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Tumor angiogenesis is a crucial step in the further growth and metastasis of solid tumors. However, its regulatory mechanism remains unclear. Here, we showed that TARBP2, an RNA‐binding protein, played a role in promoting tumor‐induced angiogenesis both in vitro and in vivo through degrading the mRNAs of antiangiogenic factors, including thrombospondin1/2 (THBS1/2), tissue inhibitor of metalloproteinases 1 (TIMP1), and serpin family F member 1 (SERPINF1), by targeting their 3′untranslated regions (3′UTRs). Overexpression of TARBP2 promotes tumor cell–induced angiogenesis, while its knockdown inhibits tumor angiogenesis. Clinical cohort analysis revealed that high expression level of TARBP2 was associated with poor survival of lung cancer and breast cancer patients. Mechanistically, TARBP2 physically interacts with the stem‐loop structure located in the 3′UTR of antiangiogenic transcripts, leading to mRNA destabilization by the dsRNA‐binding domains 1/2 (dsRBDs1/2). Notably, the expression level of TARBP2 in human tumor tissue is negatively correlated with the expression of antiangiogenic factors, including THBS1/2, and brain‐specific angiogenesis inhibitor 1 (BAI1). Moreover, TARBP2 expression is strongly associated with tumor angiogenesis in a group of human lung cancer samples. Collectively, our results highlight that TARBP2 is a novel tumor angiogenesis regulator that could promote tumor angiogenesis by selectively downregulating antiangiogenic gene expression., We demonstrated that TARBP2 was able to promote tumor angiogenesis through selectively destabilizing the mRNAs of antiangiogenic factor genes via binding to the stem‐loop structure located in the 3′UTRs. Strikingly, our basic research findings have also been confirmed by analyzing the clinical samples and data from human breast and lung cancers. Thus, TARBP2 is likely to be a useful molecular target for antiangiogenic tumor therapy.

RecQ-mediated genome instability protein 1 (RMI1) is an important component of the BLM-Topo IIIα-RMI1-RMI2 complex and plays a critical role in maintaining genome stability. However, the cellular functions of RMI1 in response to ionizing radiation (IR) are poorly understood. In this study, we found that RMI1 knockdown led to enhanced radiosensitivity and apoptosis after irradiation. To analyze the effect of RMI1 knockdown on the expression of circular RNAs (circRNAs), we performed high-throughput RNA sequencing on four groups of human embryonic kidney (HEK) 293T cells: control cells and RMI1 knockdown cells with or without IR exposure. A total of 179 and 160 differentially expressed circRNAs (DE-circRNAs) were identified under RMI1 knockdown without and with exposure to IR, respectively. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that these DE-circRNAs were involved in a variety of functions and signal pathways, including histone H3-K36 methylation, nuclear pore organization, mRNA destabilization, the mismatch repair pathway, and the apoptotic signaling pathway. Overall, our results indicate that RMI1 plays a crucial role in the response to IR and, more generally, that circRNAs are important in the regulatory mechanism of the radiation response.

Impaired insulin secretion from the pancreatic β-cells is central in the pathogenesis of type 2 diabetes (T2D), and microRNAs (miRNAs) are fundamental regulatory factors in this process. Differential expression of miRNAs contributes to β-cell adaptation to compensate for increased insulin resistance, but deregulation of miRNA expression can also directly cause β-cell impairment during the development of T2D. miRNAs are small noncoding RNAs that posttranscriptionally reduce gene expression through translational inhibition or mRNA destabilization. The nature of miRNA targeting implies the presence of complex and large miRNA–mRNA regulatory networks in every cell, including the insulin-secreting β-cell. Here we exemplify one such network using our own data on differential miRNA expression in the islets of T2D Goto-Kakizaki rat model. Several biological processes are influenced by multiple miRNAs in the β-cell, but so far most studies have focused on dissecting the mechanism of action of individual miRNAs. In this Perspective we present key islet miRNA families involved in T2D pathogenesis including miR-200, miR-7, miR-184, miR-212/miR-132, and miR-130a/b/miR-152. Finally, we highlight four challenges and opportunities within islet miRNA research, ending with a discussion on how miRNAs can be utilized as therapeutic targets contributing to personalized T2D treatment strategies.

AU-rich elements (AREs) are 3′ UTR cis-regulatory elements that regulate the stability of mRNAs. Consensus ARE motifs have been determined, but little is known about how differences in 3′ UTR sequences that conform to these motifs affect their function. Here we use functional annotation of sequences from 3′ UTRs (fast-UTR), a massively parallel reporter assay (MPRA), to investigate the effects of 41,288 3′ UTR sequence fragments from 4,653 transcripts on gene expression and mRNA stability. The library included 9,142 AREs, and incorporated a set of fragments bearing mutations in each ARE. Our analyses demonstrate that the length of an ARE and its registration (the first and last nucleotides of the repeating ARE motif) have significant effects on gene expression and stability. Based on this finding, we propose improved ARE classification and concomitant methods to categorize and predict the effect of AREs on gene expression and stability. Our new approach explains 64±13% of the contribution of AREs to the stability of human 3′ UTRs in Jurkat cells and predicts ARE activity in an unrelated cell type. Finally, to investigate the advantages of our general experimental design for annotating 3′ UTR elements we examine other motifs including constitutive decay elements (CDEs), where we show that the length of the CDE stem-loop has a significant impact on steady-state expression and mRNA stability. We conclude that fast-UTR, in conjunction with our analytical approach, can produce improved yet simple sequence-based rules for predicting the activity of human 3′ UTRs containing functional motifs.

MicroRNAs (miRNAs) are small (∼21-nucleotides) non-coding RNAs found in plant and animals. MiRNAs function as critical post-transcriptional regulators of gene expression by binding to complementary sequences in their target mRNAs, leading to mRNA destabilization and translational inhibition. Plant miRNAs have some distinct characteristics compared to their animal counterparts, including greater evolutionary conservation and unique miRNA processing methods. The lifecycle of a plant begins with embryogenesis and progresses through seed germination, vegetative growth, reproductive growth, flowering and fruiting, and finally senescence and death. MiRNAs participate in the transformation of plant growth and development and directly monitor progression of these processes and the expression of certain morphological characteristics by regulating transcription factor genes involved in cell growth and differentiation. In woody plants, a large and rapidly increasing number of miRNAs have been identified, but their biological functions are largely unknown. In this review, we summarize the progress of miRNA research in woody plants to date. In particular, we discuss the potential roles of these miRNAs in growth, development, and biotic and abiotic stresses responses in woody plants.

Gene expression strategies ensuring bacterial survival and competitiveness rely on cis- and trans-acting RNA-regulators (riboregulators). Among the cis-acting riboregulators are transcriptional and translational attenuators, and antisense RNAs (asRNAs). The trans-acting riboregulators are small RNAs (sRNAs) that bind proteins or base pairs with other RNAs. This classification is artificial since some regulatory RNAs act both in cis and in trans, or function in addition as small mRNAs. A prominent example is the archetypical, ribosome-dependent attenuator of tryptophan (Trp) biosynthesis genes. It responds by transcription attenuation to two signals, Trp availability and inhibition of translation, and gives rise to two trans-acting products, the attenuator sRNA rnTrpL and the leader peptide peTrpL. In Escherichia coli, rnTrpL links Trp availability to initiation of chromosome replication and in Sinorhizobium meliloti, it coordinates regulation of split tryptophan biosynthesis operons. Furthermore, in S. meliloti, peTrpL is involved in mRNA destabilization in response to antibiotic exposure. It forms two types of asRNA-containing, antibiotic-dependent ribonucleoprotein complexes (ARNPs), one of them changing the target specificity of rnTrpL. The posttranscriptional role of peTrpL indicates two emerging paradigms: (1) sRNA reprograming by small molecules and (2) direct involvement of antibiotics in regulatory RNPs. They broaden our view on RNA-based mechanisms and may inspire new approaches for studying, detecting, and using antibacterial compounds. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

Most targeted cancer therapies fail to achieve complete tumor regressions or attain durable remissions. To understand why these treatments fail to induce robust cytotoxic responses despite appropriately targeting oncogenic drivers, here we systematically interrogated the dependence of cancer cells on the BCL-2 family of apoptotic proteins after drug treatment. We observe that multiple targeted therapies, including BRAF or EGFR inhibitors, rapidly deplete the pro-apoptotic factor NOXA, thus creating a dependence on the anti-apoptotic protein MCL-1. This adaptation requires a pathway leading to destabilization of the NOXA mRNA transcript. We find that interruption of this mechanism of anti-apoptotic adaptive resistance dramatically increases cytotoxic responses in cell lines and a murine melanoma model. These results identify NOXA mRNA destabilization/MCL-1 adaptation as a non-genomic mechanism that limits apoptotic responses, suggesting that sequencing of MCL-1 inhibitors with targeted therapies could overcome such widespread and clinically important resistance., MAPK-targeted therapies fail to achieve complete remission. Here, the authors show that anti-apoptosis resistance is acquired in these targeted therapies through the mRNA destabilization of NOXA which leads to dependence on MCL-1, and that sequential combination of MCL-1 inhibition with targeted therapies overcomes this resistance.

Long non-coding RNAs (lncRNAs) play important roles in tumorigenesis and cancer progression. The orphan nuclear receptor subfamily 4 group A member 1 (NR4A1) acts as an oncogene, and is involved in colorectal cancer (CRC) development. However, the mechanism through which lncRNA regulates NR4A1 expression remains unknown. We aimed to identify lncRNAs that regulate NR4A1 and assess their underlying mechanisms in CRC. We first identified an antisense lncRNA of NR4A1 that was up-regulated in CRC tissues and cells with rapid amplification of cDNA ends (RACE), and designated it as NR4A1AS. Spearman correlation analysis showed that NR4A1AS was positively correlated with NR4A1 mRNA levels in 37 CRC tissues. Mechanistically, NR4A1AS stabilized NR4A1 mRNA by forming RNA–RNA complexes via partial base-pairing and up-regulated NR4A1 expression in CRC cells. RNA immunoprecipitation (RIP) assays revealed that knockdown of NR4A1AS expression by siRNA enhanced up-frameshift 1 (UPF1) recruitment to NR4A1 mRNA, thereby decreasing NR4A1 mRNA stability. Moreover, depletion of NR4A1AS was found to mimic the effect of NR4A1 knockdown, specifically by suppressing cell proliferation, migration and invasion, and inducing apoptosis and cell cycle arrest. Accordingly, restoring NR4A1 expression ameliorated the effects of NR4A1AS knockdown on tumor growth and metastasis of CRC cells in vitro and in vivo. Thus, we conclude that NR4A1AS up-regulates NR4A1 expression by forming RNA–RNA complexes and blocking UPF1-mediated mRNA destabilization, and it functions in tumor growth and metastasis of CRC cells at least partly through regulating NR4A1, suggesting that NR4A1AS might be as a potential target for RNA-based anti-CRC drug studies.

Compared to coding sequences, untranslated regions of the transcriptome are not well conserved, and functional annotation of these sequences is challenging. Global relationships between nucleotide composition of 3′ UTR sequences and their sequence conservation have been appreciated since mammalian genomes were first sequenced, but the functional relevance of these patterns remain unknown. We systematically measured the effect on gene expression of the sequences of more than 25,000 RNA-binding protein (RBP) binding sites in primary mouse T cells using a massively parallel reporter assay. GC-rich sequences were destabilizing of reporter mRNAs and come from more rapidly evolving regions of the genome. These sequences were more likely to be folded in vivo and contain a number of structural motifs that reduced accumulation of a heterologous reporter protein. Comparison of full-length 3′ UTR sequences across vertebrate phylogeny revealed that strictly conserved 3′ UTRs were GC-poor and enriched in genes associated with organismal development. In contrast, rapidly evolving 3′ UTRs tended to be GC-rich and derived from genes involved in metabolism and immune responses. Cell-essential genes had lower GC content in their 3′ UTRs, suggesting a connection between unstructured mRNA noncoding sequences and optimal protein production. By reducing gene expression, GC-rich RBP-occupied sequences act as a rapidly evolving substrate for gene regulatory interactions.

Vascular calcification is strongly associated with increased cardiovascular mortality and morbidity. C1q/TNF-related protein-13 (CTRP13) is a secreted adipokine that plays important roles in the cardiovascular system. However, the functional role of CTRP13 in the development of vascular calcification has yet to be explored. In this study, we collected blood samples from patients with chronic renal failure (CRF) and from rats with adenine-induced CRF. We found that the serum CTRP13 levels were decreased in patients and rats with CRF and were negatively associated with calcium deposition in the abdominal aorta. Compared to those of the controls, ectopic CTRP13 treatment significantly attenuated the calcium accumulation and alkaline phosphatase activity in the abdominal aorta of CRF rats, and β-glycerophosphate induced the formation of arterial rings and of vascular smooth muscle cells (VSMCs) and decreased the number of VSMCs that transitioned from a contractile to an osteogenic phenotype. The overexpression of Runx2 blocked CTRP13-reduced VSMC calcification. Mechanistically, CTRP13 repressed the phosphorylation of tristetraprolin (TTP), thereby activating TTP and increasing the TTP binding to the 3'untranslated region of the Runx2 mRNA, accelerating the Runx2 mRNA destabilization and degradation. In summary, these findings reveal that CTRP13 regulation is a novel method for the prevention of vascular calcification, representing a novel mechanism of the regulation of Runx2 expression in VSMCs.-Li, Y., Wang, W., Chao, Y., Zhang, F., Wang, C. CTRP13 attenuates vascular calcification by regulating Runx2.

Key message Here we demonstrate that the APUM9 RNA-binding protein and its co-factors play a role in mRNA destabilization and how this activity might regulate early plant development. Abstract APUM9 is a conserved PUF RNA-binding protein (RBP) under complex transcriptional control mediated by a transposable element (TE) that restricts its expression in Arabidopsis. Currently, little is known about the functional and mechanistic details of the plant PUF regulatory system and the biological relevance of the TE-mediated repression of APUM9 in plant development and stress responses. By combining a range of transient assays, we show here, that APUM9 binding to target transcripts can trigger their rapid decay via its conserved C-terminal RNA-binding domain. APUM9 directly interacts with DCP2, the catalytic subunit of the decapping complex and DCP2 overexpression induces rapid decay of APUM9 targeted mRNAs. We show that APUM9 negatively regulates the expression of ABA signaling genes during seed imbibition, and thereby might contribute to the switch from dormant stage to seed germination. By contrast, strong TE-mediated repression of APUM9 is important for normal plant growth in the later developmental stages. Finally, APUM9 overexpression plants show slightly enhanced heat tolerance suggesting that TE-mediated control of APUM9, might have a role not only in embryonic development, but also in plant adaptation to heat stress conditions. Electronic supplementary material The online version of this article (10.1007/s11103-019-00853-7) contains supplementary material, which is available to authorized users.

Background Tristetraprolin (TTP) family proteins contain conserved tandem CCCH zinc-finger binding to AU-rich elements and C-terminal NOT1-binding domain. TTP is phosphorylated extensively in cells, and its mRNA destabilization activity is regulated by protein phosphorylation. Methods We generated an antibody against phospho-Serine316 located at the C-terminal NOT1-binding site and examined TTP phosphorylation in LPS-stimulated RAW264.7 cells. Knockout of TTP was created in RAW264.7 cells using CRISPR/Cas9 gene editing to explore TTP functions. Results We demonstrated that Ser316 was phosphorylated by p90 ribosomal S6 kinase 1 (RSK1) and p38-activated protein kinase (MK2) and dephosphorylated by Protein Phosphatase 2A (PP2A). A phosphorylation-mimic mutant of S316D resulted in dissociation with the CCR4-NOT deadenylase complex through weakening interaction with CNOT1. Furthermore, Ser316 and serines 52 and 178 were independently contributed to the CCR4-NOT complex recruitment in the immunoprecipitation assay using phosphor-mimic mutants. In RAW264.7 macrophages, TTP was induced, and Ser316 was phosphorylated through RSK1 and MK2 by LPS stimulation. Knockout of TTP resulted in TNFα mRNA increased due to mRNA stabilization. Overexpression of non-phosphorylated S316A TTP mutant can restore TTP activity and lead to TNFα mRNA decreased. GST pull-down and RNA pull-down analyses demonstrated that endogenous TTP with Ser316 phosphorylation decreased the interaction with CNOT1. Conclusions Our results suggest that the TTP-mediated mRNA stability is modulated by Ser316 phosphorylation via regulating the TTP interaction with the CCR4-NOT deadenylase complex.

N6-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 responsible methyltransferase 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, remains elusive. Here, we address multiple roles of Mettl3 during Xenopus neurulation on the anteroposterior neural patterning, neural crest specification and neuronal cell differentiation. Knockdown of Mettl3 causes anteriorization of neurula 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 a 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.

The mitotic checkpoint (also called spindle assembly checkpoint, SAC) is a signaling pathway that safeguards proper chromosome segregation. Proper functioning of the SAC depends on adequate protein concentrations and appropriate stoichiometries between SAC proteins. Yet very little is known about SAC gene expression. Here, we show in fission yeast (S. pombe) that a combination of short mRNA half-lives and long protein half-lives supports stable SAC protein levels. For the SAC genes mad2+ and mad3+, their short mRNA half-lives are supported by a high frequency of non-optimal codons. In contrast, mad1+ mRNA has a short half-life despite a low frequency of non-optimal codons and despite the lack of known destabilizing motifs. Hence, different SAC genes employ different strategies of expression. We further show that Mad1 homodimers form co-translationally, which may necessitate a certain codon usage pattern. Taken together, we propose that the codon usage of SAC genes is fine-tuned for proper SAC function. Our work shines light on gene expression features that promote spindle assembly checkpoint function and suggests that synonymous mutations may weaken the checkpoint.

The lncRNA SNHG6 (small nucleolar RNA host gene 6) plays vital roles in tumorigenesis and the progression of hepatocellular carcinoma (HCC). However, the regulatory mechanisms of SNHG6 are largely unknown. In this study, we identified, via quantitative proteomics, specific cytoskeleton-associated proteins and enzyme modulators to be potential targets of SNHG6. SNHG6 reduced the mRNA levels of lysine methyltransferase, SET domain containing 7 (SETD7) and leucine zipper transcription factor-like 1 (LZTFL1) by posttranscriptional destabilization. Silencing of SETD7 or LZTFL1 reversed the suppressive effects of SNHG6 knockdown on HCC progression. Heterogeneous nuclear ribonucleoprotein L (HNRNPL) and polypyrimidine tract binding protein 1 (PTBP1) were identified as SNHG6-interacting proteins that bind to SETD7 or LZTFL1 mRNA. Forced expression of SNHG6 led to HNRNPL being competitively adsorbed by SNHG6, thereby removing its stabilizing effect on SETD7. Concurrently, the functional SNHG6-PTBP1 complex facilitated the degradation of LZTFL1 mRNA in hepatoma cells. These results indicated that SNHG6 promotes HCC progression by functioning as a "decoy plus guide" for HNRNPL and PTBP1 to facilitate mRNA decay of SETD7 and LZTFL1, thereby serving as a novel therapeutic target for HCC.

Hepatitis B virus (HBV) mRNA metabolism is dependent upon host proteins PAPD5 and PAPD7 (PAPD5/7). PAPD5/7 are cellular, noncanonical, poly(A) polymerases (PAPs) whose main function is to oligoadenylate the 3′ end of noncoding RNA (ncRNA) for exosome degradation. HBV seems to exploit these two ncRNA quality-control factors for viral mRNA stabilization, rather than degradation. RG7834 is a small-molecule compound that binds PAPD5/7 and inhibits HBV gene production in both tissue culture and animal study. We reported that RG7834 was able to destabilize multiple HBV mRNA species, ranging from the 3.5-kb pregenomic/precore mRNAs to the 2.4/2.1-kb hepatitis B virus surface protein (HBs) mRNAs, except for the smallest 0.7-kb X protein (HBx) mRNA. Compound-induced HBV mRNA destabilization was initiated by a shortening of the poly(A) tail, followed by an accelerated degradation process in both the nucleus and cytoplasm. In cells expressing HBV mRNA, both PAPD5/7 were found to be physically associated with the viral RNA, and the polyadenylating activities of PAPD5/7 were susceptible to RG7834 repression in a biochemical assay. Moreover, in PAPD5/7 double-knockout cells, viral transcripts with a regular length of the poly(A) sequence could be initially synthesized but became shortened in hours, suggesting that participation of PAPD5/7 in RNA 3′ end processing, either during adenosine oligomerization or afterward, is crucial for RNA stabilization.

Frontiers in molecular biosciences 8, 625608 (2021). doi:10.3389/fmolb.2021.625608, Published by Frontiers, Lausanne

Gene deletion and gene expression alteration can lead to growth defects that are amplified or reduced when a second mutation is present in the same cells. We performed 154 genetic interaction mapping (GIM) screens with mutants related with RNA metabolism and measured growth rates of about 700 000Saccharomyces cerevisiaedouble mutant strains. The screens used the gene deletion collection in addition to a set of 900 strains in which essential genes were affected by mRNA destabilization (DAmP). To analyze the results we developed RECAP, a strategy that validates genetic interaction profiles by comparison with gene co-citation frequency, and identified links between 1 471 genes and 117 biological processes. To validate specific results, we tested and confirmed a link between an inositol polyphosphate hydrolase complex and mRNA translation initiation. Altogether, the results and the newly developed analysis strategy should represent a useful resource for discovery of gene function in yeast.

Transfer RNAs (tRNAs) are abundantly expressed, small non-coding RNAs that have long been recognized as essential components of the protein translation machinery. The tRNA-derived small RNAs (tsRNAs), including tRNA halves (tiRNAs), and tRNA fragments (tRFs), were unexpectedly discovered and have been implicated in a variety of important biological functions such as cell proliferation, cell differentiation, and apoptosis. Mechanistically, tsRNAs regulate mRNA destabilization and translation, as well as retro-element reverse transcriptional and post-transcriptional processes. Emerging evidence has shown that tsRNAs are expressed in the heart, and their expression can be induced by pathological stress, such as hypertrophy. Interestingly, cardiac pathophysiological conditions, such as oxidative stress, aging, and metabolic disorders can be viewed as inducers of tsRNA biogenesis, which further highlights the potential involvement of tsRNAs in these conditions. There is increasing enthusiasm for investigating the molecular and biological functions of tsRNAs in the heart and their role in cardiovascular disease. It is anticipated that this new class of small non-coding RNAs will offer new perspectives in understanding disease mechanisms and may provide new therapeutic targets to treat cardiovascular disease.

The bioavailability of the major pro-inflammatory cytokines IL-1α and IL-1β is tightly controlled by transcription and post-translational processing to prevent hyperinflammation. The role of mRNA decay in maintenance of physiological IL-1 amounts remained unknown. Here we show that the down-regulation of Il1a and Il1b mRNA by the mRNA-destabilizing protein TTP (gene Zfp36) is required for immune homeostasis. The TTP deficiency syndrome, a multi organ inflammation in TTP−/− mice, was significantly ameliorated upon deletion of the IL-1 receptor. Il1a and Il1b played non-redundant roles in triggering the pathological IL-1 signaling in TTP−/− mice. Accordingly, tissues from TTP−/− animals contained increased amounts of Il1b mRNA. Unexpectedly, TTP destabilized Il1b mRNA in cell type-specific ways as evident from RNA-Seq and mRNA stability assays. These results demonstrate that TTP-driven mRNA destabilization depends on the cellular context. Moreover, such context-defined mRNA decay is essential for keeping steady state IL-1 levels in the physiological range.

Mature microRNAs (miRNAs) are small, non-coding RNA molecules that function by base-pairing with mRNAs. In multicellular organisms, miRNAs lead to mRNA destabilization and translation arrest. Importantly, the quantities and stichometry of miRNAs/mRNAs determine the miRNA regulation characteristics of specific cells. In this study, we used COMICS (Competition of miRNA Interactions in Cell Systems), a stochastic computational iterative framework to characterize genes by their sensitivity and robustness to miRNA regulation. We monitor the cell state by quantifying the retention level for all mRNAs, at the end of 100,000 simulation iterations. In HeLa cells, we partitioned all genes to five classes according to their decay rates. We show that the largest class (69% of genes) is apparently resistant to miRNA regulation. We created in silico perturbations using overexpressing of all major miRNAs (248 types) at various levels relative to the basal level (x1 to x1000). We further classified genes according to the differential behaviour for any pair of conditions of miRNA expression profile. Based on such measure (OvereXpression Ratio, OXR), we identified a small number of gene sets that are especially sensitive to OXR. Our results expose an overlooked quantitative dimension for set of genes and miRNA regulation in living cells.

Adiponectin exhibits potent antitumor activities. Herein, we examined the molecular mechanisms underlying suppression of tumor growth by globular adiponectin (gAcrp). We demonstrated that gAcrp suppressed B‐cell lymphoma 2 (Bcl‐2) expression, an anti‐apoptotic gene, by inducing its mRNA destabilization, which was accompanied with a decrease in cell viability and increased caspase‐3 activity in hepatic cancer cells. In addition, gAcrp increased expression of tristetraprolin (TTP) and AU‐rich element RNA‐binding protein 1 (AUF1), which are mRNA stability regulatory proteins. Moreover, gAcrp‐induced suppression of Bcl‐2 expression was abrogated by knockdown of TTP or AUF1. These data indicate that gAcrp induces apoptosis of hepatic cancer cells by TTP‐ and AUF1‐mediated Bcl‐2 mRNA destabilization, and further suggest that TTP and AUF1 are novel targets mediating the antitumor activity of adiponectin.

The PUF RNA-binding protein Puf5 is involved in regulation of the cell wall integrity (CWI) pathway in yeast. Puf5 negatively regulates expression of LRG1 mRNA, encoding for a GTPase-activating protein for Rho1 small GTPase. Here, we further analyzed the effect of Puf5 on LRG1 expression, together with Ccr4 and Pop2 deadenylases, Dhh1 decapping activator, and other PUF proteins. We found that the growth defect of puf5∆ mutant was enhanced by ccr4∆ mutation, which was partially suppressed by LRG1 deletion. Consistently, Lrg1 protein level was much more up-regulated in ccr4Δ puf5Δ double mutant than in each single mutant. Interestingly, LRG1 poly(A) tail length was longer in ccr4∆ mutant but not in puf5∆ mutant. Thus, Puf5 regulates LRG1 expression independently from Ccr4, although Puf5 recruits the Ccr4-Not deadenylase complex for mRNA destabilization. Unexpectedly, puf6Δ mutation suppressed the growth defect caused by ccr4Δ puf5∆ mutation. Loss of Rpl43a and Rpl43b ribosomal proteins, the previously identified Puf6 interactors, also suppressed the growth defect of ccr4Δ puf5Δ mutant. Our results suggest that Puf5 functions in the CWI pathway by regulating LRG1 expression in a deadenylase-independent manner, and that Puf6 is involved in the Ccr4- and Puf5-mediated regulation of cell growth through association with Rpl43.

MicroRNAs (miRNAs) are well known to target 3' untranslated regions (3' UTRs) in mRNAs, thereby silencing gene expression at the post-transcriptional level. Multiple reports have also indicated the ability of miRNAs to target protein-coding sequences (CDS); however, miRNAs have been generally believed to function through similar mechanisms regardless of the locations of their sites of action. Here, we report a class of miRNA-recognition elements (MREs) that function exclusively in CDS regions. Through functional and mechanistic characterization of these 'unusual' MREs, we demonstrate that CDS-targeted miRNAs require extensive base-pairing at the 3' side rather than the 5' seed; cause gene silencing in an Argonaute-dependent but GW182-independent manner; and repress translation by inducing transient ribosome stalling instead of mRNA destabilization. These findings reveal distinct mechanisms and functional consequences of miRNAs that target CDS versus the 3' UTR and suggest that CDS-targeted miRNAs may use a translational quality-control-related mechanism to regulate translation in mammalian cells.

Adiponectin, a hormone predominantly originated from adipose tissue, has exhibited potent anti-inflammatory properties. Accumulating evidence suggests that autophagy induction plays a crucial role in anti-inflammatory responses by adiponectin. However, underlying molecular mechanisms are still largely unknown. Association of Bcl-2 with Beclin-1, an autophagy activating protein, prevents autophagy induction. We have previously shown that adiponectin-induced autophagy activation is mediated through inhibition of interaction between Bcl-2 and Beclin-1. In the present study, we examined the molecular mechanisms by which adiponectin modulates association of Bcl-2 and Beclin-1 in macrophages. Herein, we demonstrated that globular adiponectin (gAcrp) induced increase in the expression of AUF1 and ZFP36L1, which act as mRNA destabilizing proteins, both in RAW 264.7 macrophages and primary peritoneal macrophages. In addition, gene silencing of AUF1 and ZFP36L1 caused restoration of decrease in Bcl-2 expression and Bcl-2 mRNA half-life by gAcrp, indicating crucial roles of AUF1 and ZFP36L1 induction in Bcl-2 mRNA destabilization by gAcrp. Moreover, knock-down of AUF1 and ZFP36L1 enhanced interaction of Bcl-2 with Beclin-1, and subsequently prevented gAcrp-induced autophagy activation, suggesting that AUF1 and ZFP36L1 induction mediates gAcrp-induced autophagy activation via Bcl-2 mRNA destabilization. Furthermore, suppressive effects of gAcrp on LPS-stimulated inflammatory mediators expression were prevented by gene silencing of AUF1 and ZFP36L1 in macrophages. Taken together, these results suggest that AUF1 and ZFP36L1 induction critically contributes to autophagy induction by gAcrp and are promising targets for anti-inflammatory responses by gAcrp.

Small non-coding RNAs including microRNAs (miRNAs) have been recently recognized as important regulators of gene expression. MicroRNAs play myriads of roles in physiological processes as well as in the pathogenesis of a number of diseases by translational repression or mRNA destabilization of numerous target genes. The miR-106b-25 cluster is highly conserved in vertebrates and consists of three members including miR-106b, miR-93 and miR-25. MiR-106b and miR-93 share the same seed sequences; however, miR-25 has only a similar seed sequence resulting in different predicted target mRNAs. In this review, we specifically focus on the role of miR-25 in healthy and diseased conditions. Many of miR-25 target mRNAs are involved in biological processes such as cell proliferation, differentiation, and migration, apoptosis, oxidative stress, inflammation, calcium handling, etc. Therefore, it is no surprise that miR-25 has been reported as a key regulator of common cancerous and non-cancerous diseases. MiR-25 plays an important role in the pathogenesis of acute myocardial infarction, left ventricular hypertrophy, heart failure, diabetes mellitus, diabetic nephropathy, tubulointerstitial nephropathy, asthma bronchiale, cerebral ischemia/reperfusion injury, neurodegenerative diseases, schizophrenia, multiple sclerosis, etc. MiR-25 is also a well-described oncogenic miRNA playing a crucial role in the development of many tumor types including brain tumors, lung, breast, ovarian, prostate, thyroid, oesophageal, gastric, colorectal, hepatocellular cancers, etc. In this review, our aim is to discuss the translational therapeutic role of miR-25 in common diseased conditions based on relevant basic research and clinical studies.

BACKGROUND AND PURPOSE Adiponectin potently suppresses inflammatory mediator production. Autophagy is known to play a critical role in the modulation of inflammatory responses by adiponectin. However, the underlying mechanisms are not clearly understood. Interaction between Beclin-1 and B-cell lymphoma 2 (Bcl-2) is a critical event in autophagy induction. We examined the effects of globular adiponectin (gAcrp) on the Beclin-1/Bcl-2 association and its underlying mechanisms. EXPERIMENTAL APPROACH The effect of gAcrp on the interaction between Beclin-1 and Bcl-2 was examined by immunoprecipitation followed by Western blotting. To elucidate the underlying mechanisms, we determined the effects of gAcrp on Beclin-1 phosphorylation and Bcl-2 mRNA stability, and investigated their role in the suppression of inflammatory mediators using pharmacological inhibitors and transient target gene knockdown. KEY RESULTS Globular adiponectin disrupted the association between Beclin-1 and Bcl-2 and increased Beclin-1 phosphorylation at Thr119 , critical residue for binding with Bcl-2, via a death-associated protein kinase-1 (DAPK1)-dependent mechanism. Moreover, gAcrp reduced Bcl-2 expression via Bcl-2 mRNA destabilization, without significantly affecting Bcl-2 promoter activity and protein degradation, which was mediated by tristetraprolin (TTP) induction. Finally, DAPK1 and TTP were shown to play key roles in gAcrp-induced autophagosome formation and suppression of LPS-stimulated TNF-α and IL-1β expression. CONCLUSION AND IMPLICATIONS Beclin-1 phosphorylation and Bcl-2 mRNA destabilization mediated by DAPK1 and TTP are crucial events leading to autophagy and the suppression of inflammatory cytokine production by gAcrp. These results provide novel mechanisms underlying adiponectin's modulation of inflammatory responses. DAPK and TTP are potential therapeutic targets for the management of inflammation.

Aromatic hydrocarbons belong to the most abundant contaminants in groundwater systems. They can serve as carbon and energy source for a multitude of indigenous microorganisms. Predictions of contaminant biodegradation and microbial growth in contaminated aquifers are often vague because the parameters of microbial activity in the mathematical models used for predictions are typically derived from batch experiments, which don't represent conditions in the field. In order to improve our understanding of key drivers of natural attenuation and the accuracy of predictive models, we conducted comparative experiments in batch and sediment flow-through systems with varying concentrations of contaminant in the inflow and flow velocities applying the aerobic Pseudomonas putida strain F1 and the denitrifying Aromatoleum aromaticum strain EbN1. We followed toluene degradation and bacterial growth by measuring toluene and oxygen concentrations and by direct cell counts. In the sediment columns, the total amount of toluene degraded by P. putida F1 increased with increasing source concentration and flow velocity, while toluene removal efficiency gradually decreased. Results point at mass transfer limitation being an important process controlling toluene biodegradation that cannot be assessed with batch experiments. We also observed a decrease in the maximum specific growth rate with increasing source concentration and flow velocity. At low toluene concentrations, the efficiencies in carbon assimilation within the flow-through systems exceeded those in the batch systems. In all column experiments the number of attached cells plateaued after an initial growth phase indicating a specific "carrying capacity" depending on contaminant concentration and flow velocity. Moreover, in all cases, cells attached to the sediment dominated over those in suspension, and toluene degradation was performed practically by attached cells only. The observed effects of varying contaminant inflow concentration and flow velocity on biodegradation could be captured by a reactive-transport model. By monitoring both attached and suspended cells we could quantify the release of new-grown cells from the sediments to the mobile aqueous phase. Studying flow velocity and contaminant concentrations as key drivers of contaminant transformation in sediment flow-through microcosms improves our system understanding and eventually the prediction of microbial biodegradation at contaminated sites.

Intracellular iron is tightly regulated by coordinated expression of iron transport and storage genes, such as transferrin receptor-1 (TfR1) and ferritin. They are primarily regulated by iron through iron-induced dissociation of iron-regulatory proteins (IRPs) from iron-responsive elements (IREs) in the 3′-UTR (untranslated region) of TfR1 or 5′-UTR of ferritin mRNA, resulting in destabilization of TfR1 mRNA and release of ferritin translation block. Thus high iron decreases iron transport via TfR1 mRNA degradation and increases iron storage via ferritin translational up-regulation. However, the molecular mechanism of TfR1 mRNA destabilization in response to iron remains elusive. Here, we demonstrate that miR-7-5p and miR-141-3p target 3′-TfR1 IREs and down-regulate TfR1 mRNA and protein expression. Conversely, miR-7-5p and miR-141-3p antagomiRs partially but significantly blocked iron- or IRP knockdown-induced down-regulation of TfR1 mRNA, suggesting the interplay between these microRNAs and IRPs along with involvement of another uncharacterized mechanism in TfR1 mRNA degradation. Luciferase reporter assays using 3′-UTR TfR1 IRE mutants suggested that the IREs C and E are targets of miR-7-5p and miR-141-3p, respectively. Furthermore, miR-7 expression was inversely correlated with TfR1 mRNA in human pancreatic adenocarcinoma patient samples. These results suggest a role of microRNAs in the TfR1 regulation in the IRP–IRE system.

Summary As core components of the microRNA-induced silencing complex (miRISC), Argonaute (AGO) proteins interact with TNRC6 proteins, recruiting other effectors of translational repression/mRNA destabilization. Here, we show that LIMD1 coordinates the assembly of an AGO-TNRC6 containing miRISC complex by binding both proteins simultaneously at distinct interfaces. Phosphorylation of AGO2 at Ser 387 by Akt3 induces LIMD1 binding, which in turn enables AGO2 to interact with TNRC6A and downstream effector DDX6. Conservation of this serine in AGO1 and 4 indicates this mechanism may be a fundamental requirement for AGO function and miRISC assembly. Upon CRISPR-Cas9-mediated knockout of LIMD1, AGO2 miRNA-silencing function is lost and miRNA silencing becomes dependent on a complex formed by AGO3 and the LIMD1 family member WTIP. The switch to AGO3 utilization occurs due to the presence of a glutamic acid residue (E390) on the interaction interface, which allows AGO3 to bind to LIMD1, AJUBA, and WTIP irrespective of Akt signaling., Graphical Abstract, Highlights • LIMD1 is necessary for the Akt3-dependent assembly of an AGO-TNRC6A functional miRISC • AGO1, 2, and 4 recruit LIMD1 in a phospho-dependent manner • AGO2 to AGO3 switching occurs upon LIMD1 ablation in HeLa cells • AGO3-WTIP replaces AGO2-LIMD1 for miRNA silencing in an Akt3-independent manner, Argonaute (AGO) proteins mediate post-transcriptional gene silencing through formation of the microRNA-induced silencing complex (miRISC). Bridge et al. identify LIM-domain-containing proteins as essential for miRISC formation through a phosphorylation-dependent mechanism. This is critical for post-transcriptional gene silencing and reveals that miRISC functionality is maintained by “AGO switching.”

MicroRNAs (miRNAs/miRs) are short non-coding RNAs (between 20 and 22 nucleotides) that regulate gene expression by binding to the 3′-untranslated region of target mRNA, and preventing protein translation or inducing mRNA destabilization. miRNAs are predicted to target ~60% of all mRNAs, therefore providing a marked degree of regulation of a number of cellular processes. In the present study, the expression of miR-548c-3p was determined by reverse transcription-quantitative polymerase chain reaction analysis and demonstrated to be markedly downregulated in clinical malignant glioma tissues and the glioma T98G cell line compared with normal human brain tissue. Transfection of miR-548c-3p inhibited cell proliferation by inducing G1 cell cycle arrest and also inhibited the migration of the T98G cells in vitro. Furthermore, a bioinformatic algorithm and a luciferase reporter assay identified proto-oncogene c-Myb (c-Myb) as a potential direct target of miR-548c-3p. Further experiments demonstrated that the inhibition of c-Myb by miR-548c-3p partially mediated the antitumor effect of miR-548c-3p. The results of the present study provide the novel insight that miR-548c-3p inhibits glioma tumorigenesis by targeting c-Myb. Therefore, miR-548c-3p may contribute to the development of improved glioma treatment.

The vertebrate-conserved RNA-binding protein (RBP) DND1 is required for survival of primordial germ cells (PGCs), as well as germ cell tumour (TGCT) suppression in mice1–5. Here we report that DND1 binds a UU[A/U] trinucleotide motif predominantly in messenger RNA (mRNA) 3′ untranslated regions (UTRs), and destabilizes target mRNAs through direct recruitment of the CCR4-NOT deadenylase (CCR4) complex. Transcriptomic analysis revealed that the extent of suppression is dependent on the number of DND1 binding sites. The DND1-dependent mRNA destabilization is required for survival of murine PGCs and spermatogonial stem cells (SSCs) by suppressing apoptosis. The target RNA spectrum includes positive regulators of apoptosis, inflammation, and modulators of signalling pathways regulating stem cell pluripotency including the TGF-β super family, all of which are aberrantly elevated in Dnd1-deficient PGCs. We propose that the induction of the posttranscriptional suppressor DND1 synergizes with concurrent transcriptional changes to sharpen developmental transitions during cellular differentiation and maintenance of the germline.

RNA-binding proteins (RBPs) play a major role during control of mRNA localization, stability, and translation and are central to most cellular processes. In the fission yeast Schizosaccharomyces pombe, the multiple K homology (KH) domain RBP Rnc1 downregulates the activity of the cell integrity pathway (CIP) via stabilization of pmp1+ mRNA, which encodes the Pmp1 phosphatase that inactivates Pmk1, the mitogen-activated protein kinase (MAPK) component of this signaling cascade. However, Rnc1 likely regulates the half-life/stability of additional mRNAs. We show that Rnc1 downregulates the activity of Sty1, the MAPK of the stress-activated MAPK pathway (SAPK), during control of cell length at division and recovery in response to acute stress. Importantly, this control strictly depends on Rnc1’s ability to bind mRNAs encoding activators (Wak1 MAPKKK, Wis1 MAPKK) and downregulators (Atf1 transcription factor, Pyp1 and Pyp2 phosphatases) of Sty1 phosphorylation through its KH domains. Moreover, Sty1 is responsible for Rnc1 phosphorylation in vivo at multiple phosphosites during growth and stress, and these modifications trigger Rnc1 for proper binding and destabilization of the above mRNA targets. Phosphorylation by Sty1 prompts Rnc1-dependent mRNA destabilization to negatively control SAPK signaling, thus revealing an additional feedback mechanism that allows precise tuning of MAPK activity during unperturbed cell growth and stress., This work was supported by the Ministerio de Ciencia, Innovación y Universidades, Spain (grant reference BFU2017-82423-P to J.C.) and European Regional Development Fund (ERDF) cofunding from the European Union.

Several types of thalassemia (including β039-thalassemia) are caused by nonsense mutations in genes controlling globin production, leading to premature translation termination and mRNA destabilization mediated by the nonsense mediated mRNA decay. Drugs (for instance, aminoglycosides) can be designed to suppress premature translation termination by inducing readthrough (or nonsense suppression) at the premature termination codon. These findings have introduced new hopes for the development of a pharmacologic approach to cure this genetic disease. In the present review, we first summarize the principle and current status of the chemical relief for the expression of functional proteins from genes otherwise unfruitful for the presence of nonsense mutations. Second, we compare data available on readthrough molecules for β0-thalassemia. The examples reported in the review strongly suggest that ribosomal readthrough should be considered as a therapeutic approach for the treatment of β0-thalassemia caused by nonsense mutations. Concluding, the discovery of molecules, exhibiting the property of inducing β-globin, such as readthrough compounds, is of great interest and represents a hope for several patients, whose survival will depend on the possible use of drugs rendering blood transfusion and chelation therapy unnecessary.

Bacterial ribosome-dependent attenuators are widespread posttranscriptional regulators. They harbour small upstream ORFs (uORFs) encoding leader peptides, for which no functions in trans are known yet. In the soil-dwelling plant symbiont Sinorhizobium meliloti, the tryptophan biosynthesis gene trpE(G) is preceded by the uORF trpL and is regulated by transcription attenuation according to tryptophan availability. However, trpLE(G) transcription is initiated independently of the tryptophan level in the cell, thereby ensuring a largely tryptophan-independent production of the leader peptide peTrpL. We provide evidence that peTrpL plays a role in the differential posttranscriptional regulation of the smeABR operon encoding the major multidrug efflux pump SmeAB and the TtgR-type transcription repressor SmeR. We show that peTrpL is involved in a tetracycline-dependent smeR mRNA destabilization and forms an antibiotic-dependent ribonucleoprotein (ARNP) complex with smeR and its antisense RNA (asRNA). Induction of asRNA transcription, ARNP formation and smeR downregulation were promoted by several antibiotics and the flavonoid genistein, and the resistance to these antimicrobial compounds was increased by peTrpL. The role of peTrpL in resistance is conserved in other soil Alphaproteobacteria.

HPV16 is the most carcinogenic human papillomavirus and causes >50% of cervical cancers, the majority of anal cancers and 30% of oropharyngeal squamous cell carcinomas. HPV carcinogenesis relies on the continuous expression of the two main viral oncoproteins E6 and E7 that target >150 cellular proteins. Among them, epigenetic modifiers, including DNA Methyl Transferases (DNMT), are dysregulated, promoting an aberrant methylation pattern in HPV-positive cancer cells. It has been previously reported that the treatment of HPV-positive cervical cancer cells with DNMT inhibitor 5-aza-2'-deoxycytidine (5azadC) caused the downregulation of E6 expression due to mRNA destabilization that was mediated by miR-375. Recently, the T-box transcription factor 2 (TBX2) has been demonstrated to repress HPV LCR activity. In the current study, the role of TBX2 in E6 repression was investigated in HPV16 cervical cancer cell lines following 5azadC treatment. A decrease of E6 expression was accompanied by p53 and p21 restoration. While TBX2 mRNA was upregulated in 5azadC-treated SiHa and Ca Ski cells, TBX2 protein was not detectable. Furthermore, the overexpression of TBX2 protein in cervical cancer cells did not allow the repression of E6 expression. The TBX2 transcription factor is therefore unlikely to be associated with the repression of E6 following 5azadC treatment of SiHa and Ca Ski cells. © 2020 Spandidos Publications. All rights reserved.

The transferrin receptor (TfR1) is the principal means of iron importation for most mammalian cells, and regulation of mRNA stability is a major mechanism through which TfR1 expression is controlled in response to changing intracellular iron levels. An endonuclease activity degrades the TfR1 mRNA during iron-repletion, which reduces iron importation and contributes to the restoration of homeostasis. Correct identification of the TfR1 mRNA endonuclease activity is important as it has the potential to be a pharmacological target for the treatment of several pathologies in which iron homeostasis is perturbed. A recent RNA article identified both miR-7-5p and miR-141-3p as mediators of TfR1 mRNA degradation during iron-repletion. However, the proposed TfR1 microRNA binding sites are inconsistent with several earlier studies. To better understand the discrepancy, we tested the proposed sites within an assay developed to detect changes to TfR1 mRNA stability. The complete disruption of both proposed binding sites failed to impact the assay in all cell lines tested, which include cell lines derived from mouse connective tissue (L-M), a human colon adenocarcinoma (SW480), and a human ovarian carcinoma (A2780). The overexpression of a miR-7-5p mimic also failed to decrease expression of both the endogenous TfR1 mRNA and a luciferase-TfR1 reporter under conditions in which the expression of a previously identified mir-7-5p target is attenuated. As a result, it is unlikely that the microRNAs are directly mediating iron-responsive degradation of the TfR1 mRNA as recently proposed. Instead, three short hairpin loops within the TfR1 3′-UTR are shown to be more consistent as endonuclease recognition elements.

The aim of this study was to investigate the mechanism of YM155 cytotoxicity in human chronic myeloid leukemia (CML) cells. YM155-induced apoptosis of human CML K562 cells was characterized by ROS-mediated p38 MAPK activation, mitochondrial depolarization, and survivin and MCL1 downregulation. Moreover, YM155-induced autophagy caused degradation of HuR mRNA and downregulation of HuR protein expression, which resulted in destabilized survivin and MCL1 mRNA. Interestingly, survivin and MCL1 suppression contributed to autophagy-mediated HuR mRNA destabilization in YM155-treated cells. Pretreatment with inhibitors of p38 MAPK or autophagy alleviated YM155-induced autophagy and apoptosis in K562 cells, as well as YM155-induced downregulation of HuR, survivin, and MCL1. Ectopic overexpression of HuR, survivin, or MCL1 attenuated the cytotoxic effect of YM155 on K562 cells. Conversely, YM155 sensitized K562 cells to ABT-199 (a BCL2 inhibitor), and circumvented K562 cell resistance to ABT-199 because of its inhibitory effect on survivin and MCL1 expression. Overall, our data indicate that YM155-induced apoptosis is mediated by inducing autophagic HuR mRNA degradation, and reveal the pathway responsible for YM155-induced downregulation of survivin and MCL1 in K562 cells. Our findings also indicate a similar pathway underlying YM155-induced death in human CML MEG-01 cells.

SummaryThe ribosome-dependent attenuator located upstream of bacterial tryptophan biosynthesis genes harbors a small ORF trpL containing tryptophan codons. When tryptophan is available, efficient trpL translation causes transcription termination and release of the attenuator RNA rnTrpL. In Sinorhizobium meliloti, rnTrpL is a trans-acting sRNA. Here, we identified an evolutionary conserved function for the trpL-encoded 14-aa leader peptide peTrpL. Upon exposure to tetracycline, the cellular peTrpL levels were increased and rnTrpL was generated independently of tryptophan availability. Both peTrpL and rnTrpL were found to be involved in tetracycline-dependent destabilization of rplUrpmA mRNA encoding ribosomal proteins L21 and L27. We provide evidence for redirection of the sRNA rnTrpL from its antibiotic-independent target trpDC to rplUrpmA by formation of an antibiotic-dependent ribonucleoprotein complex (ARNP). ARNPs comprising peTrpL, rnTrpL, rplUrpmA and antisense RNA were also observed for other translation-inhibiting antibiotics, suggesting that bacteria evolved mechanisms to utilize antibiotics for mRNA destabilization.

Deadenylate or activate? When cells are quiescent, they undergo reversible cell cycle arrest and evince low basal metabolism. Naïve T cells are normally quiescent until they recognize cognate antigens through T cell receptor–costimulatory molecule signaling. T cell quiescence appears to be an active process, but the mechanistic details are poorly understood. Hwang et al. report that the transcription factors BTG1 and BTG2 are selectively expressed in quiescent T cells. In mice, T cells conditionally knocked out for both factors showed enhanced proliferation and a lowered threshold of activation both in vitro and in response to Listeria monocytogenes infection. Deficiency of BTG1 and BTG2 resulted in increases in global messenger RNA half-life, suggesting that messenger RNA deadenylation and degradation are important processes for maintaining T cell quiescence. Science , this issue p. 1255

Adenosine N6-methylation (m6A) is one of the most pervasive mRNA modifications, and yet the physiological significance of m6A removal (demethylation) remains elusive. Here, we report that the m6A demethylase FTO functions as a conserved regulator of motile ciliogenesis. Mechanistically, FTO demethylates and thereby stabilizes the mRNA that encodes the master ciliary transcription factor FOXJ1. Depletion of Fto in Xenopus laevis embryos caused widespread motile cilia defects, and Foxj1 was identified as one of the major phenocritical targets. In primary human airway epithelium, FTO depletion also led to FOXJ1 mRNA destabilization and a severe loss of ciliated cells with an increase of neighboring goblet cells. Consistently, Fto knockout mice showed strong asthma-like phenotypes upon allergen challenge, a result owing to defective ciliated cells in the airway epithelium. Altogether, our study reveals a conserved role of the FTO-FOXJ1 axis in embryonic and homeostatic motile ciliogenesis.

The herpes simplex virus 1 (HSV-1) ICP0 protein is an E3 ubiquitin ligase that promotes the degradation of several host cell proteins. Most studies have found that ICP0 promotes the loss of IFI16 in infected cells, but one study reported that ICP0 was not necessary or sufficient for loss of IFI16 in a tumor-derived cell line. Therefore, in this study, we examined the requirement for ICP0 in promoting the loss of IFI16 in several normal and tumor-derived cell lines. HSV-1 infection resulted in an observable decrease of IFI16 protein levels in normal human foreskin fibroblasts (HFFs), normal oral keratinocytes (NOKs), and HeLa cells but not in U2OS cells. During infection with an ICP0-null virus, we observed a reduced loss of IFI16 in HFFs and NOKs but not in HeLa cells. Ectopic expression of ICP0 from a transfected plasmid was sufficient to promote the loss of IFI16 in HFFs and NOKs. In the absence of ICP0, we observed a delayed reduction of IFI16 protein that correlated with a reduction in the steady-state levels of IFI16 mRNA. In addition, we show that the ICP0-independent loss of IFI16 in HeLa cells is dependent in part on the activity of the viral virion host shutoff (vhs) tegument protein. Together, these results demonstrate that HSV-1 promotes the loss of IFI16 through at least two mechanisms: (i) by ICP0-dependent degradation of IFI16 and (ii) by vhs-dependent turnover of IFI16 mRNA. In addition, this study highlights a potential intrinsic difference between normal and tumor-derived cells for the activities of IFI16 and HSV-1 ICP0. IMPORTANCE HSV-1 is a ubiquitous virus that establishes a lifetime persistent infection in humans. The relative success of HSV-1 as a pathogen is, in part, dependent on the expression of viral proteins that counteract host intrinsic defense mechanisms and that modulate immune responses during viral infection. In this study, we examined the relative roles of two viral gene products for the ability to promote loss of the antiviral IFI16 DNA sensor. We demonstrate that the viral immediate early ICP0 protein plays a dominant role in the loss of IFI16 in normal, but not tumor-derived, human cell lines. In contrast, viral vhs-mediated loss of IFI16 by mRNA destabilization is revealed to be dominant in tumor-derived cells in which ICP0 is nonfunctional. Together, these results contribute to our understanding of how HSV-1 modulates IFI16 protein levels and highlight cell-type-dependent differences between normal and tumor-derived cells.

In mammalian post-transcriptional gene silencing, the Argonaute protein AGO2 indirectly recruits translation inhibitors, deadenylase complexes, and decapping factors to microRNA-targeted mRNAs, thereby repressing mRNA translation and accelerating mRNA decay. However, the exact composition and assembly pathway of the microRNA-induced silencing complex are not completely elucidated. As the GYF domain of human GIGYF2 was shown to bind AGO2 in pulldown experiments, we wondered whether GIGYF2 could be a novel protein component of the microRNA-induced silencing complex. Here we show that full-length GIGYF2 coimmunoprecipitates with AGO2 in human cells, and demonstrate that, upon tethering to a reporter mRNA, GIGYF2 exhibits strong, dose-dependent silencing activity, involving both mRNA destabilization and translational repression.