Exonized Alu repeats in the 3'UTR of a CYP20A1_Alu-LT transcript act as a miRNA sponge.

OBJECTIVE: Alu repeats have gained huge importance in the creation and modification of regulatory networks. We previously reported a unique isoform of human CYP20A1 i.e. CYP20A1_Alu-LT with 23 Alu repeats exonized in its 9 kb long 3'UTR with 4742 potential binding sites for 994 miRNAs. The role of this transcript was hypothesized as a potential miRNA sponge in primary neurons as its expression correlated with that of 380 genes having shared miRNA sites and enriched in neuro-coagulopathy. This study provides experimental evidence for the miRNA sponge activity of CYP20A1_Alu-LT in neuronal cell lines. RESULTS: We studied the Alu-rich fragment of the CYP20A1_Alu-LT extended 3'UTR with > 10 binding sites for miR-619-5p and miR-3677-3p. Enrichment of the Alu-rich fragment with Ago2 confirmed miRNA association of this transcript. Cloning the fragment downstream of a reporter gene led to a 90% decrease in luciferase activity. Overexpression and knockdown studies revealed a positive correlation between the expression of CYP20A1_Alu-LT and miR-619-5p / miR-3677-3p target genes. GAP43, one of the key modulators of nerve regeneration, was significantly altered by the expression of CYP20A1_Alu-LT. This study, for the first time, provides evidence for a unique regulatory function of exonized Alu repeats as miRNA sponges.

The Slowing Rate of CpG Depletion in SARS-CoV-2 Genomes Is Consistent with Adaptations to the Human Host.

Depletion of CpG dinucleotides in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genomes has been linked to virus evolution, host-switching, virus replication, and innate immune responses. Temporal variations, if any, in the rate of CpG depletion during virus evolution in the host remain poorly understood. Here, we analyzed the CpG content of over 1.4 million full-length SARS-CoV-2 genomes representing over 170 million documented infections during the first 17 months of the pandemic. Our findings suggest that the extent of CpG depletion in SARS-CoV-2 genomes is modest. Interestingly, the rate of CpG depletion is highest during early evolution in humans and it gradually tapers off, almost reaching an equilibrium; this is consistent with adaptations to the human host. Furthermore, within the coding regions, CpG depletion occurs predominantly at codon positions 2-3 and 3-1. Loss of ZAP (Zinc-finger antiviral protein)-binding motifs in SARS-CoV-2 genomes is primarily driven by the loss of the terminal CpG within the motifs. Nonetheless, majority of the CpG depletion in SARS-CoV-2 genomes occurs outside ZAP-binding motifs. SARS-CoV-2 genomes selectively lose CpGs-motifs from a U-rich context; this may help avoid immune recognition by TLR7. SARS-CoV-2 alpha-, beta-, and delta-variants of concern have reduced CpG content compared to sequences from the beginning of the pandemic. In sum, we provide evidence that the rate of CpG depletion in virus genomes is not uniform and it greatly varies over time and during adaptations to the host. This work highlights how temporal variations in selection pressures during virus adaption may impact the rate and the extent of CpG depletion in virus genomes.

Measuring lncRNA Expression by Real-Time PCR.

Long noncoding RNAs are defined as transcripts longer than 200 nt with no protein coding potential. Most lncRNAs are expressed in a tissue-specific manner and barring a few, their absolute expression is lower compared to most coding transcripts. Differential expression studies have contributed the most to the functional characterisation of the lncRNAs we know. Sensitive and specific quantification of lncRNA expression is crucial for such studies. SYBR Green dye based real time quantitative PCR is a simple and affordable method of quantitative PCR, wherein the specific binding of the dye to double stranded DNA amplicon emits fluorescence proportionate to the amount of PCR products. Here we describe a detailed protocol for successful lncRNA quantitation by reverse transcription followed by SYBR Green chemistry-based real-time PCR.

Systematic analysis of migration factors by MigExpress identifies essential cell migration control genes in non-small cell lung cancer.

Cell migration is an essential process in health and in disease, including cancer metastasis. A comprehensive inventory of migration factors is nonetheless lacking-in part due to the difficulty in assessing migration using high-throughput technologies. Hence, there are currently very few screens that systematically reveal factors controlling cell migration. Here, we introduce MigExpress as a platform for the 'identification of Migration control genes by differential Expression'. MigExpress exploits the combination of in-depth molecular profiling and the robust quantitative analysis of migration capacity in a broad panel of samples and identifies migration-associated genes by their differential expression in slow- versus fast-migrating cells. We applied MigExpress to investigate non-small cell lung cancer (NSCLC), which is the most frequent cause of cancer mortality mainly due to metastasis. In 54 NSCLC cell lines, we comprehensively determined mRNA and protein expression. Correlating the transcriptome and proteome profiles with the quantified migration properties led to the discovery and validation of FLNC, DSE, CPA4, TUBB6, and BICC1 as migration control factors in NSCLC cells, which were also negatively correlated with patient survival. Notably, FLNC was the least expressed filamin in NSCLC, but the only one controlling cell migration and correlating with patient survival and metastatic disease stage. In our study, we present MigExpress as a new method for the systematic analysis of migration factors and provide a comprehensive resource of transcriptomic and proteomic data of NSCLC cell lines related to cell migration.

Lyz2-Cre-Mediated Genetic Deletion of Septin7 Reveals a Role of Septins in Macrophage Cytokinesis and Kras-Driven Tumorigenesis.

By crossing septin7-floxed mice with Lyz2-Cre mice carrying the Cre recombinase inserted in the Lysozyme-M (Lyz2) gene locus we aimed the specific deletion of septin7 in myeloid cells, such as monocytes, macrophages and granulocytes. Septin7 flox/flox :Lyz2-Cre mice show no alterations in the myeloid compartment. Septin7-deleted macrophages (BMDMs) were isolated and analyzed. The lack of Septin7 expression was confirmed and a constitutive double-nucleation was detected in Septin7-deficient BMDMs indicating a defect in macrophage cytokinesis. However, phagocytic function of macrophages as judged by uptake of labelled E. coli particles and LPS-stimulated macrophage activation as judged by induction of TNF mRNA expression and TNF secretion were not compromised. In addition to myeloid cells, Lyz2-Cre is also active in type II pneumocytes (AT2 cells). We monitored lung adenocarcinoma formation in these mice by crossing them with the conditional knock-in Kras-LSL-G12D allele. Interestingly, we found that control mice without septin7 depletion die after 3-5 weeks, while the Septin7-deficient animals survived 11 weeks or even longer. Control mice sacrificed in the age of 4 weeks display a bronchiolo-alveolar hyperplasia with multiple adenomas, whereas the Septin7-deficient animals of the same age are normal or show only a weak multifocal brochiolo-alveolar hyperplasia. Our findings indicate an essential role of Septin7 in macrophage cytokinesis but not in macrophage function. Furthermore, septin7 seems absolutely essential for oncogenic Kras-driven lung tumorigenesis making it a potential target for anti-tumor interventions.

A pan-cancer analysis reveals nonstop extension mutations causing SMAD4 tumour suppressor degradation.

Nonstop or stop-loss mutations convert a stop into a sense codon, resulting in translation into the 3' untranslated region as a nonstop extension mutation to the next in-frame stop codon or as a readthrough mutation into the poly-A tail. Nonstop mutations have been characterized in hereditary diseases, but not in cancer genetics. In a pan-cancer analysis, we curated and analysed 3,412 nonstop mutations from 62 tumour entities, generating a comprehensive database at http://NonStopDB.dkfz.de. Six different nonstop extension mutations affected the tumour suppressor SMAD4, extending its carboxy terminus by 40 amino acids. These caused rapid degradation of the SMAD4 mutants via the ubiquitin-proteasome system. A hydrophobic degron signal sequence of ten amino acids within the carboxy-terminal extension was required to induce complete loss of the SMAD4 protein. Thus, we discovered that nonstop mutations can be functionally important in cancer and characterize their loss-of-function impact on the tumour suppressor SMAD4.

Beclin 1 Phosphorylation - at the Center of Autophagy Regulation.

Autophagy is a tightly regulated catabolic process wherein cells under stress sequester cytosolic constituents like damaged proteins and organelles in double-membrane vesicles called autophagosomes. The autophagosomes degrade their cargo by lysosomal proteolysis generating raw materials for the biosynthesis of vital macromolecules. One of the initial steps in the assembly of autophagosomes from pre-autophagic structures is the recruitment and activation of the class III phosphatidylinositol 3-kinase complex consisting of Beclin 1 (BECN1), VPS34, VPS15, and ATG14 proteins. Several pieces of evidence indicate that the phosphorylation and ubiquitination of BECN1 at an array of residues fine-tune the responses to diverse autophagy modulating stimuli and helps in maintaining the balance between pro-survival autophagy and pro-apoptotic responses. In this mini-review, we will discuss the importance of distinct BECN1 phosphorylation events, the diverse signaling pathways and kinases involved and their role in the regulation of autophagy.

Non-coding transcript variants of protein-coding genes - what are they good for?

The total number of protein-coding genes in the human genome is not significantly higher than those in much simpler eukaryotes, despite a general increase in genome size proportionate to the organismal complexity. The large non-coding transcriptome and extensive differential splicing, are increasingly being accepted as the factors contributing to the complex mammalian physiology and architecture. Recent studies reveal additional layers of functional complexity: some long non-coding RNAs have been re-defined as micropeptide or microprotein encoding transcripts, and in turn some protein-coding RNAs are bifunctional and display also non-coding functions. Moreover, several protein-coding genes express long non-coding RNA splice-forms and generate circular RNAs in addition to their canonical mRNA transcripts, revoking the strict definition of a gene as coding or non-coding. In this mini review, we discuss the current understanding of these hybrid genes and their possible roles and relevance.

LINC00261 and the Adjacent Gene FOXA2 Are Epithelial Markers and Are Suppressed during Lung Cancer Tumorigenesis and Progression.

Lung cancer continues to be the leading cause of cancer-related deaths worldwide, with little improvement in patient survival rates in the past decade. Long non-coding RNAs (lncRNAs) are gaining importance as possible biomarkers with prognostic potential. By large-scale data mining, we identified LINC00261 as a lncRNA which was significantly downregulated in lung cancer. Low expression of LINC00261 was associated with recurrence and poor patient survival in lung adenocarcinoma. Moreover, the gene pair of LINC00261 and its neighbor FOXA2 were significantly co-regulated. LINC00261 as well as FOXA2 negatively correlated with markers for epithelial-to-mesenchymal transition (EMT) and were suppressed by the EMT inducer TGFß. Hierarchical clustering of gene expression data from lung cancer cell lines could further verify the association of high LINC00261/FOXA2 expression to an epithelial gene signature. Furthermore, higher expression of the LINC00261/FOXA2 locus was associated with lung cancer cell lines with lower migratory capacity. All these data establish LINC00261 and FOXA2 as an epithelial-specific marker pair, downregulated during EMT and lung cancer progression, and associated with lower cell migration potential in lung cancer cells.

The long non-coding RNA PARROT is an upstream regulator of c-Myc and affects proliferation and translation.

Long non-coding RNAs are important regulators of gene expression and signaling pathways. The expression of long ncRNAs is dysregulated in cancer and other diseases. The identification and characterization of long ncRNAs is often challenging due to their low expression level and localization to chromatin. Here, we identify a functional long ncRNA, PARROT (Proliferation Associated RNA and Regulator Of Translation) transcribed by RNA polymerase II and expressed at a relatively high level in a number of cell lines. The PARROT long ncRNA is associated with proliferation in both transformed and normal cell lines. We characterize the long ncRNA PARROT as an upstream regulator of c-Myc affecting cellular proliferation and translation using RNA sequencing and mass spectrometry following depletion of the long ncRNA. PARROT is repressed during senescence of human mammary epithelial cells and overexpressed in some cancers, suggesting an important association with proliferation through regulation of c-Myc. With this study, we add to the knowledge of cytoplasmic functional long ncRNAs and extent the long ncRNA-Myc regulatory network in transformed and normal cells.

From junk to master regulators of invasion: lncRNA functions in migration, EMT and metastasis.

Metastasis is a multistep process that involves the dissemination of cells from the primary tumor and colonization of distant secondary organs. Epithelial cells at the invasive front of a carcinoma acquire an enhanced migratory phenotype in a process called epithelial-to-mesenchymal transition (EMT). This cellular plasticity seems to drive the initiation of metastasis. Identifying important molecules and understanding their molecular mechanisms is a key to cancer prognosis and the development of therapeutics for late stage malignancies. Recent advances in sequencing technology uncovered that the mammalian genome is pervasively transcribed into many nonprotein-coding RNAs including the class of long noncoding RNA, a.k.a. lncRNA. Several lncRNAs are differentially expressed in carcinomas and they are emerging as potent regulators of tumor progression and metastasis. Here, we review the diverse molecular mechanisms, cellular roles and regulatory patterns that are becoming apparent for the noncoding transcriptome. Chromatin modification, epigenetic regulation, alternative splicing and translational control by MALAT1, HOTAIR and TRE lncRNAs represent important examples of lncRNA-mediated control of cell migration and invasion, EMT and metastasis. Beyond these better characterized examples, numerous additional transcripts have been associated with cancer metastasis, but their functional roles await their discovery.

The problem of pyridinyl imidazole class inhibitors of MAPK14/p38α and MAPK11/p38ß in autophagy research.

In addition to its established role in inflammation, the stress-activated p38 MAP kinase pathway plays major roles in the regulation of cell cycle, senescence, and autophagy. Robust studies could establish mechanistic links between MAPK11-MAPK14/p38 signaling and macroautophagy converging at ATG9-trafficking and BECN1 phosphorylation. However, several reports seem to monitor MAPK11-MAPK14/p38-dependence of autophagy exclusively by the use of the SB203580/SB202190 class of MAPK14/MAPK11/p38α/ß inhibitors. In this "Letter to the editor" we present data to support our claim that these inhibitors interfere with autophagic flux in a MAPK11-MAPK14/p38-independent manner and hence should no longer be used as pharmacological tools in the analysis of MAPK11-MAPK14/p38-dependence of autophagy. We propose a general guideline from Autophagy with regard to this issue to avoid such misinterpretations in the future.

Interleukin-17 (IL-17) and IL-1 activate translation of overlapping sets of mRNAs, including that of the negative regulator of inflammation, MCPIP1.

Changes in gene expression during inflammation are in part caused by post-transcriptional mechanisms. A transcriptome-wide screen for changes in ribosome occupancy indicated that the inflammatory cytokine IL-17 activates translation of a group of mRNAs that overlaps partially with those affected similarly by IL-1. Included are mRNAs of IκBζ and of MCPIP1, important regulators of the quality and course of immune and inflammatory responses. Evidence for increased ribosome association of these mRNAs was also obtained in LPS-activated RAW264.7 macrophages and human peripheral blood mononuclear cells. Like IL-1, IL-17 activated translation of IκBζ mRNA by counteracting the function of a translational silencing element in its 3'-UTR defined previously. Translational silencing of MCPIP1 mRNA in unstimulated cells resulted from the combined suppressive activities of its 5'-UTR, which contains upstream open reading frames, and of its 3'-UTR, which silences independently of the 5'-UTR. Only the silencing function of the 3'-UTR was counteracted by IL-17 as well as by IL-1. Translational silencing by the 3'-UTR was dependent on a putative stem-loop-forming region previously associated with rapid degradation of the mRNA. The results suggest that translational control exerted by IL-1 and IL-17 plays an important role in the coordination of an inflammatory reaction.

The p38/MK2-driven exchange between tristetraprolin and HuR regulates AU-rich element-dependent translation.

TNF expression of macrophages is under stringent translational control that depends on the p38 MAPK/MK2 pathway and the AU-rich element (ARE) in the TNF mRNA. Here, we elucidate the molecular mechanism of phosphorylation-regulated translation of TNF. We demonstrate that translation of the TNF-precursor at the ER requires expression of the ARE-binding and -stabilizing factor human antigen R (HuR) together with either activity of the p38 MAPK/MK2 pathway or the absence of the ARE-binding and -destabilizing factor tristetraprolin (TTP). We show that phosphorylation of TTP by MK2 decreases its affinity to the ARE, inhibits its ability to replace HuR, and permits HuR-mediated initiation of translation of TNF mRNA. Since translation of TTP's own mRNA is also regulated by this mechanism, an intrinsic feedback control of the inflammatory response is ensured. The phosphorylation-regulated TTP/HuR exchange at target mRNAs provides a reversible switch between unstable/non-translatable and stable/efficiently translated mRNAs.

Interleukin-1 activates synthesis of interleukin-6 by interfering with a KH-type splicing regulatory protein (KSRP)-dependent translational silencing mechanism.

Post-transcriptional mechanisms play an important role in the control of inflammatory gene expression. The heterogeneous nuclear ribonucleoprotein K homology (KH)-type splicing regulatory protein (KSRP) triggers rapid degradation of mRNAs for various cytokines, chemokines, and other inflammation-related proteins by interacting with AU-rich elements (AREs) in the 3'-untranslated mRNA regions. In addition to destabilizing mRNAs, AU-rich elements can restrict their translation. Evidence that KSRP also participates in translational silencing was obtained in a screen comparing the polysome profiles of cells with siRNA-mediated depletion of KSRP with that of control cells. Among the group of mRNAs showing increased polysome association upon KSRP depletion are those of interleukin (IL)-6 and IL-1α as well as other ARE-containing transcripts. Redistribution of IL-6 mRNA to polysomes was associated with increased IL-6 protein secretion by the KSRP-depleted cells. Silencing of IL-6 and IL-1α mRNAs depended on their 3'-untranslated regions. The sequence essential for translational control of IL-6 mRNA and its interaction with KSRP was located to an ARE. KSRP-dependent silencing was reversed by IL-1, a strong inducer of IL-6 mRNA and protein expression. The results identify KSRP as a protein involved in ARE-mediated translational silencing. They suggest that KSRP restricts inflammatory gene expression not only by enhancing degradation of mRNAs but also by inhibiting translation, both functions that are counteracted by the proinflammatory cytokine IL-1.

IL-1-induced post-transcriptional mechanisms target overlapping translational silencing and destabilizing elements in IκBζ mRNA.

The inflammatory cytokine IL-1 induces profound changes in gene expression. This is contributed in part by activating translation of a distinct set of mRNAs, including IκBζ, as indicated by genome-wide analysis of changes in ribosomal occupancy in IL-1α-treated HeLa cells. Polysome profiling of IκBζ mRNA and reporter mRNAs carrying its 3' UTR indicated poor translation in unstimulated cells. 3' UTR-mediated translational silencing was confirmed by suppression of luciferase activity. Translational silencing was unaffected by replacing the poly(A) tail with a histone stem-loop, but lost under conditions of cap-independent internal initiation. IL-1 treatment of the cells caused profound shifts of endogenous and reporter mRNAs to polysome fractions and relieved suppression of luciferase activity. IL-1 also inhibited rapid mRNA degradation. Both translational activation and mRNA stabilization involved IRAK1 and -2 but occurred independently of the p38 MAPK pathway, which is known to target certain other post-transcriptional mechanisms. The translational silencing RNA element contains the destabilizing element but requires additional 5' sequences and is impaired by mutations that leave destabilization unaffected. These differences in function are associated with differential changes in protein binding in vitro. Thus, rapid degradation occurs independently of the translational silencing effect. The results provide evidence for a novel mode of post-transcriptional control by IL-1, which impinges on the time course and pattern of IL-1-induced gene expression.

Functional analysis of KSRP interaction with the AU-rich element of interleukin-8 and identification of inflammatory mRNA targets.

mRNA stability is a major determinant of inflammatory gene expression. Rapid degradation of interleukin-8 (IL-8) mRNA is imposed by a bipartite AU-rich element (ARE) in the 3' untranslated region (R. Winzen et al., Mol. Cell. Biol. 24:4835-4847, 2004). Small interfering RNA-mediated knockdown of the ARE-binding protein KSRP resulted in stabilization of IL-8 mRNA or of a beta-globin reporter mRNA containing the IL-8 ARE. Rapid deadenylation was impaired, indicating a crucial role for KSRP in this step of mRNA degradation. The two IL-8 ARE domains both contribute to interaction with KSRP, corresponding to the importance of both domains for rapid degradation. Exposure to the inflammatory cytokine IL-1 has been shown to stabilize IL-8 mRNA through p38 mitogen-activated protein (MAP) kinase and MK2. IL-1 treatment impaired the interaction of KSRP with the IL-8 ARE in a manner dependent on p38 MAP kinase but apparently independent of MK2. Instead, evidence that TTP, a target of MK2, can also destabilize the IL-8 ARE reporter mRNA is presented. In a comprehensive approach to identify mRNAs controlled by KSRP, two criteria were evaluated by microarray analysis of (i) association of mRNAs with KSRP in pulldown assays and (ii) increased amounts in KSRP knockdown cells. According to both criteria, a group of 100 mRNAs is controlled by KSRP, many of which are unstable and encode proteins involved in inflammation. These results indicate that KSRP functions as a limiting factor in inflammatory gene expression.