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The estrogen signalling pathway is highly dynamic and primarily mediated by estrogen receptors (ERs) that transcriptionally regulate the expression of target genes. While transcriptional functions of ERs have been widely studied, their roles in RNA biology have not been extensively explored. Here, we reveal a novel biological role of ER alpha (ERα) in mRNA 3' end processing in breast cancer cells, providing an alternative mechanism in regulating gene expression at the post-transcriptional level. We show that ERα activates poly(A) specific ribonuclease (PARN) deadenylase using in vitro assays, and that this activation is further increased by tumour suppressor p53, a factor involved in mRNA processing. Consistent with this, we confirm ERα-mediated activation of nuclear deadenylation by PARN in samples from MCF7 and T47D breast cancer cells that vary in expression of ERα and p53. We further show that ERα can form complex(es) with PARN and p53. Lastly, we identify and validate expression of common mRNA targets of ERα and PARN known to be involved in cell invasion, metastasis and angiogenesis, supporting the functional overlap of these factors in regulating gene expression in a transactivation-independent manner. Together, these results show a new regulatory mechanism by which ERα regulates mRNA processing and gene expression post-transcriptionally, highlighting its contribution to unique transcriptomic profiles and breast cancer progression.

SARS-CoV-2 non-structural protein 10 (nsp10) is essential for the stimulation of enzymatic activities of nsp14 and nsp16, acting as both an activator and scaffolding protein. Nsp14 is a bifunctional enzyme with the N-terminus containing a 3'-5' exoribonuclease (ExoN) domain that allows the excision of nucleotide mismatches at the virus RNA 3'-end, and a C-terminal N7-methyltransferase (N7-MTase) domain. Nsp10 is required for stimulating both ExoN proofreading and the nsp16 2'-O-methyltransferase activities. This makes nsp10 a central player in both viral resistance to nucleoside-based drugs and the RNA cap methylation machinery that helps the virus evade innate immunity. We characterised the interactions between full-length nsp10 (139 residues), N- and C-termini truncated nsp10 (residues 10-133), and nsp10 with a C-terminal truncation (residues 1-133) with nsp14 using microscale thermophoresis, multi-detection SEC, and hydrogen-deuterium (H/D) exchange mass spectrometry. We describe the functional role of the C-terminal region of nsp10 for binding to nsp14 and show that full N- and C-termini of nsp10 are important for optimal binding. In addition, our H/D exchange experiments suggest an intermediary interaction of nsp10 with the N7-MTase domain of nsp14. In summary, our results suggest intermediary steps in the process of association or dissociation of the nsp10-nsp14 complex, involving contacts between the two proteins in regions not identifiable by X-ray crystallography alone.

Telomeres are terminal structures that define the ends of linear chromosomes. They harbour specialized ribonucleoprotein complexes which play a major role in genome integrity by preventing unscheduled DNA damage repair events. Genes located adjacent to telomere repeat sequences are repressed by a phenomenon called telomere position effect (TPE) via epigenetic silencing. RNA surveillance pathways post-transcriptionally regulate any leaky transcripts arising from the telomeres. Recently, multiple non-coding RNA species originate from telomere ends, namely, TERRA (telomeric repeat-containing RNA), ARRET, sub-telomeric XUTs and sub-telomeric CUTs have been identified. In this study, we report a role for the transcription termination complex (Rtt103-Rai1-Rat1) in regulating the abundance of the sub-telomeric transcripts in a transcription-dependent manner. We show that the Rtt103 mutants have elevated levels of TERRA and other sub-telomeric transcripts that are usually silenced. Our study suggests that Rtt103 potentially recruits the exonuclease, Rat1 in a RNA polymerase II dependent manner to degrade these transcripts and regulate their levels in the cell.

Immune checkpoints play various important roles in tumour immunity, which usually contribute to T cells' exhaustion, leading to immunosuppression in the tumour microenvironment. However, the roles of immune checkpoints in infectious diseases, especially fungal infection, remain elusive. Here, we reanalyzed a recent published single-cell RNA-sequencing (scRNA-seq) data of peripheral blood mononuclear cells (PBMCs) stimulated with Candida albicans ( C. albicans ), to explore the expression patterns of immune checkpoints after C. albicans bloodstream infection. We characterized the heterogeneous pathway activities among different immune cell subpopulations after C. albicans infection. The CTLA-4 pathway was up-regulated in stimulated CD4 + and CD8 + T cells, while the PD-1 pathway showed high activity in stimulated plasmacytoid dendritic cell (pDC) and monocytes. Importantly, we found that immunosuppressive checkpoints HAVCR2 and LAG3 were only expressed in stimulated NK and CD8 + T cells, respectively. Their viabilities were validated by flow cytometry. We also identified three overexpressed genes ( ISG20 , LY6E , ISG15 ) across all stimulated cells. Also, two monocyte-specific overexpressed genes ( SNX10 , IDO1 ) were screened out in this study. Together, these results supplemented the landscape of immune checkpoints in fungal infection, which may serve as potential therapeutic targets for C. albicans infection. Moreover, the genes with the most relevant for C. albicans infection were identified in this study.

Cholangiocarcinoma (CCA) is one of the most common histological types of primary hepatic malignancy and is associated with poor overall prognosis, causing a ponderous burden on human life. Hence, it is necessary to elucidate the pathogenesis of CCA. The objective of our research was to shed light on the mechanism through which long non-coding RNA titin-antisense RNA1 (lncRNA TTN-AS1) is involved in the development of CCA. Reverse transcription quantitative polymerase chain reaction was used to detect TTN-AS1 expression in CCA samples and cells. Functional experiments were performed using the Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine, transwell, and in vivo tumor growth assays. The relationship between TTN-AS1, miR-513a-5p, and stratifin (SFN) was explored using a dual luciferase reporter assay, RNA immunoprecipitation (RIP) experiment, and Pearson correlation analysis. The result showed that TTN-AS1 and SFN are highly expressed in CCA tissues. Bioinformatics analysis, luciferase reporter and RIP experiments revealed the correlation between TTN-AS1, miR-513a-5p, and SFN. In addition, silencing TTN-AS1 mitigated CCA cell proliferation and migration. Mechanistically, miR-513a-5p is sponged by TTN-AS1. The miR-513a-5p inhibitor abolished the effect of TTN-AS1 silencing on the aggressive behaviors of CCA cells. Furthermore, we showed that miR-513a-5p is a regulator of CCA by targeting SFN. TTN-AS1 induced CCA cell growth and metastasis via the miR-513a-5p/SFN pathway, which offers a new strategy for therapeutic interventions for CCA.

After infection by flaviviruses like Zika and West Nile virus, eukaryotic hosts employ the well-conserved endoribonuclease Xrn1 to degrade the viral genomic RNA. Within the 3' untranslated regions, this enzyme encounters intricate Xrn1-resistant structures. This results in the accumulation of subgenomic flaviviral RNAs, an event that improves viral growth and aggravates viral pathogenicity. Xrn1-resistant RNAs have been established throughout the flaviviral genus, but not yet throughout the entire Flaviviridae family. In this work, we use previously determined characteristics of these structures to identify homologous sequences in many members of the genera pegivirus, hepacivirus and pestivirus. We used structural alignment and mutational analyses to establish that these sequences indeed represent Xrn1-resistant RNA and that they employ the general features of the flaviviral xrRNAs, consisting of a double pseudoknot formed by five base-paired regions stitched together by a crucial triple base interaction. Furthermore, we demonstrate that the pestivirus Bungowannah virus produces subgenomic RNA in vivo . Altogether, these results indicate that viruses make use of a universal Xrn1-resistant RNA throughout the Flaviviridae family.

A new paradigm has emerged proposing that the crosstalk between nuclear transcription and cytoplasmic mRNA stability keeps robust mRNA levels in cells under steady-state conditions. A key piece in this crosstalk is the highly conserved 5'-3' RNA exonuclease Xrn1, which degrades most cytoplasmic mRNAs but also associates with nuclear chromatin to activate transcription by not well-understood mechanisms. Here, we investigated the role of Xrn1 in the transcriptional response of Saccharomyces cerevisiae cells to osmotic stress. We show that a lack of Xrn1 results in much lower transcriptional induction of the upregulated genes but in similar high levels of their transcripts because of parallel mRNA stabilization. Unexpectedly, lower transcription in xrn1 occurs with a higher accumulation of RNA polymerase II (RNAPII) at stress-inducible genes, suggesting that this polymerase remains inactive backtracked. Xrn1 seems to be directly implicated in the formation of a competent elongation complex because Xrn1 is recruited to the osmotic stress-upregulated genes in parallel with the RNAPII complex, and both are dependent on the mitogen-activated protein kinase Hog1. Our findings extend the role of Xrn1 in preventing the accumulation of inactive RNAPII at highly induced genes to other situations of rapid and strong transcriptional upregulation.

mRNA homoeostasis is favoured by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae . We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5' exhibited significant alterations that were compatible with decreased elongation rates in the absence of Xrn1. Nucleosome mapping detected altered chromatin configuration in the gene bodies. We also detected accumulation of RNA pol II shortly upstream of polyadenylation sites by CRAC, although not by BioGRO-seq, suggesting higher frequency of backtracking before pre-mRNA cleavage. This phenomenon was particularly linked to genes with poorly positioned nucleosomes at this position. Accumulation of RNA pol II at 3' was also detected in other mRNA decay mutants. According to these and other pieces of evidence, Xrn1 seems to influence transcription elongation at least in two ways: by directly favouring elongation rates and by a more general mechanism that connects mRNA decay to late elongation.

The recent pandemic caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) calls the whole world into a medical emergency. For tackling Coronavirus Disease 2019 (COVID-19), researchers from around the world are swiftly working on designing and identifying inhibitors against all possible viral key protein targets. One of the attractive drug targets is guanine-N7 methyltransferase which plays the main role in capping the 5'-ends of viral genomic RNA and sub genomic RNAs, to escape the host's innate immunity. We performed homology modeling and molecular dynamic (MD) simulation, in order to understand the molecular architecture of Guanosine-P3-Adenosine-5',5'-Triphosphate (G3A) binding with C-terminal N7-MTase domain of nsp14 from SARS-CoV-2. The residue Asn388 is highly conserved in present both in N7-MTase from SARS-CoV and SARS-CoV-2 and displays a unique function in G3A binding. For an in-depth understanding of these substrate specificities, we tried to screen and identify inhibitors from the Traditional Chinese Medicine (TCM) database. The combination of several computational approaches, including screening, MM/GBSA, MD simulations, and PCA calculations, provides the screened compounds that readily interact with the G3A binding site of homology modeled N7-MTase domain. Compounds from this screening will have strong potency towards inhibiting the substrate-binding and efficiently hinder the viral 5'-end RNA capping mechanism. We strongly believe the final compounds can become COVID-19 therapeutics, with huge international support.[Formula: see text]The focus of this study is to screen for antiviral inhibitors blocking guanine-N7 methyltransferase (N7-MTase), one of the key drug targets involved in the first methylation step of the SARS-CoV-2 RNA capping mechanism. Compounds binding the substrate-binding site can interfere with enzyme catalysis and impede 5'-end cap formation, which is crucial to mimic host RNA and evade host cellular immune responses. Therefore, our study proposes the top hit compounds from the Traditional Chinese Medicine (TCM) database using a combination of several computational approaches.Communicated by Ramaswamy H. Sarma.

Subgenomic RNAs are produced by several RNA viruses through incomplete degradation of their genomic RNA by the exoribonuclease Xrn1, and have been shown to be essential for viral growth and pathogenicity. Within the flavivirus genus of the Flaviviridae family, two distinct classes of Xrn1-resistant RNA motifs have been proposed; one for mosquito-borne and insect-specific flaviviruses, and one for tick-borne flaviviruses and no-known-vector flaviviruses. We investigated tick-borne and no-known-vector flavivirus Xrn1-resistant RNA motifs through systematic in vitro mutational analysis and showed that both classes actually possess very similar structural configurations, including a double pseudoknot and a base-triple at identical, conserved locations. For the no-known-vector flavivirus Modoc virus, we show that in vivo generation of subgenomic flaviviral RNA was affected by mutations targeted at nucleotides involved in the structural features of flaviviral Xrn1-resistant RNA motifs that were defined in this work. Our results suggest that throughout the genus flavivirus Xrn1-resistant RNA motifs adopt the same topologically conserved structure.

The Coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2 virus, is now causing a tremendous global health concern. Since its first appearance in December 2019, the outbreak has already caused over 5.8 million infections worldwide (till 29 May 2020), with more than 0.35 million deaths. Early virus-mediated immune suppression is believed to be one of the unique characteristics of SARS-CoV-2 infection and contributes at least partially to the viral pathogenesis. In this study, we identified the key viral interferon antagonists of SARS-CoV-2 and compared them with two well-characterized SARS-CoV interferon antagonists, PLpro and orf6. Here we demonstrated that the SARS-CoV-2 nsp13, nsp14, nsp15 and orf6, but not the unique orf8, could potently suppress primary interferon production and interferon signalling. Although SARS-CoV PLpro has been well-characterized for its potent interferon-antagonizing, deubiquitinase and protease activities, SARS-CoV-2 PLpro, despite sharing high amino acid sequence similarity with SARS-CoV, loses both interferon-antagonising and deubiquitinase activities. Among the 27 viral proteins, SARS-CoV-2 orf6 demonstrated the strongest suppression on both primary interferon production and interferon signalling. Orf6-deleted SARS-CoV-2 may be considered for the development of intranasal live-but-attenuated vaccine against COVID-19.

Shortening of mRNA poly(A) tails (deadenylation) to trigger their decay is mediated mainly by the CCR4-NOT deadenylase complex. While four catalytic subunits (CNOT6, 6L 7, and 8) have been identified in the mammalian CCR4-NOT complex, their individual biological roles are not fully understood. In this study, we addressed the contribution of CNOT7/8 to viability of primary mouse embryonic fibroblasts (MEFs). We found that MEFs lacking CNOT7/8 expression [ Cnot7/8 -double knockout (dKO) MEFs] undergo cell death, whereas MEFs lacking CNOT6/6L expression ( Cnot6/6l -dKO MEFs) remain viable. Co-immunoprecipitation analyses showed that CNOT6/6L are also absent from the CCR4-NOT complex in Cnot7/8 -dKO MEFs. In contrast, either CNOT7 or CNOT8 still interacts with other subunits in the CCR4-NOT complex in Cnot6/6l -dKO MEFs. Exogenous expression of a CNOT7 mutant lacking catalytic activity in Cnot7/8 -dKO MEFs cannot recover cell viability, even though CNOT6/6L exists to some extent in the CCR4-NOT complex, confirming that CNOT7/8 is essential for viability. Bulk poly(A) tail analysis revealed that mRNAs with longer poly(A) tails are more numerous in Cnot7/8 -dKO MEFs than in Cnot6/6l -dKO MEFs. Consistent with elongated poly(A) tails, more mRNAs are upregulated and stabilized in Cnot7/8 -dKO MEFs than in Cnot6/6l -dKO MEFs. Importantly, Cnot6/6l -dKO mice are viable and grow normally to adulthood. Taken together, the CNOT7/8 catalytic subunits are essential for deadenylation, which is necessary to maintain cell viability, whereas CNOT6/6L are not.

Xrn1 is a major 5'-3' exoribonuclease involved in the RNA metabolism of many eukaryotic species. RNA viruses have evolved ways to thwart Xrn1 in order to produce subgenomic non-coding RNA that affects the hosts RNA metabolism. The 3' untranslated region of several beny- and cucumovirus RNAs harbors a so-called 'coremin' motif that is required for Xrn1 stalling. The structural features of this motif have not been studied in detail yet. Here, by using in vitro Xrn1 degradation assays, we tested over 50 different RNA constructs based on the Beet necrotic yellow vein virus sequence to deduce putative structural features responsible for Xrn1 stalling. We demonstrated that the minimal benyvirus stalling site consists of two hairpins of 3 and 4 base pairs respectively. The 5' proximal hairpin requires a YGAD (Y = U/C, D = G/A/U) consensus loop sequence, whereas the 3' proximal hairpin loop sequence is variable. The sequence of the 10-nucleotide spacer that separates the hairpins is highly conserved and potentially involved in tertiary interactions. Similar coremin motifs were identified in plant virus isolates from other families including Betaflexiviridae, Virgaviridae, Potyviridae and Secoviridae (order of the Picornavirales). We conclude that Xrn1-stalling motifs are more widespread among RNA viruses than previously realized.

DIS3L2, in which mutations have been linked to Perlman syndrome, is an RNA-binding protein with 3'-5' exoribonuclease activity. It contains two CSD domains and one S1 domain, all of which are RNA-binding domains, and one RNB domain that is responsible for the exoribonuclease activity. The 3' polyuridine of RNA substrates can serve as a degradation signal for DIS3L2. Because DIS3L2 is predominantly localized in the cytoplasm, it can recognize, bind, and mediate the degradation of cytoplasmic uridylated RNA, including pre-microRNA, mature microRNA, mRNA, and some other non-coding RNAs. Therefore, DIS3L2 plays an important role in cytoplasmic RNA surveillance and decay. DIS3L2 is involved in multiple biological and physiological processes such as cell division, proliferation, differentiation, and apoptosis. Nonetheless, the function of DIS3L2, especially its association with cancer, remains largely unknown. We summarize here the RNA substrates degraded by DIS3L2 with its exonucleolytic activity, together with the corresponding biological functions it is implicated in. Furthermore, we discuss whether DIS3L2 can function independently of its 3'-5' exoribonuclease activity, as well as its potential tumor-suppressive or oncogenic roles during cancer progression.

Ribosomes are macromolecular machines that carry out protein synthesis. After each round of translation, ribosome recycling is essential for reinitiating protein synthesis. Ribosome recycling factor (RRF), together with elongation factor G (EF-G), catalyse the transient split of the 70S ribosome into subunits. This splitting is then stabilized by initiation factor 3 (IF3), which functions as an anti-association factor. The correct amount of these factors ensures the precise level of 70S ribosomes in the cell. RNase R is a highly conserved exoribonuclease involved in the 3' to 5' degradation of RNAs. In this work we show that pneumococcal RNase R directly controls the expression levels of frr, fusA and infC mRNAs, the corresponding transcripts of RRF, EF-G and IF3, respectively. We present evidences showing that accumulation of these factors leads to a decreased amount of 70S active particles, as demonstrated by the altered sucrose gradient ribosomal pattern in the RNase R mutant strain. Furthermore, the single deletion of RNase R is shown to have a global impact on protein synthesis and cell viability, leading to a ~50% reduction in bacterial CFU/ml. We believe that the fine-tuned regulation of these transcripts by RNase R is essential for maintaining the precise amount of active ribosomal complexes required for proper mRNA translation and thus we propose RNase R as a new auxiliary factor in ribosome reassociation. Considering the overall impact of RNase R on protein synthesis, one of the main targets of antibiotics, this enzyme may be a promising target for antimicrobial treatment.

Nucleolin (NCL) is an abundant stress-responsive, RNA-binding phosphoprotein that controls gene expression by regulating either mRNA stability and/or translation. NCL binds to the AU-rich element (ARE) in the 3'UTR of target mRNAs, mediates miRNA functions in the nearby target sequences, and regulates mRNA deadenylation. However, the mechanism by which NCL phosphorylation affects these functions and the identity of the deadenylase involved, remain largely unexplored. Earlier we demonstrated that NCL phosphorylation is vital for cell cycle progression and proliferation, whereas phosphorylation-deficient NCL at six consensus CK2 sites confers dominant-negative effect on proliferation by increasing p53 expression, possibly mimicking cellular DNA damage conditions. In this study, we show that NCL phosphorylation at those CK2 consensus sites in the N-terminus is necessary to induce deadenylation upon oncogenic stimuli and UV stress. NCL-WT, but not hypophosphorylated NCL-6/S*A, activates poly (A)-specific ribonuclease (PARN) deadenylase activity. We further demonstrate that NCL interacts directly with PARN, and under non-stress conditions also forms (a) complex (es) with factors that regulate deadenylation, such as p53 and the ARE-binding protein HuR. Upon UV stress, the interaction of hypophosphorylated NCL-6/S*A with these proteins is favored. As an RNA-binding protein, NCL interacts with PARN deadenylase substrates such as TP53 and BCL2 mRNAs, playing a role in their downregulation under non-stress conditions. For the first time, we show that NCL phosphorylation offers specificity to its protein-protein, protein-RNA interactions, resulting in the PARN deadenylase regulation, and hence gene expression, during cellular stress responses.

Plants have developed an internal timing mechanism, the circadian system, that serves to synchronise physiological and metabolic functions with daily cues such as dawn and dusk, and provides plants with an advantage in adapting to changing and challenging conditions. We have recently shown that the SAL1-PAP-XRN retrograde signalling pathway, which is proposed to regulate plant responses under stress conditions, also acts within the circadian system. Here we provide further evidence of circadian regulation by SAL1-PAP-XRN signalling, thereby affirming a link between molecular timekeeping and abiotic stress response mechanisms.

Macroautophagy/autophagy is a conserved catabolic process that promotes survival during stress. Autophagic dysfunction is associated with pathologies such as cancer and neurodegenerative diseases. Thus, autophagy must be strictly modulated at multiple levels (transcriptional, post-transcriptional, translational and post-translational) to prevent deregulation. Relatively little is known about the post-transcriptional control of autophagy. Here we report that the exoribonuclease Xrn1/XRN1 functions as a negative autophagy factor in the yeast Saccharomyces cerevisiae and in mammalian cells. In yeast, chromosomal deletion of XRN1 enhances autophagy and the frequency of autophagosome formation. Loss of Xrn1 results in the upregulation of autophagy-related (ATG) transcripts under nutrient-replete conditions, and this effect is dependent on the ribonuclease activity of Xrn1. Xrn1 expression is regulated by the yeast transcription factor Ash1 in rich conditions. In mammalian cells, siRNA depletion of XRN1 enhances autophagy and the replication of 2 picornaviruses. This work provides insight into the role of the RNA decay factor Xrn1/XRN1 as a post-transcriptional regulator of autophagy.

Every transcription cycle ends in termination when RNA polymerase dissociates from the DNA. Although conceptually simple, the mechanism has proven somewhat elusive in eukaryotic systems. Gene-editing and high resolution polymerase mapping now offer clarification of important steps preceding transcriptional termination by RNA polymerase II in human cells.

Ribosome biogenesis drives cell growth and proliferation, but mechanisms that modulate this process remain poorly understood. For a long time, small rRNA sequences have been widely treated as non-specific degradation products and neglected as garbage sequences. Recently, we identified a new class of antisense ribosomal siRNAs (risiRNAs) that downregulate pre-rRNA through the nuclear RNAi pathway in C. elegans. risiRNAs exhibit sequence characteristics similar to 22G RNA while complement to 18S and 26S rRNA. risiRNAs elicit the translocation of the nuclear Argonaute protein NRDE-3 from the cytoplasm to nucleus and nucleolus, in which the risiRNA/NRDE complex binds to pre-rRNA and silences rRNA expression. Interestingly, when C. elegans is exposed to environmental stimuli, such as cold shock and ultraviolet illumination, risiRNAs accumulate and further turn on the nuclear RNAi-mediated gene silencing pathway. risiRNA may act in a quality control mechanism of rRNA homeostasis. When the exoribonuclease SUSI-1(ceDis3L2) is mutated, risiRNAs are dramatically increased. In this Point of View article, we will summarize our understanding of the small antisense ribosomal siRNAs in a variety of organisms, especially C. elegans, and their possible roles in the quality control mechanism of rRNA homeostasis.

Deadenylases belong to an expanding family of exoribonucleases involved mainly in mRNA stability and turnover, with the exception of PARN which has additional roles in the biogenesis of several important non-coding RNAs, including miRNAs and piRNAs. Recently, PARN in C. elegans and its homolog PNLDC1 in B. mori were reported as the elusive trimmers mediating piRNA biogenesis. In addition, characterization of mammalian PNLDC1 in comparison to PARN, showed that is specifically expressed in embryonic stem and germ cells, as well as during early embryo development. Moreover, its expression is correlated with epigenetic events mediated by the de novo DNMT3b methyltransferase and knockdown in stem cells upregulates important genes that regulate multipotency. The recent data suggest that at least some new deadenylases may have expanded roles in cell metabolism as regulators of gene expression, through mRNA deadenylation, ncRNAs biogenesis and ncRNA-mediated mRNA targeting, linking essential mechanisms that regulate epigenetic control and transition events during differentiation. The possible roles of mammalian PNLDC1 along those dynamic networks are discussed in the light of new extremely important findings.

Loss-of-function mutations in 3'-to-5' exoribonucleases have been implicated in hereditary human diseases. For example, PARN mutations cause a severe form of dyskeratosis congenita (DC), wherein PARN deficiency leads to human telomerase RNA instability. Since the DC phenotype in PARN patients is even more severe than that of loss-of-function alleles in telomerase components, we hypothesized that PARN would also be required for the stability of other RNAs. Here, we show that PARN depletion reduces the levels of abundant human Y RNAs, which might contribute to the severe phenotype of DC observed in patients. Depletion of PAPD5 or the cytoplasmic exonuclease DIS3L rescues the effect of PARN depletion on Y RNA levels, suggesting that PARN stabilizes Y RNAs by removing oligoadenylated tails added by PAPD5, which would otherwise recruit DIS3L for Y RNA degradation. Through deep sequencing of 3' ends, we provide evidence that PARN can also deadenylate the U6 and RMRP RNAs without affecting their levels. Moreover, we observed widespread posttranscriptional oligoadenylation, uridylation, and guanylation of U6 and Y RNA 3' ends, suggesting that in mammalian cells, the formation of a 3' end for noncoding RNAs can be a complex process governed by the activities of various 3'-end polymerases and exonucleases., (Copyright © 2017 American Society for Microbiology.)

Transcription by RNA polymerase (RNAP) II is regulated at multiple steps by phosphorylation, catalyzed mainly by members of the cyclin-dependent kinase (CDK) family. The CDKs involved in transcription have overlapping substrate specificities, but play largely non-redundant roles in coordinating gene expression. Novel functions and targets of CDKs have recently emerged at the end of the transcription cycle, when the primary transcript is cleaved, and in most cases polyadenylated, and transcription is terminated by the action of the "torpedo" exonuclease Xrn2, which is a CDK substrate. Collectively, various functions have been ascribed to CDKs or CDK-mediated phosphorylation: recruiting cleavage and polyadenylation factors, preventing premature termination within gene bodies while promoting efficient termination of full-length transcripts, and preventing extensive readthrough transcription into intergenic regions or neighboring genes. The assignment of precise functions to specific CDKs is still in progress, but recent advances suggest ways in which the CDK network and RNAP II machinery might cooperate to ensure timely exit from the transcription cycle.

Eukaryotic protein synthesis is a multifaceted process that requires coordination of a set of translation factors in a particular cellular state. During normal growth and proliferation, cells generally make their proteome via conventional translation that utilizes canonical translation factors. When faced with environmental stress such as growth factor deprivation, or in response to biological cues such as developmental signals, cells can reduce canonical translation. In this situation, cells adapt alternative modes of translation to make specific proteins necessary for required biological functions under these distinct conditions. To date, a number of alternative translation mechanisms have been reported, which include non-canonical, cap dependent translation and cap independent translation such as IRES mediated translation. Here, we discuss one of the alternative modes of translation mediated by a specialized microRNA complex, FXR1a-microRNP that promotes non-canonical, cap dependent translation in quiescent conditions, where canonical translation is reduced due to low mTOR activity.

In a complex organism, cell proliferation and apoptosis need to be precisely controlled in order for tissues to develop correctly. Excessive cell proliferation can lead to diseases such as cancer. We have shown that the exoribonuclease Dis3L2 is required for the correct regulation of proliferation in a natural tissue within the model organism Drosophila melanogaster. Dis3L2 is a member of a highly conserved family of exoribonucleases that degrade RNA in a 3'-5' direction. We show that knockdown of dis3L2 in the Drosophila wing imaginal discs results in substantial wing overgrowth due to increased cellular proliferation rather than an increase in cell size. Imaginal discs are specified in the embryo before proliferating and differentiating to form the adult structures of the fly. Using RNA-seq we identified a small set of mRNAs that are sensitive to Dis3L2 activity. Of the mRNAs which increase in levels and are therefore potential targets of Dis3L2, we identified 2 that change at the post-transcriptional level but not at the transcriptional level, namely CG2678 (a transcription factor) and pyrexia (a TRP cation channel). We also demonstrate a compensatory effect between Dis3L2 and the 5'-3' exoribonuclease Pacman demonstrating that these 2 exoribonucleases function to regulate opposing pathways within the developing tissue. This work provides the first description of the molecular and developmental consequences of Dis3L2 inactivation in a non-human animal model. The work is directly relevant to the understanding of human overgrowth syndromes such as Perlman syndrome.

The importance of telomere function for human health is exemplified by a collection of Mendelian disorders referred to as the telomere biology disorders (TBDs), telomeropathies, or syndromes of telomere shortening. Collectively, the TBDs cover a spectrum of conditions from multisystem disease presenting in infancy to isolated disease presentations in adulthood, most notably idiopathic pulmonary fibrosis. Eleven genes have been found mutated in the TBDs to date, each of which is linked to some aspect of telomere maintenance. This review summarizes the molecular defects that result from mutations in these genes, highlighting recent advances, including the addition of PARN to the TBD gene family and the discovery of heterozygous mutations in RTEL1 as a cause of familial pulmonary fibrosis.

The co-transcriptional biogenesis of export-competent messenger ribonucleoprotein particles (mRNPs) in yeast is under the surveillance of quality control (QC) steps. Aberrant mRNPs resulting from inappropriate or inefficient processing and packaging reactions are detected by the QC system and retained in the nucleus with ensuing elimination of their mRNA component by a mechanism that requires the catalytic activity of Rrp6p, a 3'-5' exonuclease associated with the RNA exosome. In previous studies, we implemented a new experimental approach in which the production of aberrant mRNPs is massively increased upon perturbation of mRNP biogenesis by the RNA-dependent helicase/translocase activity of the bacterial Rho factor expressed in S. cerevisiae. The analyses of a subset of transcripts such as PMA1 led us to substantiate the essential role of Rrp6p in the nuclear mRNP QC and to reveal a functional coordination of the process by Nrd1p. Here, we extended those results by showing that, in contrast to PMA1, Rho-induced aberrant HXK1 mRNPs are targeted for destruction by an Nrd1p- and Rrp6p-independent alternative QC pathway that relies on the 5'-3' exonuclease activity of Rat1p. We show that the degradation of aberrant HXK1 mRNPs by Rat1p occurs co-transcriptionally following decapping by Dcp2p and leads to premature transcription termination. We discuss the possibility that this alternative QC pathway might be linked to the well-known specific features of the HXK1 gene transcription such as its localization at the nuclear periphery and gene loop formation.

Arthropod-borne flaviviruses (FVs) are a growing world-wide health threat whose incidence and range are increasing. The pathogenicity and cytopathicity of these single-stranded RNA viruses are influenced by viral subgenomic non-protein-coding RNAs (sfRNAs) that the viruses produce to high levels during infection. To generate sfRNAs the virus co-opts the action of the abundant cellular exonuclease Xrn1, which is part of the cell's normal RNA turnover machinery. This exploitation of the cellular machinery is enabled by discrete, highly structured, Xrn1-resistant RNA elements (xrRNAs) in the 3'UTR that interact with Xrn1 to halt processive 5' to 3' decay of the viral genomic RNA. We recently solved the crystal structure of a functional xrRNA, revealing a novel fold that provides a mechanistic model for Xrn1 resistance. Continued analysis and interpretation of the structure reveals that the tertiary contacts that knit the xrRNA fold together are shared by a wide variety of arthropod-borne FVs, conferring robust Xrn1 resistance in all tested. However, there is some variability in the structures that correlates with unexplained patterns in the viral 3' UTRs. Finally, examination of these structures and their behavior in the context of viral infection leads to a new hypothesis linking RNA tertiary structure, overall 3' UTR architecture, sfRNA production, and host adaptation.

Dis3 is a highly conserved exoribonuclease which degrades RNAs in the 3'-5' direction. Mutations in Dis3 are associated with a number of human cancers including multiple myeloma and acute myeloid leukemia. In this work, we have assessed the effect of a Dis3 knockdown on Drosophila imaginal disc development and on expression of mature microRNAs. We find that Dis3 knockdown severely disrupts the development of wing imaginal discs in that the flies have a "no wing" phenotype. Use of RNA-seq to quantify the effect of Dis3 knockdown on microRNA expression shows that Dis3 normally regulates a small subset of microRNAs, with only 11 (10.1%) increasing in level ≥ 2-fold and 6 (5.5%) decreasing in level ≥ 2-fold. Of these microRNAs, miR-252-5p is increased 2.1-fold in Dis3-depleted cells compared to controls while the level of the miR-252 precursor is unchanged, suggesting that Dis3 can act in the cytoplasm to specifically degrade this mature miRNA. Furthermore, our experiments suggest that Dis3 normally interacts with the exosomal subunit Rrp40 in the cytoplasm to target miR-252-5p for degradation during normal wing development. Another microRNA, miR-982-5p, is expressed at lower levels in Dis3 knockdown cells, while the miR-982 precursor remains unchanged, indicating that Dis3 is involved in its processing. Our study therefore reveals an unexpected specificity for this ribonuclease toward microRNA regulation, which is likely to be conserved in other eukaryotes and may be relevant to understanding its role in human disease.

Poikiloderma with neutropenia (PN) is a rare inherited disorder characterized by poikiloderma, facial dysmorphism, pachyonychia, short stature and neutropenia. The molecular testing of PN patients has identified mutations in the C16orf57 gene, which encodes a protein referred to as USB1 (U Six Biogenesis 1). In this study, we developed a zebrafish model of PN by the microinjection of morpholino antisense oligos to suppress usb1 gene function. Severe morphological defects, including a bent tail, thin yolk extension and reduced body length, were predominant in the Usb1-suppressed embryos (morphants). We also observed significantly decreased number of neutrophils in the morphants by Sudan Black staining. Interestingly, the splicing of genes involved in neutrophil differentiation and development, such as mpx, ncf1, ela3l and npsn, was aberrant in the morphants. However, the splicing of haematopoietic precursors and erythroid-specific genes was unaltered. Importantly, the neutrophil defects were almost completely rescued by co-injection of ela3l mRNA, the most markedly affected gene in the morphants. Our study demonstrated a possible role of USB1 in modulating the tissue-specific gene splicing that eventually leads to the impaired development of neutrophils. This zebrafish model could serve as a valuable tool to investigate the causative role of USB1 in PN pathogenesis.

Malaria is caused by a unicellular protozoan pathogen of the genus Plasmodium. Although genes represent monocistronic units that are expressed in a life cycle stage-specific manner, post-transcriptional regulation via translational repression of mRNA has been observed in parasite stages that transition from the vertebrate host to the Anopheles vector. An interesting new type of post-transcriptional control was recently discovered in Plasmodium falciparum stages that infect human erythrocytes. A subgroup of genes that were thought to be transcriptionally silent are actually transcribed but degraded immediately by an RNase II that is recruited to these gene loci. This cryptic RNA is not detectable in steady-state RNA but has been detected using nuclear run-on techniques and in mutant RNase II parasites. Nascent RNA degradation controls virulence genes expressed in a monoallelic fashion and noncoding RNAs (ncRNAs), but also a number of housekeeping-like of genes. More studies on other life cycle stages may reveal the full extent of this type of gene regulation in malaria parasites. It is tempting to speculate that RNase II-mediated gene control may exist in other eukaryotic organisms.

What has been will be again, what has been done will be done again; there is nothing new under the sun. -Ecclesiastes 1:9 (New International Version) Posttranscriptional regulation of gene expression has an important role in defining the phenotypic characteristics of an organism. Well-defined steps in mRNA metabolism that occur in the nucleus-capping, splicing, and polyadenylation-are mechanistically linked to the process of transcription. Recent evidence suggests another link between RNA polymerase II (Pol II) and a posttranscriptional process that occurs in the cytoplasm-mRNA decay. This conclusion appears to represent a conundrum. How could mRNA synthesis in the nucleus and mRNA decay in the cytoplasm be mechanistically linked? After a brief overview of mRNA processing, we will review the recent evidence for transcription-coupled mRNA decay and the possible involvement of Snf1, the Saccharomyces cerevisiae ortholog of AMP-activated protein kinase, in this process., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Poly(A) status is the major determinant of mRNA stability, even in endosymbiotic organelles. Poly(A) specific ribonuclease (PARN) is distributed widely among eukaryotes and has been shown to regulate the poly(A) status of cytoplasmic mRNA in various organisms. Surprisingly, our recent study revealed that PARN also directly regulates poly(A) status of mitochondrial mRNA in Arabidopsis. In this addendum, we discuss whether this mitochondrial function of PARN is common in plants and why PARN has been assigned such a unique function.

All bacteria contain multiple exoribonucleases to ensure a fast breakdown of different RNA molecules, either for maturation or for complete degradation to the level of mononucleotides. This efficient RNA degradation plays pivotal roles in the post-transcriptional gene regulation, in RNA processing and maturation as well as in RNA quality control mechanisms and global adaption to stress conditions. Besides different 3'-to-5' exoribonucleases mostly with overlapping functions in vivo many bacteria additionally possess the 5'-to-3' exoribonuclease, RNase J, to date the only known bacterial ribonuclease with this activity. An RNA-seq approach was applied to identify specific targets of RNase J in the α-proteobacterium Rhodobacter sphaeroides. Only few transcripts were strongly affected by the lack of RNase J implying that its function is mostly required for specific processing/degradation steps in this bacterium. The accumulation of diverse RNA fragments in the RNase J deletion mutant points to RNA features that apparently cannot be targeted by the conventional 3'-exoribonucleases in Gram-negative bacteria.

Pacman/Xrn1 is a highly conserved exoribonuclease known to play a critical role in gene regulatory events such as control of mRNA stability, RNA interference and regulation via miRNAs. Although Pacman has been well studied in Drosophila tissue culture cells, the biologically relevant cellular pathways controlled by Pacman in natural tissues are unknown. This study shows that a hypomorphic mutation in pacman (pcm (5)) results in smaller wing imaginal discs. These tissues, found in the larva, are known to grow and differentiate to form wing and thorax structures in the adult fly. Using microarray analysis, followed by quantitative RT-PCR, we show that eight mRNAs were increased in level by>2-fold in the pcm5 mutant wing discs compared with the control. The levels of pre-mRNAs were tested for five of these mRNAs; four did not increase in the pcm (5) mutant, showing that they are regulated at the post-transcriptional level and, therefore, could be directly affected by Pacman. These transcripts include one that encodes the heat shock protein Hsp67Bc, which is upregulated 11.9-fold at the post-transcriptional level and 2.3-fold at the protein level. One miRNA, miR-277-3p, is 5.6-fold downregulated at the post-transcriptional level in mutant discs, suggesting that Pacman affects its processing in this tissue. Together, these data show that a relatively small number of mRNAs and miRNAs substantially change in abundance in pacman mutant wing imaginal discs. Since Hsp67Bc is known to regulate autophagy and protein synthesis, it is possible that Pacman may control the growth of wing imaginal discs by regulating these processes.

RNA enables the material interpretation of genetic information through time and in space. The creation, destruction and activity of RNA must be well controlled and tightly synchronized with numerous cellular processes. We discuss here the pathways and mechanism of bacterial RNA turnover, and describe how RNA itself modulates these processes as part of decision-making networks. The central roles of RNA decay and other aspects of RNA metabolism in cellular control are also suggested by their vulnerability to sabotage by phages; nonetheless, RNA can be used in defense against phage infection, and these processes are described here. Salient aspects of RNA turnover are drawn together to suggest how it could affect complex effects such as phenotypic diversity in populations and responses that persist for multiple generations.

Discovered in eukaryotes as a modification essential for mRNA function, polyadenylation was then identified as a means used by all cells to destabilize RNA. In Escherichia coli, most accessible 3' RNA extremities are believed to be potential targets of poly(A) polymerase I. However, some RNAs might be preferentially adenylated. After a short statement of the current knowledge of poly(A) metabolism, we discuss how Hfq could affect recognition and polyadenylation of RNA terminated by Rho-independent terminators. Comparison of RNA terminus leads to the proposal that RNAs harboring 3' terminal features required for Hfq binding are not polyadenylated, whereas those lacking these structural elements can gain the oligo(A) tails that initiate exonucleolytic degradation. We also speculate that Hfq stimulates the synthesis of longer tails that could be used as Hfq-binding sites involved in non-characterized functions of Hfq-dependent sRNAs.

Transcription elongation is now recognized as an important mechanism of gene regulation in eukaryotes. A large number of genes undergo an early step in transcription that is rate limiting for expression. Genome-wide studies showing that RNA polymerase II accumulates to high densities near the promoters of many genes has led to the idea that promoter-proximal pausing of transcription is a widespread, rate-limiting step in early elongation. Recent evidence suggests that much of this paused RNA polymerase II is competent for transcription elongation. Here, we discuss recent studies suggesting that RNA polymerase II that accumulates nearby the promoter of a subset of genes is undergoing premature termination of transcription.

RNA helicases are compact, machine-like proteins that can harness the energy of nucleoside triphosphate binding and hydrolysis to dynamically remodel RNA structures and protein-RNA complexes. Through such activities, helicases participate in virtually every process associated with the expression of genetic information. Often found as components of multi-enzyme assemblies, RNA helicases facilitate the processivity of RNA degradation, the remodeling of protein interactions during maturation of structured RNA precursors, and fidelity checks of RNA quality. In turn, the assemblies modulate and guide the activities of the helicases. We describe the roles of RNA helicases with a conserved "DExD/H box" sequence motif in representative examples of such machineries from bacteria, archaea and eukaryotes. The recurrent occurrence of such helicases in complex assemblies throughout the course of evolution suggests a common requirement for their activities to meet cellular demands for the dynamic control of RNA metabolism.

microRNAs (miRNAs) are 20-24 nucleotide RNAs that regulate a variety of developmental and metabolic processes. The accumulation of miRNAs in vivo can be controlled at multiple levels. In addition to miRNA biogenesis, mechanisms that lead to RNA degradation, such as 3' uridylation and 3' truncation, also affect the steady-state levels of miRNAs. On the other hand, 2'-O-methylation in plant miRNAs protects their 3' ends from truncation and uridylation. The recent identification of HESO1 as the key enzyme responsible for miRNA uridylation in Arabidopsis was a first step toward a full understanding of the mechanisms underlying miRNA turnover. Analyses of the heso1 mutant predicted the existence of another uridylation activity and a previously unknown nuclease that act on miRNAs. The future identification of these enzymes will enrich our understanding of miRNA turnover.

Ribosome synthesis entails the formation of mature rRNAs from long precursor molecules, following a complex pre-rRNA processing pathway. Why the generation of mature rRNA ends is so complicated is unclear. Nor is it understood how pre-rRNA processing is coordinated at distant sites on pre-rRNA molecules. Here we characterized, in budding yeast and human cells, the evolutionarily conserved protein Las1. We found that, in both species, Las1 is required to process ITS2, which separates the 5.8S and 25S/28S rRNAs. In yeast, Las1 is required for pre-rRNA processing at both ends of ITS2. It is required for Rrp6-dependent formation of the 5.8S rRNA 3' end and for Rat1-dependent formation of the 25S rRNA 5' end. We further show that the Rat1-Rai1 5'-3' exoribonuclease (exoRNase) complex functionally connects processing at both ends of the 5.8S rRNA. We suggest that pre-rRNA processing is coordinated at both ends of 5.8S rRNA and both ends of ITS2, which are brought together by pre-rRNA folding, by an RNA processing complex. Consistently, we note the conspicuous presence of ~7- or 8-nucleotide extensions on both ends of 5.8S rRNA precursors and at the 5' end of pre-25S RNAs suggestive of a protected spacer fragment of similar length.

The GIR1 branching ribozyme constitutes a separate class of naturally occurring ribozymes. Most studies have been performed with the single GIR1 known from the myxomycete Didymium iridis whereas the large number of GIR1s found in the amoeboflagellate Naegleria has remained largely uncharacterized. Here, we investigate ribozyme cleavage properties of a collection of Naegleria GIR1 ribozymes and define the variant from N. pringsheimi as a suitable model due to its superior activity in vitro. We identify the minimal ribozyme by deletion analysis applying a new RNase R based assay for the branching reaction, and by mutational analysis we demonstrate a surprising effect on the activity of structural elements J2/10 and L9 located outside the core of the ribozyme. These elements are located in regions that differ mostly from the Didymium ribozyme and illustrate the usefulness of comparative ribozyme studies.

Organelle DNA in plastids and mitochondria is present in multiple copies and undergoes degradation developmentally. For example, organelle DNA that is detectable cytologically using DNA-fluorescent dye disappears during pollen development. Nevertheless, nucleases involved in this degradation process remain unknown. Our recent study identified the organelle nuclease, DPD1, which has Mg2+ -dependent exonuclease activity in vitro. The discovery of DPD1 emerged from Arabidopsis mutant screening and concomitant isolation of dpd1 mutants that retain organelle DNA in mature pollen. DPD1 is conserved only in angiosperms: not in other photosynthetic organisms. Despite these findings, the physiological significance of organelle DNA degradation during pollen development remains unclear because dpd1 exhibits no apparent defects in pollen viability or in the maternal inheritance of organelle DNA. We discuss a possible role of organelle DNA degradation mediated by DPD1, based on a DPD1 expression profile studied using in silico analyses.

Exosome-like protein complexes are essential 3'−>5' ribonucleases involved in processing and degradation of many RNAs. They are conserved in the three domains of life and share a common architecture comprised of a ring-like core structure organized around a central channel. RNA degradation by bacterial and archaeal exosome-like complexes requires threading through this single-stranded RNA specific channel to reach the phosphorolytic active sites buried deep within the barrel-shaped complex. In contrast most eukaryotic exosomes appear to have lost phosphorolytic activity and instead rely on hydrolytic RNases for catalytic activity raising the question of the degree of conservation of RNA recruitment mechanisms between prokaryotic and eukaryotic complexes. Recent single particle electron microscopy reconstructions of apo and RNA bound yeast exosomes provide the first direct structural evidence for a channeling mechanism by a eukaryotic exosome suggesting that this mechanism is conserved between all exosome-like complexes., (© 2011 Landes Bioscience)

Temperature downshift from 37 °C to 15 °C results in the exertion of cold shock response in Escherichia coli, which induces cold shock proteins, such as CsdA. Previously, we showed that the helicase activity of CsdA is critical for its function in the cold acclimation of cells and its primary role is mRNA degradation. Only RhlE (helicase), CspA (RNA chaperone) and RNase R (exoribonuclease) were found to complement the cold shock function of CsdA. RNase R has two independent activities, helicase and ribonuclease, only helicase being essential for the functional complementation of CsdA. Here, we discuss the significance of above findings as these emphasize the importance of the unwinding activity of cold-shock-inducible proteins in the RNA metabolism at low temperature, which may be different than that at 37 °C. It requires assistance of proteins to destabilize the secondary structures in mRNAs that are stabilized upon temperature downshift, hindering the activity of ribonucleases., (© 2011 Landes Bioscience)

Nidoviruses employ unique strategies to replicate and express their exceptionally large RNA genomes. The viruses use a variety of enzymes to synthesize, modify and process an extensive set of viral RNAs of both genome and subgenome length, including RNA polymerase, primase, helicase, ribose 2'-O and guanosine-N7 methyltransferases and several types of nuclease activities. In this review, the recent progress in the structural and functional characterization of nidovirus nuclease activities is discussed, focusing on a nidovirus-wide conserved uridylate-specific endoribonuclease, NendoU, and a 3'-to-5' exoribonuclease called ExoN. The latter enzyme is related to members of the DEDD exoribonuclease superfamily and conserved in all nidovirus families with genome sizes approaching 30 kilobases. Recent evidence implicates ExoN in reduced mutation rates during viral RNA replication and, possibly, superior fidelity of nidovirus replicases, leading to the suggestion that ExoN may be a key factor in the expansion of nidovirus genomes to sizes not seen in other RNA viruses.

mRNA turnover is a critical step in the control of gene expression. In mammalian cells, a subset of mRNAs regulated at the level of mRNA turnover contain destabilizing AU-rich elements (AREs) in their 3' untranslated regions. These transcripts are bound by a suite of ARE-binding proteins (AUBPs) that receive information from cell signaling events to modulate rates of ARE mRNA decay. Here we show that a key destabilizing AUBP, tristetraprolin (TTP), is repressed by the p38 mitogen-activated protein kinase (MAPK)-activated kinase MK2 due to the inability of phospho-TTP to recruit deadenylases to target mRNAs. TTP is tightly associated with cytoplasmic deadenylases and promotes rapid deadenylation of target mRNAs both in vitro and in cells. TTP can direct the deadenylation of substrate mRNAs when tethered to a heterologous mRNA, yet its ability to do so is inhibited upon phosphorylation by MK2. Phospho-TTP is not impaired in mRNA binding but does fail to recruit the major cytoplasmic deadenylases. These observations suggest that phosphorylation of TTP by MK2 primarily affects mRNA decay downstream of RNA binding by preventing recruitment of the deadenylation machinery. Thus, TTP may remain poised to rapidly reactivate deadenylation of bound transcripts to downregulate gene expression once the p38 MAPK pathway is deactivated.

mRNA polyadenylation and deadenylation are important processes that allow rapid regulation of gene expression in response to different cellular conditions. Almost all eukaryotic mRNA precursors undergo a co-transcriptional cleavage followed by polyadenylation at the 3' end. After the signals are selected, polyadenylation occurs to full extent, suggesting that this first round of polyadenylation is a default modification for most mRNAs. However, the length of these poly(A) tails changes by the activation of deadenylation, which might regulate gene expression by affecting mRNA stability, mRNA transport, or translation initiation. The mechanisms behind deadenylation activation are highly regulated and associated with cellular conditions such as development, mRNA surveillance, DNA damage response, cell differentiation and cancer. After deadenylation, depending on the cellular response, some mRNAs might undergo an extension of the poly(A) tail or degradation. The polyadenylation/deadenylation machinery itself, miRNAs, or RNA binding factors are involved in the regulation of polyadenylation/deadenylation. Here, we review the mechanistic connections between polyadenylation and deadenylation and how the two processes are regulated in different cellular conditions. It is our conviction that further studies of the interplay between polyadenylation and deadenylation will provide critical information required for a mechanistic understanding of several diseases, including cancer development.

One of the many important consequences that temperature down-shift has on cells is stabilization of secondary structures of RNAs. This stabilization has wide-spread effects, such as inhibition of expression of several genes due to termination of their transcription and inefficient RNA degradation that adversely affect cell growth at low temperature. Several cold shock proteins are produced to counteract these effects and thus allow cold acclimatization of the cell. The main RNA modulating cold shock proteins of E. coli can be broadly divided into two categories, (1) the CspA family proteins, which mainly affect the transcription and possibly translation at low temperature through their RNA chaperoning function and (2) RNA helicases and exoribonucleases that stimulate RNA degradation at low temperature through their RNA unwinding activity.

In eukaryotic cells, degradation of many mRNAs is initiated by removal of the poly(A) tail followed by decapping and 5'-3' exonucleolytic decay. Although the order of these events is well established, we are still lacking a mechanistic understanding of how deadenylation and decapping are linked. In this report we identify human Pat1b as a protein that is tightly associated with the Ccr4-Caf1-Not deadenylation complex as well as with the Dcp1-Dcp2 decapping complex. In addition, the RNA helicase Rck and Lsm1 proteins interact with human Pat1b. These interactions are mediated via at least three independent domains within Pat1b, suggesting that Pat1b serves as a scaffold protein. By tethering Pat1b to a reporter mRNA, we further provide evidence that Pat1b is also functionally linked to both deadenylation and decapping. Finally, we report that Pat1b strongly induces the formation of processing (P) bodies, cytoplasmic foci that contain most enzymes of the RNA decay machinery. An amino-terminal region within Pat1b serves as an aggregation-prone domain that nucleates P bodies, whereas an acidic domain controls the size of P bodies. Taken together, these findings provide evidence that human Pat1b is a central component of the RNA decay machinery by physically connecting deadenylation with decapping.