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Hepatokines, secretory proteins from the liver, mediate inter-organ communication to maintain a metabolic balance between food intake and energy expenditure. However, molecular mechanisms by which hepatokine levels are rapidly adjusted following stimuli are largely unknown. Here, we unravel how CNOT6L deadenylase switches off hepatokine expression after responding to stimuli (e.g., exercise and food) to orchestrate energy intake and expenditure. Mechanistically, CNOT6L inhibition stabilizes hepatic Gdf15 and Fgf21 mRNAs, increasing corresponding serum protein levels. The resulting upregulation of GDF15 stimulates the hindbrain to suppress appetite, while increased FGF21 affects the liver and adipose tissues to induce energy expenditure and lipid consumption. Despite the potential of hepatokines to treat metabolic disorders, their administration therapies have been challenging. Using small-molecule screening, we identified a CNOT6L inhibitor enhancing GDF15 and FGF21 hepatokine levels, which dramatically improves diet-induced metabolic syndrome. Our discovery, therefore, lays the foundation for an unprecedented strategy to treat metabolic syndrome., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The role of N6-methyladenosine (m 6 A) modifications has increasingly been associated with a diverse set of roles in modulating viruses and influencing the outcomes of viral infection. Here, we report that the landscape of m 6 A deposition is drastically shifted during Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection for both viral and host transcripts. In line with previous reports, we also saw an overall decrease in host methylation in favor of viral messenger RNA (mRNA), along with 5' hypomethylation and 3' hypermethylation. During KSHV lytic infection, a major shift in overall mRNA abundance is driven by the viral endoribonuclease SOX, which induces the decay of greater than 70% of transcripts. Here, we reveal that interlukin-6 (IL-6) mRNA, a well-characterized, SOX-resistant transcript, is m 6 A modified during lytic infection. Furthermore, we show that this modification falls within the IL-6 SOX resistance element, an RNA element in the IL-6 3' untranslated region (UTR) that was previously shown to be sufficient for protection from SOX cleavage. We show that the presence of this m 6 A modification is essential to confer SOX resistance to the IL-6 mRNA. We next show that this modification recruits the m 6 A reader YTHDC2 and found that YTHDC2 is necessary for the escape of the IL-6 transcript. These results shed light on how the host cell has evolved to use RNA modifications to circumvent viral manipulation of RNA fate during KSHV infection., Competing Interests: The authors declare no competing interest.

Accurate control of the cell cycle is critical for development and tissue homeostasis, and requires precisely timed expression of many genes. Cell cycle gene expression is regulated through transcriptional and translational control, as well as through regulated protein degradation. Here, we show that widespread and temporally controlled mRNA decay acts as an additional mechanism for gene expression regulation during the cell cycle in human cells. We find that two waves of mRNA decay occur sequentially during the mitosis-to-G1 phase transition, and we identify the deadenylase CNOT1 as a factor that contributes to mRNA decay during this cell cycle transition. Collectively, our data show that, akin to protein degradation, scheduled mRNA decay helps to reshape cell cycle gene expression as cells move from mitosis into G1 phase., Competing Interests: LK, SS, MT No competing interests declared, (© 2022, Krenning et al.)

Detailed studies of the Gram-negative model bacterium, Escherichia coli , have demonstrated that post-transcriptional events exert important and possibly greater control over gene regulation than transcription initiation or effective translation. Thus, over the past 30 years, considerable effort has been invested in understanding the pathways of mRNA turnover in E. coli . Although it is assumed that most of the ribonucleases and accessory proteins involved in mRNA decay have been identified, our understanding of the regulation of mRNA decay is still incomplete. Furthermore, the vast majority of the studies on mRNA decay have been conducted on exponentially growing cells. Thus, the mechanism of mRNA decay as currently outlined may not accurately reflect what happens when cells find themselves under a variety of stress conditions, such as, nutrient starvation, changes in pH and temperature, as well as a host of others. While the cellular machinery for degradation is relatively constant over a wide range of conditions, intracellular levels of specific ribonucleases can vary depending on the growth conditions. Substrate competition will also modulate ribonucleolytic activity. Post-transcriptional modifications of transcripts by polyadenylating enzymes may favor a specific ribonuclease activity. Interactions with small regulatory RNAs and RNA binding proteins add additional complexities to mRNA functionality and stability. Since many of the ribonucleases are found at the inner membrane, the physical location of a transcript may help determine its half-life. Here we discuss the properties and role of the enzymes involved in mRNA decay as well as the multiple factors that may affect mRNA decay under various in vivo conditions.

Non-small cell lung cancer (NSCLC) is one of the prevalent and deadly cancers worldwide. Cisplatin (CDDP) has been used as a standard adjuvant therapy for advanced NSCLC patients, while chemoresistance is one of the most challenging problems to limit its clinical application. Our data showed that the expression of visfatin was significantly increased in CDDP resistant NSCLC cells as compared with that in their parental cells, while knockdown of visfatin or its neutralization antibody can restore the CDDP sensitivity of resistant NSCLC cells. The upregulation of visfatin in CDDP resistant NSCLC cells was due to the increased mRNA stability and promoter activity. Further, we found that signal transducer and activator of transcription 3 (STAT3), which was increased in chemoresistant cells, can increase the transcription of visfatin. While tristetraprolin (TTP), which can decease mRNA stability of visfatin, was decreased in chemoresistant cells. Inhibition of STAT3 or over expression of TTP can restore CDDP sensitivity of resistant NSCLC cells. Collectively, our data showed that STAT3 and TTP-regulated expression of visfatin was involved in CDDP resistance of NSCLC cells. It indicated that targeted inhibition of visfatin should be a potential approach to overcome CDDP resistance of NSCLC treatment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The genome is pervasively transcribed across various species, yielding numerous non-coding RNAs. As a counterbalance for pervasive transcription, various organisms have a nuclear RNA exosome complex, whose structure is well conserved between yeast and mammalian cells. The RNA exosome not only regulates the processing of stable RNA species, such as rRNAs, tRNAs, small nucleolar RNAs, and small nuclear RNAs, but also plays a central role in RNA surveillance by degrading many unstable RNAs and misprocessed pre-mRNAs. In addition, associated cofactors of RNA exosome direct the exosome to distinct classes of RNA substrates, suggesting divergent and/or multi-layer control of RNA quality in the cell. While the RNA exosome is essential for cell viability and influences various cellular processes, mutations and alterations in the RNA exosome components are linked to the collection of rare diseases and various diseases including cancer, respectively. The present review summarizes the relationships between pervasive transcription and RNA exosome, including evolutionary crosstalk, mechanisms of RNA exosome-mediated RNA surveillance, and physiopathological effects of perturbation of RNA exosome.

Nicotine addiction and the occurrence of lymph node spread are two major significant factors associated with esophageal cancer's poor prognosis; however, nicotine's role in inducing lymphatic metastasis of esophageal cancer remains unclear. Here we show that OTU domain-containing protein 3 (OTUD3) is downregulated by nicotine and correlates with poor prognosis in heavy-smoking esophageal cancer patients. OTUD3 directly interacts with ZFP36 ring finger protein (ZFP36) and stabilizes it by inhibiting FBXW7-mediated K48-linked polyubiquitination. ZFP36 binds with the VEGF-C 3-'UTR and recruits the RNA degrading complex to induce its rapid mRNA decay. Downregulation of OTUD3 and ZFP36 is essential for nicotine-induced VEGF-C production and lymphatic metastasis in esophageal cancer. This study establishes that the OTUD3/ZFP36/VEGF-C axis plays a vital role in nicotine addiction-induced lymphatic metastasis, suggesting that OTUD3 may serve as a prognostic marker, and induction of the VEGF-C mRNA decay might be a potential therapeutic strategy against human esophageal cancer., (© 2021. The Author(s).)

This study aims to investigate the biological role of 6-methyladenine (m6A) methylation in inducing the carcinogenesis of glioma and its proliferation. Relative levels of ALKBH5 and glucose-6-phosphate dehydrogenase (G6PD) in glioma tissues and cell lines were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Gain-of-function and loss-of-function approaches were used to investigate the role of ALKBH5 in mediating proliferation and energy metabolism of glioma cells. The regulatory effect of ALKBH5 on G6PD was analyzed using m6A-qRT-PCR. Our results showed that ALKBH5 was upregulated in glioma, which stimulated glioma cells to proliferate. Serving as a m6A eraser, ALKBH5 demethylated the target transcript G6PD and enhanced its mRNA stability, thereby promoting G6PD translation and activating the pentose phosphate pathway (PPP). Collectively, ALKBH5 stimulates glioma cells to proliferate through erasing the m6A methylation of G6PD, which can be utilized as a potential therapeutic target for glioma., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

One of the many challenges faced by RNA viruses is the maintenance of their genomes during infections of host cells. Members of the family Tombusviridae are plus-strand RNA viruses with unmodified triphosphorylated genomic 5' termini. The tombusvirus Carnation Italian ringspot virus was used to investigate how it protects its RNA genome from attack by 5'-end-targeting degradation enzymes. In vivo and in vitro assays were employed to determine the role of genomic RNA structure in conferring protection from the 5'-to-3' exoribonuclease Xrn. The results revealed that (i) the CIRV RNA genome is more resistant to Xrn than its sg mRNAs, (ii) the genomic 5'-untranslated region (UTR) folds into a compact RNA structure that effectively and independently prevents Xrn access, (iii) the RNA structure limiting 5' access is formed by secondary and tertiary interactions that function cooperatively, (iv) the structure is also able to block access of RNA pyrophosphohydrolase to the genomic 5' terminus, and (v) the RNA structure does not stall an actively digesting Xrn. Based on its proficiency at impeding Xrn 5' access, we have termed this 5'-terminal structure an X rn- e vading RNA, or xeRNA. These and other findings demonstrate that the 5'UTR of the CIRV RNA genome folds into a complex structural conformation that helps to protect its unmodified 5' terminus from enzymatic decay during infections. IMPORTANCE The plus-strand RNA genomes of plant viruses in the large family Tombusviridae are not 5' capped. Here, we explored how a species in the type genus Tombusvirus protects its genomic 5' end from cellular nuclease attack. Our results revealed that the 5'-terminal sequence of the CIRV genome folds into a complex RNA structure that limits access of the 5'-to-3' exoribonuclease Xrn, thereby protecting it from processive degradation. The RNA conformation also impeded access of RNA pyrophosphohydrolase, which converts 5'-triphosphorylated RNA termini into 5'-monophosphorylated forms, the preferred substrate for Xrn. This study represents the first report of a higher-order RNA structure in an RNA plant virus genome independently conferring resistance to 5'-end-attacking cellular enzymes.

Trans-active response DNA-binding protein of 43 kDa (TDP-43) promotes tau mRNA instability and tau exon 10 inclusion. Aggregation of phosphorylated TDP-43 is associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. Casein kinase 1ε (CK1ε) phosphorylates TDP-43 at multiple sites, enhances its cytoplasmic aggregation, and modulates its function in tau mRNA processing. To determine roles of TDP-43 site-specific phosphorylation in its localization, aggregation, and function in tau mRNA processing, TDP-43 was mutated to alanine or aspartic acid at Ser379, Ser403/404, or Ser409/410 to block or mimic phosphorylation. Site-specific phosphorylation of TDP-43 and its mutants by CK1ε was studied in vitro and in cultured cells. Cytoplasmic and nuclear TDP-43 and phospho-TDP-43 were analyzed by western blots. Aggregation of TDP-43 was assessed by immunostaining and level of radioimmunoprecipitation assay buffer-insoluble TDP-43. Green florescent protein tailed with tau 3'-untranslated region and mini-tau gene pCI/SI9-LI10 were used to study tau mRNA stability and alternative splicing of tau exon 10. We found that phospho-blocking mutations of TDP-43 at Ser379, Ser403/404, or Ser409/410 were not effectively phosphorylated by CK1ε. Compared with TDP-43, higher level of phosphorylated TDP-43 in the cytoplasm was observed. Phospho-mimicking mutations at these sites enhanced cytoplasmic aggregation of TDP-43. Green florescent protein expression was not inhibited by phospho-blocking mutants of TDP-43, but tau exon 10 inclusion was further enhanced by phospho-blocking mutations at Ser379 and Ser403/404. Phosphorylation of TDP-43 at Ser379, Ser403/404, or Ser409/410 primes its phosphorylation by CK1ε, promotes TDP-43 cytoplasmic aggregation, and modulates its function in tau mRNA processing in site-specific manner., (© 2021 International Society for Neurochemistry.)

Cellular RNA levels typically fluctuate and are influenced by different transcription rates and RNA degradation rates. However, the understanding of the fundamental relationships between RNA abundance, environmental stimuli, RNA activities, and RNA age distributions is incomplete. Furthermore, the rates of RNA degradation and transcription are difficult to measure in transcriptomic experiments in living organisms, especially in studies involving humans. A model based on activity demands and RNA age was developed to explore the mechanisms of RNA level fluctuations. Using single-cell time-series gene expression experimental data, we assessed the transcription rates, RNA degradation rates, RNA life spans, RNA demand, accumulated transcription levels, and accumulated RNA degradation levels. This model could also predict RNA levels under simulation backgrounds, such as stimuli that induce regular oscillations in RNA abundance, stable RNA levels over time that result from long-term shortage of total RNA activity or from uncontrollable transcription, and relationships between RNA/protein levels and metabolic rates. This information contributes to existing knowledge., Competing Interests: The authors have declared that no competing interests exist.

N 6 -Methyladenosine (m6A) is the most prevalent internal RNA modification and has a widespread impact on mRNA stability and translation. Methyltransferase-like 3 (Mettl3) is a methyltransferase responsible for RNA m6A modification, and it is essential for early embryogenesis before or during gastrulation in mice and zebrafish. However, due to the early embryonic lethality, loss-of-function phenotypes of Mettl3 beyond gastrulation, especially during neurulation stages when spatial neural patterning takes place, remain elusive. Here, we address multiple roles of Mettl3 during Xenopus neurulation in anteroposterior neural patterning, neural crest specification, and neuronal cell differentiation. Knockdown of Mettl3 causes anteriorization of neurulae and tailbud embryos along with the loss of neural crest and neuronal cells. Knockdown of the m6A reader Ythdf1 and mRNA degradation factors, such as 3' to 5' exonuclease complex component Lsm1 or mRNA uridylation enzyme Tut7, also show similar neural patterning defects, suggesting that m6A-dependent mRNA destabilization regulates spatial neural patterning in Xenopus . We also address that canonical WNT signaling is inhibited in Mettl3 morphants, which may underlie the neural patterning defects of the morphants. Altogether, this study reveals functions of Mettl3 during spatial neural patterning in Xenopus .

Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3' untranslated region (3'-UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3'-UTR reporter transgene to Ca 2+ , the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3'-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca 2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node.

TENT4A (PAPD7) is a non-canonical poly(A) polymerase, of which little is known. Here, we show that TENT4A regulates multiple biological pathways and focuses on its multilayer regulation of translesion DNA synthesis (TLS), in which error-prone DNA polymerases bypass unrepaired DNA lesions. We show that TENT4A regulates mRNA stability and/or translation of DNA polymerase η and RAD18 E3 ligase, which guides the polymerase to replication stalling sites and monoubiquitinates PCNA, thereby enabling recruitment of error-prone DNA polymerases to damaged DNA sites. Remarkably, in addition to the effect on RAD18 mRNA stability via controlling its poly(A) tail, TENT4A indirectly regulates RAD18 via the tumor suppressor CYLD and via the long non-coding antisense RNA PAXIP1-AS2 , which had no known function. Knocking down the expression of TENT4A or CYLD , or overexpression of PAXIP1-AS2 led each to reduced amounts of the RAD18 protein and DNA polymerase η, leading to reduced TLS, highlighting PAXIP1-AS2 as a new TLS regulator. Bioinformatics analysis revealed that TLS error-prone DNA polymerase genes and their TENT4A -related regulators are frequently mutated in endometrial cancer genomes, suggesting that TLS is dysregulated in this cancer.

The current SARS-CoV-2 pandemic underscores the importance of understanding the evolution of RNA genomes. While RNA is subject to the formation of similar lesions as DNA, the evolutionary and physiological impacts RNA lesions have on viral genomes are yet to be characterized. Lesions that may drive the evolution of RNA genomes can induce breaks that are repaired by recombination or can cause base substitution mutagenesis, also known as base editing. Over the past decade or so, base editing mutagenesis of DNA genomes has been subject to many studies, revealing that exposure of ssDNA is subject to hypermutation that is involved in the etiology of cancer. However, base editing of RNA genomes has not been studied to the same extent. Recently hypermutation of single-stranded RNA viral genomes have also been documented though its role in evolution and population dynamics. Here, we will summarize the current knowledge of key mechanisms and causes of RNA genome instability covering areas from the RNA world theory to the SARS-CoV-2 pandemic of today. We will also highlight the key questions that remain as it pertains to RNA genome instability, mutations accumulation, and experimental strategies for addressing these questions.

To replicate efficiently and evade the antiviral immune response of the host, some viruses degrade host mRNA to induce host gene shutoff via encoding shutoff factors. In this study, we found that feline calicivirus (FCV) infection promotes the degradation of endogenous and exogenous mRNAs and induces host gene shutoff, which results in global inhibition of host protein synthesis. Screening assays revealed that proteinase-polymerase (PP) is a most effective factor in reducing mRNA expression. Moreover, PP from differently virulent strains of FCV could induce mRNA degradation. Further, we found that the key sites of the PP protein required for its proteinase activity are also essential for its shutoff activity but also required for viral replication. The mechanism analysis showed that PP mainly targets Pol II-transcribed RNA in a ribosome-, 5' cap-, and 3' poly(A) tail-independent manner. Moreover, purified glutathione S -transferase (GST)-PP fusion protein exhibits RNase activity in vitro in assays using green fluorescent protein (GFP) RNA transcribed in vitro as a substrate in the absence of other viral or cellular proteins. Finally, PP-induced shutoff requires host Xrn1 to complete further RNA degradation. This study provides a newly discovered strategy in which FCV PP protein induces host gene shutoff by promoting the degradation of host mRNAs. IMPORTANCE Virus infection-induced shutoff is the result of targeted or global manipulation of cellular gene expression and leads to efficient viral replication and immune evasion. FCV is a highly contagious pathogen that persistently infects cats. It is unknown how FCV blocks the host immune response and persistently exists in cats. In this study, we found that FCV infection promotes the degradation of host mRNAs and induces host gene shutoff via a common strategy. Further, PP protein for different FCV strains is a key factor that enhances mRNA degradation. An in vitro assay showed that the GST-PP fusion protein possesses RNase activity in the absence of other viral or cellular proteins. This study demonstrates that FCV induces host gene shutoff by promoting the degradation of host mRNAs, thereby introducing a potential mechanism by which FCV infection inhibits the immune response.

Amyloid precursor protein (APP) is a transmembrane protein that plays a crucial role in the production of amyloid-β peptides. Any disruption in APP protein production, its mRNA decay rate or processing may result in abnormal production of amyloid-β peptides and subsequent development of protein aggregation diseases. Therefore, the equilibrium is crucial for neuronal function. An association study of heterogeneous nuclear ribonucleoprotein (hnRNP)-F and hnRNP H1 with APP was carried out in Neuro-2a (N2a) cells. In the present study, we found that hnRNP F and hnRNP H1 were significantly upregulated in the hippocampus of APP/PS1 mice. The changes in APP expression were positively associated with hnRNP F and hnRNP H1 when hnRNP F and hnRNP H1 were depleted or increased in N2a cells. Importantly, cross-linked RNA immunoprecipitation demonstrated binding affinities of hnRNP F and hnRNP H1 for App mRNA. Mechanistically, mRNA stability assay revealed that overexpression of hnRNP F or hnRNP H1 increases the APP level by stabilizing App mRNA half-life, implying that levels of hnRNP F and hnRNP H1 can change the production of APP. Further understanding of the regulatory mechanism of APP expression in association with hnRNP F and hnRNP H1 would provide insights into the mechanism underlying the maintenance of brain health and cognition. This study provides a theoretical basis for the development of hnRNP-stabilizing compounds to regulate APP., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

SARS-CoV-2 infection and the resulting COVID-19 have afflicted millions of people in an ongoing worldwide pandemic. Safe and effective vaccination is needed urgently to protect not only the general population but also vulnerable subjects such as patients with cancer. Currently approved mRNA-based SARS-CoV-2 vaccines seem suitable for patients with cancer based on their mode of action, efficacy, and favorable safety profile reported in the general population. Here, we provide an overview of mRNA-based vaccines including their safety and efficacy. Extrapolating from insights gained from a different preventable viral infection, we review existing data on immunity against influenza A and B vaccines in patients with cancer. Finally, we discuss COVID-19 vaccination in light of the challenges specific to patients with cancer, such as factors that may hinder protective SARS-CoV-2 immune responses in the context of compromised immunity and the use of immune-suppressive or immune-modulating drugs., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Extracellular vesicles (EVs) have emerged as promising candidates in biomarker discovery and diagnostics. Protected by the lipid bilayer, the molecular content of EVs in diverse biofluids are protected from RNases and proteases in the surrounding environment that may rapidly degrade targets of interests. Nonetheless, cryopreservation of EV-containing samples to -80°C may expose the lipid bilayer to physical and biological stressors which may result in cryoinjury and contribute to changes in EV yield, function, or molecular cargo. In the present work, we systematically evaluate the effect of cryopreservation at -80°C for a relatively short duration of storage (up to 12 days) on plasma- and media-derived EV particle count and/or RNA yield/quality, as compared to paired fresh controls. On average, we found that the plasma-derived EV concentration of stored samples decreased to 23% of fresh samples. Further, this significant decrease in EV particle count was matched with a corresponding significant decrease in RNA yield whereby plasma-derived stored samples contained only 47-52% of the total RNA from fresh samples, depending on the extraction method used. Similarly, media-derived EVs showed a statistically significant decrease in RNA yield whereby stored samples were 58% of the total RNA from fresh samples. In contrast, we did not obtain clear evidence of decreased RNA quality through analysis of RNA traces. These results suggest that samples stored for up to 12 days can indeed produce high-quality RNA; however, we note that when directly comparing fresh versus cryopreserved samples without cryoprotective agents there are significant losses in total RNA. Finally, we demonstrate that the addition of the commonly used cryoprotectant agent, DMSO, alongside greater control of the rate of cooling/warming, can rescue EVs from damaging ice formation and improve RNA yield., Competing Interests: Drs. Stott, Tessier, and Toner are inventors on several provisional patents on the topic of blood stabilization and cryopreservation of EVs, cells, tissues, and organs (W02016022433A1, W02018005802A1, W02020163774A1). These do not alter our adherence to PLOS ONE policies on sharing data and materials. There are no restrictions on sharing of data and/or materials as a result of these competing interests. Researcher’s interests are managed by the MGH and Partners HealthCare in accordance with their conflict-of-interest policies.

Accurate relative gene expression analysis by reverse transcription-quantitative polymerase chain reaction relies on the usage of suitable reference genes for data normalization. The RNA content of small extracellular vesicles including exosomes is growingly considered as cancer biomarkers. So, reliable relative quantification of exosomal messenger RNA (mRNA) is essential for cancer diagnosis and prognosis applications. However, suitable reference genes for accurate normalization of a target gene in exosomes derived from cancer cells are not depicted yet. Here, we analyzed the expression and stability of eight well-known reference genes namely GAPDH, B2M, HPRT1, ACTB, YWHAZ, UBC, RNA18S, and TBP in exosomes-isolated from the liver (Huh7, HepG2, PLC/PRF/5) and breast (SK-BR-3) cancer cell lines using five different algorithms including geNorm, BestKeeper, Delta Ct, NormFinder, and RefFinder. Our results showed that ACTB, TBP, and HPRT1 were not expressed in exosomes-isolated from studied liver and breast cancer cell lines. The geNorm and BestKeeper algorithms indicated GAPDH and UBC as the most stable candidates. Moreover, Delta Ct and NormFinder algorithms showed YWHAZ as the most stable reference genes. Comprehensive ranking calculated by the RefFinder algorithm also pointed out GAPDH, YWHAZ, and UBC as the first three stable reference genes. Taken together, this study validated the common reference genes stability in exosomal mRNA derived from liver and breast cancer cell lines for the first time. We believe that this study would be the first step in finding more stable reference genes in exosomes that triggers more accurate detection of exosomal biomarkers., (© 2021 International Federation for Cell Biology.)

Eukaryotic cells integrate multiple quality control (QC) responses during protein synthesis in the cytoplasm. These QC responses are signaled by slow or stalled elongating ribosomes. Depending on the nature of the delay, the signal may lead to translational repression, messenger RNA decay, ribosome rescue, and/or nascent protein degradation. Here, we discuss how the structure and composition of an elongating ribosome in a troubled state determine the downstream quality control pathway(s) that ensue. We highlight the intersecting pathways involved in RNA decay and the crosstalk that occurs between RNA decay and ribosome rescue., Competing Interests: Declaration of interests R.G. is a member of the scientific advisory board at the journal Molecular Cell., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Growing evidences suggest that cancer stem cells exhibit many molecular characteristics and phenotypes similar to their ancestral progenitor cells. In the present study, human embryonic stem cells are induced to differentiate into hepatocytes along hepatic lineages to mimic liver development in vitro. A liver progenitor specific gene, RALY RNA binding protein like (RALYL), is identified. RALYL expression is associated with poor prognosis, poor differentiation, and metastasis in clinical HCC patients. Functional studies reveal that RALYL could promote HCC tumorigenicity, self-renewal, chemoresistance, and metastasis. Moreover, molecular mechanism studies show that RALYL could upregulate TGF-β2 mRNA stability by decreasing N6-methyladenosine (m 6 A) modification. TGF-β signaling and the subsequent PI3K/AKT and STAT3 pathways, upregulated by RALYL, contribute to the enhancement of HCC stemness. Collectively, RALYL is a liver progenitor specific gene and regulates HCC stemness by sustaining TGF-β2 mRNA stability. These findings may inspire precise therapeutic strategies for HCC.

Multicellular organisms coordinate tissue specific responses to environmental information via both cell-autonomous and non-autonomous mechanisms. In addition to secreted ligands, recent reports implicated release of small RNAs in regulating gene expression across tissue boundaries. Here, we show that the conserved poly-U specific endoribonuclease ENDU-2 in C. elegans is secreted from the soma and taken-up by the germline to ensure germline immortality at elevated temperature. ENDU-2 binds to mature mRNAs and negatively regulates mRNA abundance both in the soma and the germline. While ENDU-2 promotes RNA decay in the soma directly via its endoribonuclease activity, ENDU-2 prevents misexpression of soma-specific genes in the germline and preserves germline immortality independent of its RNA-cleavage activity. In summary, our results suggest that the secreted RNase ENDU-2 regulates gene expression across tissue boundaries in response to temperature alterations and contributes to maintenance of stem cell immortality, probably via retaining a stem cell specific program of gene expression.

Inhibition of the host RNA polyadenylating polymerases, PAPD5 and PAPD7 (PAPD5/7), with dihydroquinolizinone, a small orally available, molecule, results in a rapid and selective degradation of hepatitis B virus (HBV) RNA, and hence reduction in the amounts of viral gene products. DHQ, is a first in class investigational agent and could represent an entirely new category of HBV antivirals. PAPD5 and PAPD7 are non-canonical, cell specified, polyadenylating polymerases, also called terminal nucleotidyl transferases 4B and 4A (TENT4B/A), respectively. They are involved in the degradation of poor-quality cell transcripts, mostly non-coding RNAs and in the maturation of a sub-set of transcripts. They also appear to play a role in shielding some mRNA from degradation. The results of studies with DHQ, along with other recent findings, provide evidence that repression of the PAPD5/7 arm of the cell "RNA quality control" pathway, causes a profound (multi-fold) reduction rather than increase, in the amount of HBV pre-genomic, pre-core and HBsAg mRNA levels in tissue culture and animal models, as well. In this review we will briefly discuss the need for new HBV therapeutics and provide background about HBV transcription. We also discuss cellular degradation of host transcripts, as it relates to a new family of anti-HBV drugs that interfere with these processes. Finally, since HBV mRNA maturation appears to be selectively sensitive to PAPD5/7 inhibition in hepatocytes, we discuss the possibility of targeting host RNA "quality control" as an antiviral strategy., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Infection with kinetoplastid parasites, including Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania can cause serious disease in humans. Like other kinetoplastid species, mRNAs of these disease-causing parasites must undergo posttranscriptional editing in order to be functional. mRNA editing is directed by gRNAs, a large group of small RNAs. Similar to mRNAs, gRNAs are also precisely regulated. In T. brucei, overexpression of RNase D ribonuclease (TbRND) leads to substantial reduction in the total gRNA population and subsequent inhibition of mRNA editing. However, the mechanisms regulating gRNA binding and cleavage by TbRND are not well defined. Here, we report a thorough structural study of TbRND. Besides Apo- and NMP-bound structures, we also solved one TbRND structure in complexed with single-stranded RNA. In combination with mutagenesis and in vitro cleavage assays, our structures indicated that TbRND follows the conserved two-cation-assisted mechanism in catalysis. TbRND is a unique RND member, as it contains a ZFD domain at its C-terminus. In addition to T. brucei, our studies also advanced our understanding on the potential gRNA degradation pathway in T. cruzi, Leishmania, as well for as other disease-associated parasites expressing ZFD-containing RNDs., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The luciferase reporter assay is a widely used tool to study the cis and trans factors controlling regulation of gene expression. In this assay, regulatory elements can be fused to the luciferase gene, and as a result effect protein output by changing rates of transcription, rates of translation, or mRNA stability. This protocol focuses on probing the function of RNA-binding proteins (RBPs) through their interactions with the 3' untranslated region (UTR), thus examining gene expression regulation on the mRNA level. Assessment of 3' UTR sequence requirements, as well as single and co-regulatory roles of RBPs in regulation of mRNAs will be discussed.

miRNA markers have been an area of forensic interest to identify body fluid sources in recent years. In this study, reverse transcription and quantitative real time polymerase chain reaction (RT-qPCR) were performed to detect the existence of blood-specific miRNA markers in bloodstained samples under different environmental conditions, Blood samples from 6 individuals were deposited onto glass plates and exposed to different temperature, humidity, ultraviolet light intensity, and natural condition. When samples were stored to a series of estimated test times, total RNA was extracted and the Ct values of the target RNAs were detected, targets included two miRNA markers (hsa-miR-16-5p, hsa-miR-451a) and one reference gene (U6 snRNA). Analysis results showed that miR-451a represented strong stability and could be detected at all detection points. Meanwhile, each RNAs exhibited unique degradation characteristics, compared to U6, miRNAs showed stronger stability. Additionally, rain had an adverse effect on RNAs stability and accelerates its degradation rate., Competing Interests: Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, “The persistence and stability of miRNA in bloodstained samples under different environmental conditions”, (Copyright © 2020 Elsevier B.V. All rights reserved.)

Glioblastoma is one of the most frequent and aggressive primary tumor of glial brain tumors. Long non-coding RNA Prostate cancer-associated ncRNA transcript 6 (PCAT6) has been identified to influence the progression of many cancers, but its expression and functions in glioblastoma remain unclear. In this study, we intended to investigate the expression, functions and the corresponding mechanisms of PCAT6 in glioblastoma. We observed that PCAT6 expression was upregulated in glioblastoma tissues and cell lines and its high expression was due to the transcriptional activation by Yin Yang 1. miR-513 was a target of PCAT6 and Insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) was a target of miR-513. Hence, PCAT6 upregulated IGF2BP1 expression via miR-513 in a competing endogenous RNAs manner. PCAT6 and IGF2BP1 functioned as oncogenes while miR-513 acted as a tumor suppressor gene in glioblastoma. PCAT6 and miR-513 modulated the proliferation and survival of glioblastoma cells via AKT signaling by mediating IGF2BP1. IGF2BP1 raised the expression of PCAT6 by increasing its stability. In conclusion, our results indicate that PCAT6/miR-513/IGF2BP1 positive feedback loop plays a crucial role in facilitating glioblastoma progression.

Decapping is the first committed step in 5'-to-3' RNA decay, and in the cytoplasm of human cells, multiple decapping enzymes regulate the stabilities of distinct subsets of cellular transcripts. However, the complete set of RNAs regulated by any individual decapping enzyme remains incompletely mapped, and no consensus sequence or property is currently known to unambiguously predict decapping enzyme substrates. Dcp2 was the first-identified and best-studied eukaryotic decapping enzyme, but it has been shown to regulate the stability of <400 transcripts in mammalian cells to date. Here, we globally profile changes in the stability of the human transcriptome in Dcp2 knockout cells via TimeLapse-seq. We find that P-body enrichment is the strongest correlate of Dcp2-dependent decay and that modification with m 6 A exhibits an additive effect with P-body enrichment for Dcp2 targeting. These results are consistent with a model in which P-bodies represent sites where translationally repressed transcripts are sorted for decay by soluble cytoplasmic decay complexes through additional molecular marks.

RNA turnover is a general process that maintains appropriate mRNA abundance at the posttranscriptional level. Although long thought to be antagonistic to translation, discovery of the 5' to 3' cotranslational mRNA decay pathway demonstrated that both processes are intertwined. Cotranslational mRNA decay globally shapes the transcriptome in different organisms and in response to stress; however, the dynamics of this process during plant development is poorly understood. In this study, we used a multiomics approach to reveal the global landscape of cotranslational mRNA decay during Arabidopsis ( Arabidopsis thaliana ) seedling development. We demonstrated that cotranslational mRNA decay is regulated by developmental cues. Using the EXORIBONUCLEASE4 (XRN4) loss-of-function mutant, we showed that XRN4 poly(A + ) mRNA targets are largely subject to cotranslational decay during plant development. As cotranslational mRNA decay is interconnected with translation, we also assessed its role in translation efficiency. We discovered that clusters of transcripts were specifically subjected to cotranslational decay in a developmental-dependent manner to modulate their translation efficiency. Our approach allowed the determination of a cotranslational decay efficiency that could be an alternative to other methods to assess transcript translation efficiency. Thus, our results demonstrate the prevalence of cotranslational mRNA decay in plant development and its role in translational control., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Maternal mRNA clearance is an essential process that occurs during maternal-to-zygotic transition (MZT). However, the dynamics, functional importance, and pathological relevance of maternal mRNA decay in human preimplantation embryos have not yet been analyzed. Here we report the zygotic genome activation (ZGA)-dependent and -independent maternal mRNA clearance processes during human MZT and demonstrate that subgroups of human maternal transcripts are sequentially removed by maternal (M)- and zygotic (Z)-decay pathways before and after ZGA. Key factors regulating M-decay and Z-decay pathways in mouse have similar expression pattern during human MZT, suggesting that YAP1-TEAD4 transcription activators, TUT4/7-mediated mRNA 3'-oligouridylation, and BTG4/CCR4-NOT-induced mRNA deadenylation may also be involved in the regulation of human maternal mRNA stability. Decreased expression of these factors and abnormal accumulation of maternal transcripts are observed in the development-arrested embryos of patients who seek assisted reproduction. Defects of M-decay and Z-decay are detected with high incidence in embryos that are arrested at the zygote and 8-cell stages, respectively. In addition, M-decay is not found to be affected by maternal TUBB8 mutations, although these mutations cause meiotic cell division defects and zygotic arrest, which indicates that mRNA decay is regulated independent of meiotic spindle assembly. Considering the correlations between maternal mRNA decay defects and early developmental arrest of in vitro fertilized human embryos, M-decay and Z-decay pathway activities may contribute to the developmental potential of human preimplantation embryos.

RNA decay is an important regulatory mechanism for gene expression at the posttranscriptional level. Although the main pathways and major enzymes that facilitate this process are well defined, global analysis of RNA turnover remains under-investigated. Recent advances in the application of next-generation sequencing technology enable its use in order to examine various RNA decay patterns at the genome-wide scale. In this study, we investigated human RNA decay patterns using parallel analysis of RNA end-sequencing (PARE-seq) data from XRN1 -knockdown HeLa cell lines, followed by a comparison of steady state and degraded mRNA levels from RNA-seq and PARE-seq data, respectively. The results revealed 1103 and 1347 transcripts classified as stable and unstable candidates, respectively. Of the unstable candidates, we found that a subset of the replication-dependent histone transcripts was polyadenylated and rapidly degraded. Additionally, we identified 380 endonucleolytically cleaved candidates by analyzing the most abundant PARE sequence on a transcript. Of these, 41.4% of genes were classified as unstable genes, which implied that their endonucleolytic cleavage might affect their mRNA stability. Furthermore, we identified 1877 decapped candidates, including HSP90B1 and SWI5, having the most abundant PARE sequences at the 5'-end positions of the transcripts. These results provide a useful resource for further analysis of RNA decay patterns in human cells.

Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway that is important for the elimination of faulty, and the regulation of normal, mRNAs. The molecular details of the early steps in NMD are not fully understood but previous work suggests that NMD activation occurs as a consequence of ribosome stalling at the termination codon (TC). To test this hypothesis, we established an in vitro translation-coupled toeprinting assay based on lysates from human cells that allows monitoring of ribosome occupancy at the TC of reporter mRNAs. In contrast to the prevailing NMD model, our in vitro system reveals similar ribosomal occupancy at the stop codons of NMD-sensitive and NMD-insensitive reporter mRNAs. Moreover, ribosome profiling reveals a similar density of ribosomes at the TC of endogenous NMD-sensitive and NMD-insensitive mRNAs in vivo. Together, these data show that NMD activation is not accompanied by stable stalling of ribosomes at TCs.

Transfer RNAs (tRNA) are quintessential in deciphering the genetic code; disseminating nucleic acid triplets into correct amino acid identity. While this decoding function is clear, an emerging theme is that tRNA abundance and functionality can powerfully impact protein production rate, folding, activity, and messenger RNA stability. Importantly, however, the expression pattern of tRNAs is obliquely known. Here we present Quantitative Mature tRNA sequencing (QuantM-tRNA seq), a technique to monitor tRNA abundance and sequence variants secondary to RNA modifications. With QuantM-tRNA seq, we assess the tRNA transcriptome in mammalian tissues. We observe dramatic distinctions in isodecoder expression and known tRNA modifications between tissues. Remarkably, despite dramatic changes in tRNA isodecoder gene expression, the overall anticodon pool of each tRNA family is similar across tissues. These findings suggest that while anticodon pools appear to be buffered via an unknown mechanism, underlying transcriptomic and epitranscriptomic differences suggest a more complex tRNA regulatory landscape.

The N 6 -methyladenosine (m 6 A) RNA modification is essential during embryonic development of various organs. However, its role in embryonic and early postnatal islet development remains unknown. Mice in which RNA methyltransferase-like 3/14 (Mettl3/14) were deleted in Ngn3 + endocrine progenitors ( Mettl3/14 nKO ) developed hyperglycemia and hypoinsulinemia at 2 weeks after birth. We found that Mettl3/14 specifically regulated both functional maturation and mass expansion of neonatal β-cells before weaning. Transcriptome and m 6 A methylome analyses provided m 6 A-dependent mechanisms in regulating cell identity, insulin secretion, and proliferation in neonatal β-cells. Importantly, we found that Mettl3/14 were dispensable for β-cell differentiation but directly regulated essential transcription factor MafA expression at least partially via modulating its mRNA stability. Failure to maintain this modification impacted the ability to fulfill β-cell functional maturity. In both diabetic db/db mice and patients with type 2 diabetes (T2D), decreased Mettl3/14 expression in β-cells was observed, suggesting its possible role in T2D. Our study unraveled the essential role of Mettl3/14 in neonatal β-cell development and functional maturation, both of which determined functional β-cell mass and glycemic control in adulthood., (© 2020 by the American Diabetes Association.)

Many cellular noncoding RNAs contain chemically modified nucleotides that are essential for their function. The Epstein-Barr virus expresses two highly abundant noncoding RNAs called EBV-encoded RNA 1 (EBER1) and EBER2. To examine whether these viral RNAs contain modified nucleotides, we purified native EBERs from EBV-infected cells and performed mass spectrometry analysis. While EBER2 contains no modified nucleotides at stoichiometric amounts, EBER1 was found to carry 5-methylcytosine (m 5 C) modification. Bisulfite sequencing indicated that a single cytosine of EBER1 is methylated in ∼95% of molecules, and the RNA methyltransferase NSUN2 was identified as the EBER1-specific writer. Intriguingly, ablation of NSUN2 and thus loss of m 5 C modification resulted in an increase in EBER1 levels. We further found that EBER1 is a substrate for the RNase Angiogenin and cleavage in vivo is dependent on the presence of m 5 C, providing an explanation as to why loss of m 5 C increases EBER1 levels. Taken together, our observations indicate that m 5 C, a modification previously shown for tRNAs to oppose Angiogenin-mediated degradation, can also adversely affect RNA stability., (© 2020 Henry et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Vertebrate segmentation is regulated by the segmentation clock, a biological oscillator that controls periodic formation of somites, or embryonic segments, which give rise to many mesodermal tissue types. This molecular oscillator generates cyclic gene expression with the same periodicity as somite formation in the presomitic mesoderm (PSM), an area of mesenchymal cells that give rise to mature somites. Molecular components of the clock include the Hes/her family of genes that encode transcriptional repressors, but additional genes cycle. Cyclic gene transcripts are cleared rapidly, and clearance depends upon the pnrc2 (proline-rich nuclear receptor co-activator 2) gene that encodes an mRNA decay adaptor. Previously, we showed that the her1 3'UTR confers instability to otherwise stable transcripts in a Pnrc2-dependent manner, however, the molecular mechanism(s) by which cyclic gene transcripts are cleared remained largely unknown. To identify features of the her1 3'UTR that are critical for Pnrc2-mediated decay, we developed an array of transgenic inducible reporter lines carrying different regions of the 3'UTR. We find that the terminal 179 nucleotides (nts) of the her1 3'UTR are necessary and sufficient to confer rapid instability. Additionally, we show that the 3'UTR of another cyclic gene, deltaC (dlc), also confers Pnrc2-dependent instability. Motif analysis reveals that both her1 and dlc 3'UTRs contain terminally-located Pumilio response elements (PREs) and AU-rich elements (AREs), and we show that the PRE and ARE in the last 179 ​nts of the her1 3'UTR drive rapid turnover of reporter mRNA. Finally, we show that mutation of Pnrc2 residues and domains that are known to facilitate interaction of human PNRC2 with decay factors DCP1A and UPF1 reduce the ability of Pnrc2 to restore normal cyclic gene expression in pnrc2 mutant embryos. Our findings suggest that Pnrc2 interacts with decay machinery components and cooperates with Pumilio (Pum) proteins and ARE-binding proteins to promote rapid turnover of cyclic gene transcripts during somitogenesis., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In human cells, the expression of ∼1,000 genes is modulated throughout the cell cycle. Although some of these genes are controlled by specific transcriptional programs, very little is known about their post-transcriptional regulation. Here, we analyze the expression signature associated with all 687 RNA-binding proteins (RBPs) and identify 39 that significantly correlate with cell cycle mRNAs. We find that NF45 and NF90 play essential roles in mitosis, and transcriptome analysis reveals that they are necessary for the expression of a subset of mitotic mRNAs. Using proteomics, we identify protein clusters associated with the NF45-NF90 complex, including components of Staufen-mediated mRNA decay (SMD). We show that depletion of SMD components increases the binding of mitotic mRNAs to the NF45-NF90 complex and rescues cells from mitotic defects. Together, our results indicate that the NF45-NF90 complex plays essential roles in mitosis by competing with the SMD machinery for a common set of mRNAs., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Eukaryotic 5'-3' mRNA decay plays important roles during development and in response to stress, regulating gene expression post-transcriptionally. In Caenorhabditis elegans , deficiency of DCAP-1/DCP1, the essential co-factor of the major cytoplasmic mRNA decapping enzyme, impacts normal development, stress survival and ageing. Here, we show that overexpression of dcap-1 in neurons of worms is sufficient to increase lifespan through the function of the insulin/IGF-like signaling and its effector DAF-16/FOXO transcription factor. Neuronal DCAP-1 affects basal levels of INS-7, an ageing-related insulin-like peptide, which acts in the intestine to determine lifespan. Short-lived dcap-1 mutants exhibit a neurosecretion-dependent upregulation of intestinal ins-7 transcription, and diminished nuclear localization of DAF-16/FOXO. Moreover, neuronal overexpression of DCP1 in Drosophila melanogaster confers longevity in adults, while neuronal DCP1 deficiency shortens lifespan and affects wing morphogenesis, cell non-autonomously. Our genetic analysis in two model-organisms suggests a critical and conserved function of DCAP-1/DCP1 in developmental events and lifespan modulation., Competing Interests: FB, JR, GK, NK, AK, CV, KV, GD, DS, CZ, PS No competing interests declared, (© 2020, Borbolis et al.)

In Drosophila, the deposition of the germ plasm at the posterior pole of the oocyte is essential for the abdomen and germ cell formation during embryogenesis. To assemble the germ plasm, oskar (osk) mRNA, produced by nurse cells, should be localized and anchored on the posterior cortex of the oocyte. Processing bodies (P-bodies) are cytoplasmic RNA granules responsible for the 5'-3' mRNA degradation. Evidence suggests that the components of P-bodies, such as Drosophila decapping protein 1 and Ge-1, are involved in the posterior localization of osk. However, whether the decapping core enzyme, Drosophila decapping protein 2 (dDcp2), is also involved remains unclear. Herein, we generated a dDcp2 null allele and showed that dDcp2 was required for the posterior localization of germ plasm components including osk. dDcp2 was distributed on the oocyte cortex and was localized posterior to the osk. In the posterior pole of dDcp2 mutant oocytes, osk was mislocalized and colocalized with F-actin detached from the cortex; moreover, considerably fewer F-actin projections were observed. Using the F-actin cosedimentation assay, we proved that dDcp2 interacted with F-actin through its middle region. In conclusion, our findings explored a novel function of dDcp2 in assisting osk localization by modulating the formation of F-actin projections on the posterior cortex., (Copyright © 2020 Elsevier Inc. All rights reserved.)

IL-23 and IL-12, two structurally related heterodimeric cytokines sharing a common subunit, divergently promote Th cell development and expansion. Both cytokines have been implicated in the pathogenesis of thyroid-associated ophthalmopathy (TAO), an autoimmune component of Graves disease. In TAO, CD34 + fibrocytes, putatively derived from bone marrow, can be identified in the orbit. There they masquerade as CD34 + orbital fibroblasts (OF) (CD34 + OF) and cohabitate with CD34 - OF in a mixed fibroblast population (GD-OF). Slit2, a neural axon repellent, is expressed and released by CD34 - OF and dampens the inflammatory phenotype of fibrocytes and CD34 + OF. In this study we report that thyrotropin (TSH) and the pathogenic, GD-specific monoclonal autoantibody, M22, robustly induce IL-23 in human fibrocytes; however, IL-12 expression is essentially undetectable in these cells under basal conditions or following TSH-stimulation. In contrast, IL-12 is considerably more inducible in GD-OF, cells failing to express IL-23. This divergent expression and induction of cytokines appears to result from cell type-specific regulation of both gene transcription and mRNA stabilities. It appears that the JNK pathway activity divergently attenuates IL-23p19 expression while enhancing that of IL-12p35. The shift from IL-23p19 expression in fibrocytes to that of IL-12p35 in their derivative CD34 + OF results from the actions of Slit2. Thus, Slit2 might represent a molecular determinant of balance between IL-23 and IL-12 expression, potentially governing immune responses in TAO., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Carbon catabolite repression 4 (CCR4) is a conserved mRNA deadenylase regulating posttranscriptional gene expression. However, regulation of CCR4 in virus infections is less understood. Here, we characterized a pro-viral role of CCR4 in replication of a plant cytorhabdovirus , Barley yellow striate mosaic virus (BYSMV). The barley ( Hordeum vulgare ) CCR4 protein (HvCCR4) was identified to interact with the BYSMV phosphoprotein (P). The BYSMV P protein recruited HvCCR4 from processing bodies (PBs) into viroplasm-like bodies. Overexpression of HvCCR4 promoted BYSMV replication in plants. Conversely, knockdown of the small brown planthopper CCR4 inhibited viral accumulation in the insect vector. Biochemistry experiments revealed that HvCCR4 was recruited into N-RNA complexes by the BYSMV P protein and triggered turnover of N-bound cellular mRNAs, thereby releasing RNA-free N protein to bind viral genomic RNA for optimal viral replication. Our results demonstrate that the co-opted CCR4-mediated RNA decay facilitates cytorhabdovirus replication in plants and insects., Competing Interests: ZZ, QG, XF, ZD, DG, WX, QC, JQ, YY, CH, YW, XY, DL, XW No competing interests declared, (© 2020, Zhang et al.)

In animals, miRNAs are the most prevalent small non-coding RNA molecules controlling posttranscriptional gene regulation. The Argonaute proteins (AGO) mediate miRNA-guided gene silencing by recruiting multiple factors involved in translational repression, deadenylation, and decapping. Here, we report that CSDE1, an RNA-binding protein linked to stem cell maintenance and metastasis in cancer, interacts with AGO2 within miRNA-induced silencing complex and mediates gene silencing through its N-terminal domains. We show that CSDE1 interacts with LSM14A, a constituent of P-body assembly and further associates to the DCP1-DCP2 decapping complex, suggesting that CSDE1 could promote the decay of miRNA-induced silencing complex-targeted mRNAs. Together, our findings uncover a hitherto unknown mechanism used by CSDE1 in the control of gene expression mediated by the miRNA pathway., (© 2020 Kakumani et al.)

Lower cultivation temperature dramatically affects cell growth and cellular productivity in Chinese hamster ovary cells (CHO) and is often used in industrial applications with the aim to enhance productivity. Cold-inducible proteins whose activity is induced at lower temperature play an important role in understanding the mechanisms of cold-induced changes in gene expression. One of these mechanisms is increased transcription of specific target genes controlled by sequence elements in cold-inducible promoters. This study provides a list of cold-inducible genes and endogenous cold-inducible promoters of CHO cells. Transcriptome data before and after a temperature shift from 37 to 33 °C are analyzed to identify 94 cold-inducible genes, which are highly expressed and have a high fold change in expression after the temperature shift. Cold-inducible promoters are identified from the top ten cold-inducible genes, showing up to 11-fold increased luciferase expression at lowered temperature in transient transfections. Additionally, several common transcription factor binding sites are identified in the top cold-inducible promoter sequences and expression of their corresponding transcription factors over temperature-shift cultivation is evaluated. These may be responsible for enhanced promoter activity under lower temperature and can be used as engineering targets., (© 2019 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hepatic stellate cells (HSCs) are the main effectors for various types of hepatic fibrosis, including Schistosome-induced hepatic fibrosis. Multiple inflammatory cytokines/chemokines, such as transforming growth factor-β1 (TGF-β1), activate HSCs, and contribute to the development of hepatic fibrosis. MicroRNAs regulate gene expression at the posttranscriptional level and are involved in regulation of inflammatory cytokine/chemokine synthesis. In this study, we showed that soluble egg antigen (SEA) stimulation and Schistosoma japonicum infection downregulate miR-27b expression and increase KH-type splicing regulatory protein (KSRP) mRNA and protein levels in vitro and in vivo. miR-27b regulates the stabilization of TGF-β1 mRNA through targeting KSRP by interacting with their AU-rich elements in hepatocytes and non-parenchymal cells, which has an effect on the activation of HSCs. Importantly, our results have shown that either knockdown miR-27b or overexpression of KSRP attenuates S. japonicum-induced hepatic fibrosis in vivo. Therefore, our study highlights the crucial role of miR-27b and KSRP in the negative regulation of immune reactions in hepatocyte and non-parenchymal cells in response to SEA stimulation and S. japonicum infection. It reveals that manipulation of miR-27b or KSRP might be a useful strategy not only for treating Schistosome-induced hepatic fibrosis but also for curing hepatic fibrosis in general., (© 2020 Federation of American Societies for Experimental Biology.)

The role of circular RNAs (circRNAs) as biomarkers remains poorly characterized. Here, we investigated the performance of the circRNA hsa&#95;circ&#95;0001445 as a biomarker of coronary artery disease (CAD) in a real-world clinical practice setting. Plasma hsa&#95;circ&#95;0001445 was measured in a study population of 200 consecutive patients with suspected stable CAD who had undergone coronary computed tomographic angiography (CTA). Multivariable logistic models were constructed combining conventional risk factors with established biomarkers and hsa&#95;circ&#95;0001445. Model robustness was internally validated by the bootstrap technique. Biomarker accuracy was evaluated using the C-index. The integrated discrimination improvement (IDI) and net reclassification improvement (NRI) were also calculated. Risk groups were developed via classification tree models. The stability of plasma hsa&#95;circ&#95;0001445 was evaluated under different clinical conditions. hsa&#95;circ&#95;0001445 levels were associated with higher coronary atherosclerosis extent and severity with a 2-fold increase across tertiles (28.4%-50.0%). Levels of hsa&#95;circ&#95;0001445 were proportional to coronary atherosclerotic burden, even after comprehensive adjustment for cardiovascular risk factors, medications, and established biomarkers (fully adjusted OR = 0.432 for hsa&#95;circ&#95;0001445 as a continuous variable and fully adjusted OR = 0.277 for hsa&#95;circ&#95;0001445 as a binary variable). The classification of patients was improved with the incorporation of hsa&#95;circ&#95;0001445 into a base clinical model (CM) composed of conventional cardiovascular risk factors, showing an IDI of 0.047 and NRI of 0.482 for hsa&#95;circ&#95;0001445 as a continuous variable and an IDI of 0.056 and NRI of 0.373 for hsa&#95;circ&#95;0001445 as a binary variable. A trend toward higher discrimination capacity was also observed (C-index CM  = 0.833, C-index CM+continuous hsa&#95;circ&#95;0001445  = 0.856 and C-index CM+binary hsa&#95;circ&#95;0001445  = 0.855). Detailed analysis of stability showed that hsa&#95;circ&#95;0001445 was present in plasma in a remarkably stable form. In vitro, hsa&#95;circ&#95;0001445 was downregulated in extracellular vesicles secreted by human coronary smooth muscle cells upon exposure to atherogenic conditions. In patients with suspected stable CAD referred for coronary CTA, plasma hsa&#95;circ&#95;0001445 improves the identification of coronary artery atherosclerosis., (© 2020 Federation of American Societies for Experimental Biology.)

Much of the mammalian genome is transcribed, generating long non-coding RNAs (lncRNAs) that can undergo post-transcriptional surveillance whereby only a subset of the non-coding transcripts is allowed to attain sufficient stability to persist in the cellular milieu and control various cellular functions. Paralleling protein turnover by the proteasome complex, lncRNAs are also likely to exist in a dynamic equilibrium that is maintained through constant monitoring by the RNA surveillance machinery. In this Review, we describe the RNA surveillance factors and discuss the vital role of lncRNA surveillance in orchestrating various biological processes, including the protection of genome integrity, maintenance of pluripotency of embryonic stem cells, antibody-gene diversification, coordination of immune cell activation and regulation of heterochromatin formation. We also discuss examples of human diseases and developmental defects associated with the failure of RNA surveillance mechanisms, further highlighting the importance of lncRNA surveillance in maintaining cell and organism functions and health.

Metabolic turnover of mRNA is fundamental to the control of gene expression in all organisms, notably in fast-adapting prokaryotes. In many bacteria, RNase Y initiates global mRNA decay via an endonucleolytic cleavage, as shown in the Gram-positive model organism Bacillus subtilis This enzyme is tethered to the inner cell membrane, a pseudocompartmentalization coherent with its task of initiating mRNA cleavage/maturation of mRNAs that are translated at the cell periphery. Here, we used total internal reflection fluorescence microscopy (TIRFm) and single-particle tracking (SPT) to visualize RNase Y and analyze its distribution and dynamics in living cells. We find that RNase Y diffuses rapidly at the membrane in the form of dynamic short-lived foci. Unlike RNase E, the major decay-initiating RNase in Escherichia coli , the formation of foci is not dependent on the presence of RNA substrates. On the contrary, RNase Y foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. The Y-complex of three proteins (YaaT, YlbF, and YmcA) has previously been shown to play an important role for RNase Y activity in vivo We demonstrate that Y-complex mutations have an effect similar to but much stronger than that of depletion of RNA in increasing the number and size of RNase Y foci at the membrane. Our data suggest that the Y-complex shifts the assembly status of RNase Y toward fewer and smaller complexes, thereby increasing cleavage efficiency of complex substrates like polycistronic mRNAs. IMPORTANCE All living organisms must degrade mRNA to adapt gene expression to changing environments. In bacteria, initiation of mRNA decay generally occurs through an endonucleolytic cleavage. In the Gram-positive model organism Bacillus subtilis and probably many other bacteria, the key enzyme for this task is RNase Y, which is anchored at the inner cell membrane. While this pseudocompartmentalization appears coherent with translation occurring primarily at the cell periphery, our knowledge on the distribution and dynamics of RNase Y in living cells is very scarce. Here, we show that RNase Y moves rapidly along the membrane in the form of dynamic short-lived foci. These foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. This contrasts with RNase E, the major decay-initiating RNase in E. coli , where it was shown that formation of foci is dependent on the presence of RNA substrates. We also show that a protein complex (Y-complex) known to influence the specificity of RNase Y activity in vivo is capable of shifting the assembly status of RNase Y toward fewer and smaller complexes. This highlights fundamental differences between RNase E- and RNase Y-based degradation machineries., (Copyright © 2020 Hamouche et al.)

K-homology (KH)-type splicing regulatory protein (KHSRP) is an RNA binding protein that participates in RNA variable splicing and stability, and facilitates the biogenesis of miRNAs that target mRNA. However, to date, the role of KHSRP in colorectal cancer (CRC) progression has not been reported. In this study, the function of KHSRP in CRC proliferation and 5-fluoruracil (5-FU) resistance was investigated. The upregulation of KHSRP expression was confirmed in CRC patient tissues and two CRC cell lines. Manipulating KHSRP expression altered cell proliferation and 5-FU resistance in CRC cells. ERRFI1, a downstream effector of KHSRP in CRC cells, reduced CRC cell proliferation. Sensitivity to 5-FU mediated by KHSRP knockdown was reversed by ERRFI1 knockdown. We found that KHSRP decreased ERRFI1 mRNA expression indirectly. By screening KHSRP-regulated miRNAs, we further found that miR-501-5p directly combines with KHSRP in CRC cells. Mechanistically, the results of a luciferase assay suggested that miR-501-5p directly binds to the ERRFI1 3'-untranslated region. Taken together, our data indicated that modification of ERRFI1 by KHSRP occurs through miR-501-5p, an essential mechanism driving CRC proliferation and 5-FU resistance. Insight into this mechanism may provide novel targets for overcoming drug resistance in CRC., (© 2019 Wiley Periodicals, Inc.)

The RNA exosome processes a wide variety of RNA and mediates RNA maturation, quality control and decay. In marked contrast to its high processivity in vivo, the purified exosome exhibits only weak activity on RNA substrates in vitro. Its activity is regulated by several auxiliary proteins, and protein complexes. In budding yeast, the activity of exosome is enhanced by the polyadenylation complex referred to as TRAMP. TRAMP oligoadenylates precursors and aberrant forms of RNAs to promote their trimming or complete degradation by exosomes. This chapter provides protocols for the purification of TRAMP and exosome complexes from yeast and the in vitro evaluation of exosome activation by the TRAMP complex. The protocols can be used for different purposes, such as the assessment of the role of individual subunits, protein domains or particular mutations in TRAMP-exosome RNA processing in vitro.