic cell cycle activity and upregulation of processes associated with meiosis. We identify the Caenorhabditis elegans SCF(PROM-1) E3 ubiquitin-ligase complex as functioning to downregulate mitotic cell cycle protein levels including cyclin E, WAPL-1, and KNL-2 at meiotic entry and, independently, promoting homologous chromosome pairing as a positive regulator of the CHK-2 kinase. SCF(PROM-1) is thus a novel regulator of meiotic entry, coordinating downregulation of mitotic cell cycle proteins and promoting homolog pairing. We further show that SCF(PROM-1) functions redundantly, in parallel to the previously described GLD-1 and GLD-2 meiotic entry pathways, downstream of and inhibited by GLP-1 Notch signaling, which specifies the stem cell fate. Accordingly, C. elegans employs three post-transcriptional pathways, SCF(PROM-1)-mediated protein degradation, GLD-1-mediated translational repression, and GLD-2-mediated translational activation, to control and coordinate the initiation of meiotic development.

Cytoplasmic polyadenylation is a widespread mechanism to regulate mRNA translation. In vertebrates, this process requires two sequence elements in target 3' UTRs: the U-rich cytoplasmic polyadenylation element and the AAUAAA hexanucleotide. In Drosophila melanogaster, cytoplasmic polyadenylation of Toll mRNA occurs independently of these canonical elements and requires a machinery that remains to be characterized. Here we identify Dicer-2 as a component of this machinery. Dicer-2, a factor previously involved in RNA interference (RNAi), interacts with the cytoplasmic poly(A) polymerase Wispy. Depletion of Dicer-2 from polyadenylation-competent embryo extracts and analysis of wispy mutants indicate that both factors are necessary for polyadenylation and translation of Toll mRNA. We further identify r2d2 mRNA, encoding a Dicer-2 partner in RNAi, as a Dicer-2 polyadenylation target. Our results uncover a novel function of Dicer-2 in activation of mRNA translation through cytoplasmic polyadenylation. This work was supported by MINECO and the European Regional Development Fund (ERDF) under BFU2012-37135, BFU2015-68741, and Consolider CSD2009-00080 grants to F.G., and by the CNRS UMR9002, ANR (ANR-2010-BLAN-1201-01 and ANR-15-CE12-0019-01), and FRM (Equipe FRM 2013 DEQ20130326534) to M.S. We acknowledge support of MINECO “Centro de Excelencia Severo Ochoa 2013-2017,” SEV-2012-0208.