Application of the iPLUS non-coding sequence in improving biopharmaceuticals production.

The biotechnological landscape has witnessed significant growth in biological therapeutics particularly in the field of recombinant protein production. Here we investigate the function of 3'UTR cis-regulatory elements in increasing mRNA and protein levels in different biological therapeutics and model systems, spanning from monoclonal antibodies to mRNA vaccines. We explore the regulatory function of iPLUS - a universal sequence capable of consistently augmenting recombinant protein levels. By incorporating iPLUS in a vector to express a monoclonal antibody used in immunotherapy, in a mammalian cell line used by the industry (ExpiCHO), trastuzumab production increases by 2-fold. As yeast Pichia pastoris is widely used in the manufacture of industrial enzymes and pharmaceuticals, we then used iPLUS in tandem (3x) and iPLUSv2 (a variant of iPLUS) to provide proof-of-concept data that it increases the production of a reporter protein more than 100-fold. As iPLUS functions by also increasing mRNA levels, we hypothesize that these sequences could be used as an asset in the mRNA vaccine industry. In fact, by including iPLUSv2 downstream of Spike we were able to double its production. Moreover, the same effect was observed when we introduced iPLUSv2 downstream of MAGEC2, a tumor-specific antigen tested for cancer mRNA vaccines. Taken together, our study provides data (TLR4) showing that iPLUS may be used as a valuable asset in a variety of systems used by the biotech and biopharmaceutical industry. Our results underscore the critical role of non-coding sequences in controlling gene expression, offering a promising avenue to accelerate, enhance, and cost-effectively optimize biopharmaceutical production processes.

Pro-inflammatory polarization and colorectal cancer modulate alternative and intronic polyadenylation in primary human macrophages.

Introduction: Macrophages are essential cells of the immune system that alter their inflammatory profile depending on their microenvironment. Alternative polyadenylation in the 3'UTR (3'UTR-APA) and intronic polyadenylation (IPA) are mechanisms that modulate gene expression, particularly in cancer and activated immune cells. Yet, how polarization and colorectal cancer (CRC) cells affect 3'UTR-APA and IPA in primary human macrophages was unclear. Methods: In this study, we isolated primary human monocytes from healthy donors, differentiated and polarized them into a pro-inflammatory state and performed indirect co-cultures with CRC cells. ChrRNA-Seq and 3'RNA-Seq was performed to quantify gene expression and characterize new 3'UTR-APA and IPA mRNA isoforms. Results: Our results show that polarization of human macrophages from naïve to a pro-inflammatory state causes a marked increase of proximal polyA site selection in the 3'UTR and IPA events in genes relevant to macrophage functions. Additionally, we found a negative correlation between differential gene expression and IPA during pro-inflammatory polarization of primary human macrophages. As macrophages are abundant immune cells in the CRC microenvironment that either promote or abrogate cancer progression, we investigated how indirect exposure to CRC cells affects macrophage gene expression and 3'UTR-APA and IPA events. Co-culture with CRC cells alters the inflammatory phenotype of macrophages, increases the expression of pro-tumoral genes and induces 3'UTR-APA alterations. Notably, some of these gene expression differences were also found in tumor-associated macrophages of CRC patients, indicating that they are physiologically relevant. Upon macrophage pro-inflammatory polarization, SRSF12 is the pre-mRNA processing gene that is most upregulated. After SRSF12 knockdown in M1 macrophages there is a global downregulation of gene expression, in particular in genes involved in gene expression regulation and in immune responses. Discussion: Our results reveal new 3'UTR-APA and IPA mRNA isoforms produced during pro-inflammatory polarization of primary human macrophages and CRC co-culture that may be used in the future as diagnostic or therapeutic tools. Furthermore, our results highlight a function for SRSF12 in pro-inflammatory macrophages, key cells in the tumor response.

MCL1 alternative polyadenylation is essential for cell survival and mitochondria morphology.

Alternative polyadenylation in the 3' UTR (3' UTR-APA) is a mode of gene expression regulation, fundamental for mRNA stability, translation and localization. In the immune system, it was shown that upon T cell activation, there is an increase in the relative expression of mRNA isoforms with short 3' UTRs resulting from 3' UTR-APA. However, the functional significance of 3' UTR-APA remains largely unknown. Here, we studied the physiological function of 3' UTR-APA in the regulation of Myeloid Cell Leukemia 1 (MCL1), an anti-apoptotic member of the Bcl-2 family essential for T cell survival. We found that T cells produce two MCL1 mRNA isoforms (pA1 and pA2) by 3' UTR-APA. We show that upon T cell activation, there is an increase in both the shorter pA1 mRNA isoform and MCL1 protein levels. Moreover, the less efficiently translated pA2 isoform is downregulated by miR-17, which is also more expressed upon T cell activation. Therefore, by increasing the expression of the more efficiently translated pA1 mRNA isoform, which escapes regulation by miR-17, 3' UTR-APA fine tunes MCL1 protein levels, critical for activated T cells' survival. Furthermore, using CRISPR/Cas9-edited cells, we show that depletion of either pA1 or pA2 mRNA isoforms causes severe defects in mitochondria morphology, increases apoptosis and impacts cell proliferation. Collectively, our results show that MCL1 alternative polyadenylation has a key role in the regulation of MCL1 protein levels upon T cell activation and reveal an essential function for MCL1 3' UTR-APA in cell viability and mitochondria dynamics.

On the function and relevance of alternative 3'-UTRs in gene expression regulation.

Messanger RNA (mRNA) isoforms with alternative 3'-untranslated regions (3'-UTRs) are produced by alternative polyadenylation (APA), which occurs during transcription in most eukaryotic genes. APA fine-tunes gene expression in a cell-type- and cellular state-dependent manner. Selection of an APA site entails the binding of core cleavage and polyadenylation factors to a particular polyadenylation site localized in the pre-mRNA and is controlled by multiple regulatory determinants, including transcription, pre-mRNA cis-regulatory sequences, and protein factors. Alternative 3'-UTRs serve as platforms for specific RNA binding proteins and microRNAs, which regulate gene expression in a coordinated manner by controlling mRNA fate and function in the cell. Genome-wide studies illustrated the full extent of APA prevalence and revealed that specific 3'-UTR profiles are associated with particular cellular states and diseases. Generally, short 3'-UTRs are associated with proliferative and cancer cells, and long 3'-UTRs are mostly found in polarized and differentiated cells. Fundamental new insights on the physiological consequences of this widespread event and the molecular mechanisms involved have been revealed through single-cell studies. Publicly available comprehensive databases that cover all APA mRNA isoforms identified in many cellular states and diseases reveal specific APA signatures. Therapies tackling APA mRNA isoforms or APA regulators may be regarded as innovative and attractive tools for diagnostics or treatment of several pathologies. We highlight the function of APA and alternative 3'-UTRs in gene expression regulation, the control of these mechanisms, their physiological consequences, and their potential use as new biomarkers and therapeutic tools. This article is categorized under: RNA Processing > 3' End Processing RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA in Disease and Development > RNA in Disease.

Epithelial Keratins Modulate cMet Expression and Signaling and Promote InlB-Mediated Listeria monocytogenes Infection of HeLa Cells.

The host cytoskeleton is a major target for bacterial pathogens during infection. In particular, pathogens usurp the actin cytoskeleton function to strongly adhere to the host cell surface, to induce plasma membrane remodeling allowing invasion and to spread from cell to cell and disseminate to the whole organism. Keratins are cytoskeletal proteins that are the major components of intermediate filaments in epithelial cells however, their role in bacterial infection has been disregarded. Here we investigate the role of the major epithelial keratins, keratins 8 and 18 (K8 and K18), in the cellular infection by Listeria monocytogenes. We found that K8 and K18 are required for successful InlB/cMet-dependent L. monocytogenes infection, but are dispensable for InlA/E-cadherin-mediated invasion. Both K8 and K18 accumulate at InlB-mediated internalization sites following actin recruitment and modulate actin dynamics at those sites. We also reveal the key role of K8 and K18 in HGF-induced signaling which occurs downstream the activation of cMet. Strikingly, we show here that K18, and at a less extent K8, controls the expression of cMet and other surface receptors such TfR and integrin ß1, by promoting the stability of their corresponding transcripts. Together, our results reveal novel functions for major epithelial keratins in the modulation of actin dynamics at the bacterial entry sites and in the control of surface receptors mRNA stability and expression.

Thymic epithelial cells require p53 to support their long-term function in thymopoiesis in mice.

Thymic epithelial cells (TECs) provide crucial microenvironments for T-cell development and tolerance induction. As the regular function of the thymus declines with age, it is of fundamental and clinical relevance to decipher new determinants that control TEC homeostasis in vivo. Beyond its recognized tumor suppressive function, p53 controls several immunoregulatory pathways. To study the cell-autonomous role of p53 in thymic epithelium functioning, we developed and analyzed mice with conditional inactivation of Trp53 in TECs (p53cKO). We report that loss of p53 primarily disrupts the integrity of medullary TEC (mTEC) niche, a defect that spreads to the adult cortical TEC compartment. Mechanistically, we found that p53 controls specific and broad programs of mTEC differentiation. Apart from restraining the expression and responsiveness of the receptor activator of NF-κB (RANK), which is central for mTEC differentiation, deficiency of p53 in TECs altered multiple functional modules of the mTEC transcriptome, including tissue-restricted antigen expression. As a result, p53cKO mice presented premature defects in mTEC-dependent regulatory T-cell differentiation and thymocyte maturation, which progressed to a failure in regular and regenerative thymopoiesis and peripheral T-cell homeostasis in the adulthood. Lastly, peripheral signs of altered immunological tolerance unfold in mutant mice and in immunodeficient mice that received p53cKO-derived thymocytes. Our findings position p53 as a novel molecular determinant of thymic epithelium function throughout life.

Expression of Rac1 alternative 3' UTRs is a cell specific mechanism with a function in dendrite outgrowth in cortical neurons.

The differential expression of mRNAs containing tandem alternative 3' UTRs, achieved by mechanisms of alternative polyadenylation and post-transcriptional regulation, has been correlated with a variety of cellular states. In differentiated cells and brain tissues there is a general use of distal polyadenylation signals, originating mRNAs with longer 3' UTRs, in contrast with proliferating cells and other tissues such as testis, where most mRNAs contain shorter 3' UTRs. Although cell type and state are relevant in many biological processes, how these mechanisms occur in specific brain cell types is still poorly understood. Rac1 is a member of the Rho family of small GTPases with essential roles in multiple cellular processes, including cell differentiation and axonal growth. Here we used different brain cell types and tissues, including oligodendrocytes, microglia, astrocytes, cortical and hippocampal neurons, and optical nerve, to show that classical Rho GTPases express mRNAs with alternative 3' UTRs differently, by gene- and cell- specific mechanisms. In particular, we show that Rac1 originate mRNA isoforms with longer 3' UTRs specifically during neurite growth of cortical, but not hippocampal neurons. Furthermore, we demonstrate that the longest Rac1 3' UTR is necessary for driving the mRNA to the neurites, and also for neurite outgrowth in cortical neurons. Our results indicate that the expression of Rac1 longer 3' UTR is a gene and cell-type specific mechanism in the brain, with a new physiological function in cortical neuron differentiation.

Evolution of the NET (NocA, Nlz, Elbow, TLP-1) protein family in metazoans: insights from expression data and phylogenetic analysis.

The NET (for NocA, Nlz, Elbow, TLP-1) protein family is a group of conserved zinc finger proteins linked to embryonic development and recently associated with breast cancer. The members of this family act as transcriptional repressors interacting with both class I histone deacetylases and Groucho/TLE co-repressors. In Drosophila, the NET family members Elbow and NocA are vital for the development of tracheae, eyes, wings and legs, whereas in vertebrates ZNF703 and ZNF503 are important for the development of the nervous system, eyes and limbs. Despite the relevance of this protein family in embryogenesis and cancer, many aspects of its origin and evolution remain unknown. Here, we show that NET family members are present and expressed in multiple metazoan lineages, from cnidarians to vertebrates. We identified several protein domains conserved in all metazoan species or in specific taxonomic groups. Our phylogenetic analysis suggests that the NET family emerged in the last common ancestor of cnidarians and bilaterians and that several rounds of independent events of gene duplication occurred throughout evolution. Overall, we provide novel data on the expression and evolutionary history of the NET family that can be relevant to understanding its biological role in both normal conditions and disease.

CD5 expression is regulated during human T-cell activation by alternative polyadenylation, PTBP1, and miR-204.

T lymphocytes stimulated through their antigen receptor (TCR) preferentially express mRNA isoforms with shorter 3´ untranslated regions (3´-UTRs) derived from alternative pre-mRNA cleavage and polyadenylation (APA). However, the physiological relevance of APA programs remains poorly understood. CD5 is a T-cell surface glycoprotein that negatively regulates TCR signaling from the onset of T-cell activation. CD5 plays a pivotal role in mediating outcomes of cell survival or apoptosis, and may prevent both autoimmunity and cancer. In human primary T lymphocytes and Jurkat cells we found three distinct mRNA isoforms encoding CD5, each derived from distinct poly(A) signals (PASs). Upon T-cell activation, there is an overall increase in CD5 mRNAs with a specific increase in the relative expression of the shorter isoforms. 3´-UTRs derived from these shorter isoforms confer higher reporter expression in activated T cells relative to the longer isoform. We further show that polypyrimidine tract binding protein (PTB/PTBP1) directly binds to the proximal PAS and PTB siRNA depletion causes a decrease in mRNA derived from this PAS, suggesting an effect on stability or poly(A) site selection to circumvent targeting of the longer CD5 mRNA isoform by miR-204. These mechanisms fine-tune CD5 expression levels and thus ultimately T-cell responses.

Extensive regulation of nicotinate phosphoribosyltransferase (NAPRT) expression in human tissues and tumors.

Nicotinamide adenine dinucleotide (NAD) is a cofactor in redox reactions and a substrate for NAD-consuming enzymes, such as PARPs and sirtuins. As cancer cells have increased NAD requirements, the main NAD salvage enzymes in humans, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinate phosphoribosyltransferase (NAPRT), are involved in the development of novel anti-cancer therapies. Knowledge of the expression patterns of both genes in tissues and tumors is critical for the use of nicotinic acid (NA) as cytoprotective in therapies using NAMPT inhibitors. Herein, we provide a comprehensive study of NAPRT and NAMPT expression across human tissues and tumor cell lines. We show that both genes are widely expressed under normal conditions and describe the occurrence of novel NAPRT transcripts. Also, we explore some of the NAPRT gene expression mechanisms. Our findings underline that the efficiency of NA in treatments with NAMPT inhibitors is dependent on the knowledge of the expression profiles and regulation of both NAMPT and NAPRT.

The mitochondrial genome of Prays oleae (Insecta: Lepidoptera: Praydidae).

Prays oleae is one of the most important olive tree pests and a species of interest in evolutionary studies, as it belongs to one of the oldest extant superfamilies of Ditrysian Lepidoptera. We determined its mitogenome sequence, and found it has common features for Lepidoptera, e.g. an >80% A + T content, an apparent CGA start codon for COX1 and an ATAGA(T)n motif in the control region, which also contains several copies of a 163-164 bp repeat. Importantly, the mitogenome displays the Met-Ile-Gln tRNA gene order typical of Ditrysia, consistent with the hypothesis that this is a synapomorphy of that clade.

Marked Genetic Differentiation between Western Iberian and Italic Populations of the Olive Fly: Southern France as an Intermediate Area.

The olive fly, Bactrocera oleae, is the most important pest affecting the olive industry, to which it is estimated to cause average annual losses in excess of one billion dollars. As with other insects with a wide distribution, it is generally accepted that the understanding of B. oleae population structure and dynamics is fundamental for the design and implementation of effective monitoring and control strategies. However, and despite important advances in the past decade, a clear picture of B. oleae's population structure is still lacking. In the Mediterranean basin, where more than 95% of olive production is concentrated, evidence from several studies suggests the existence of three distinct sub-populations, but the geographical limits of their distributions, and the level of interpenetration and gene flow among them remain ill-characterized. Here we use mitochondrial haplotype analysis to show that one of the Mediterranean mitochondrial lineages displays geographically correlated substructure and demonstrate that Italic populations, though markedly distinct from their Iberian and Levantine counterparts are more diverse than previously described. Finally, we show that this distinction does not result from extant hypothetical geographic limits imposed by the Alps or the Pyrenees nor, more generally, does it result from any sharp boundary, as intermixing is observed in a broad area, albeit at variable levels. Instead, Bayesian phylogeographic analysis suggests the interplay between isolation-mediated differentiation during glacial periods and bi-directional dispersal and population intermixing in the interglacials has played a major role in shaping current olive fly population structure.

CDX2 homeoprotein is involved in the regulation of ST6GalNAc-I gene in intestinal metaplasia.

De novo expression of Sialyl-Tn (STn) antigen is one of the most common features of intestinal metaplasia (IM) and gastric carcinomas, and its biosynthesis has been mostly attributed to ST6GalNAc-I activity. However, the regulation of this glycosyltransferase expression is not elucidated. In IM lesions and in the intestine, CDX2 homeobox transcription factor is co-expressed with STn and ST6GalNAc-I. We therefore hypothesized that CDX2 might induce STn expression by positive regulation of ST6GalNAc-I. We showed that ST6GalNAc-I transcript levels and CDX2 have a coordinated expression upon Caco-2 in vitro differentiation, and overexpression of CDX2 in MKN45 gastric cells increases ST6GalNAc-I transcript levels. Nine putative CDX-binding sites in the ST6GalNAc-I-regulatory sequence were identified and analyzed by chromatin immunoprecipitation in Caco-2 cells and in IM. The results showed that CDX2 protein is recruited to all regions, being the most proximal sites preferentially occupied in vivo. Luciferase assays demonstrated that CDX2 is able to transactivate ST6GalNac-I-regulatory region. The induction was stronger for the regions mapped in the neighbourhood of ATG start codon and site-directed mutagenesis of these sites confirmed their importance. In conclusion, we show that CDX2 transcriptionally regulates ST6GalNAc-I gene expression, specifically in the preneoplastic IM lesion.

Implications of polyadenylation in health and disease.

Polyadenylation is the RNA processing step that completes the maturation of nearly all eukaryotic mRNAs. It is a two-step nuclear process that involves an endonucleolytic cleavage of the pre-mRNA at the 3'-end and the polymerization of a polyadenosine (polyA) tail, which is fundamental for mRNA stability, nuclear export and efficient translation during development. The core molecular machinery responsible for the definition of a polyA site includes several recognition, cleavage and polyadenylation factors that identify and act on a given polyA signal present in a pre-mRNA, usually an AAUAAA hexamer or similar sequence. This mechanism is tightly regulated by other cis-acting elements and trans-acting factors, and its misregulation can cause inefficient gene expression and may ultimately lead to disease. The majority of genes generate multiple mRNAs as a result of alternative polyadenylation in the 3'-untranslated region. The variable lengths of the 3' untranslated regions created by alternative polyadenylation are a recognizable target for differential regulation and clearly affect the fate of the transcript, ultimately modulating the expression of the gene. Over the past few years, several studies have highlighted the importance of polyadenylation and alternative polyadenylation in gene expression and their impact in a variety of physiological conditions, as well as in several illnesses. Abnormalities in the 3'-end processing mechanisms thus represent a common feature among many oncological, immunological, neurological and hematological disorders, but slight imbalances can lead to the natural establishment of a specific cellular state. This review addresses the key steps of polyadenylation and alternative polyadenylation in different cellular conditions and diseases focusing on the molecular effectors that ensure a faultless pre-mRNA 3' end formation.

The mitochondrial genome of the pinewood nematode (Bursaphelenchus xylophilus) lineage introduced in Europe.

The pinewood nematode (PWN) Bursaphelenchus xylophilus is the causative agent of pine wilt disease and the greatest biological threat to conifer forests worldwide. Here we describe the near-complete mitochondrial DNA (mtDNA) sequence (12,945 bp) of the PWN lineage recently introduced in Europe. The absence of polymorphisms across the mtDNA of three Portuguese isolates suggests that a single mitochondrial lineage was introduced in southwestern Europe. We also found that Portuguese isolates have an incomplete stop codon (TA) at COX3, while the reference mtDNA from a South Korean isolate has a complete stop codon (TAA). Moreover, two insertion/deletion polymorphisms change the ND4 protein in a stretch of seven amino acids, and a polymorphic mononucleotide repeat alters the predicted structure of the tyrosine tRNA in different geographical isolates. Overall, the new PWN mtDNA sequence provides a basis for studying the European dispersion of this important invasive species.

GRG5/AES interacts with T-cell factor 4 (TCF4) and downregulates Wnt signaling in human cells and zebrafish embryos.

Transcriptional control by TCF/LEF proteins is crucial in key developmental processes such as embryo polarity, tissue architecture and cell fate determination. TCFs associate with ß-catenin to activate transcription in the presence of Wnt signaling, but in its absence act as repressors together with Groucho-family proteins (GRGs). TCF4 is critical in vertebrate intestinal epithelium, where TCF4-ß-catenin complexes are necessary for the maintenance of a proliferative compartment, and their abnormal formation initiates tumorigenesis. However, the extent of TCF4-GRG complexes' roles in development and the mechanisms by which they repress transcription are not completely understood. Here we characterize the interaction between TCF4 and GRG5/AES, a Groucho family member whose functional relationship with TCFs has been controversial. We map the core GRG interaction region in TCF4 to a 111-amino acid fragment and show that, in contrast to other GRGs, GRG5/AES-binding specifically depends on a 4-amino acid motif (LVPQ) present only in TCF3 and some TCF4 isoforms. We further demonstrate that GRG5/AES represses Wnt-mediated transcription both in human cells and zebrafish embryos. Importantly, we provide the first evidence of an inherent repressive function of GRG5/AES in dorsal-ventral patterning during early zebrafish embryogenesis. These results improve our understanding of TCF-GRG interactions, have significant implications for models of transcriptional repression by TCF-GRG complexes, and lay the groundwork for in depth direct assessment of the potential role of Groucho-family proteins in both normal and abnormal development.

Characterization of human NLZ1/ZNF703 identifies conserved domains essential for proper subcellular localization and transcriptional repression.

NET family members have recently emerged as important players in the development of multiple structures, from the trachea of fly larvae to the vertebrate eye and human breast cancers. However, their mechanisms of action are still poorly understood, and we lack a detailed characterization of their functional domains, as well as gene expression patterns-particularly in adult mammals. Here, we present a characterization of human NLZ1/ZNF703 (NocA-like zinc finger 1/Zinc finger 703), one of the two human NET family member genes. We show that the gene is ubiquitously expressed in adult human and mouse tissues, that three mRNA species with the same coding sequence are generated by alternative polyadenylation, and that the encoded protein contains six evolutionarily conserved domains, three of which are specific to NET proteins. Finally, we present functional evidence that these domains are necessary for proper subcellular distribution of and transcription repression by the NLZ1 protein, but not for its interaction with Groucho family co-repressors.

Mitochondrial haplotypes reveal olive fly (Bactrocera oleae) population substructure in the Mediterranean.

The olive fly (Bactrocera oleae) is the most important olive tree (Olea europaea) pest. In the Mediterranean basin, where 98 % of its main hosts are concentrated, it causes major agricultural losses, due to its negative effect on production and quality of both olive and olive oil. Previous phylogeographic analyses have established that Mediterranean olive fly populations are distinct from other Old World populations, but did not agree on the specific population substructure within this region. In order to achieve a higher resolution of the diversity of olive fly populations, particularly in Central and Western Mediterranean (home to 70 % of the world production), we comparatively analyzed a set of samples from Portugal in the context of published mitochondrial sequences across the species' worldwide range. Strong evidence of population substructure was found in the Central and Western Mediterranean area, with two clearly separate phylogenetic branches. Together with previously published data, our results strongly support the existence of at least three distinct Mediterranean populations of the olive fly, raise the possibility of additional regional substructure and suggest specific avenues for future research. This knowledge can be instrumental in the development of better management and control strategies for a major pest of Mediterranean agriculture.

Transcriptional regulation of the human mitochondrial peptide deformylase (PDF).

The last years of research have been particularly dynamic in establishing the importance of peptide deformylase (PDF), a protein of the N-terminal methionine excision (NME) pathway that removes formyl-methionine from mitochondrial-encoded proteins. The genomic sequence of the human PDF gene is shared with the COG8 gene, which encodes a component of the oligomeric golgi complex, a very unusual case in Eukaryotic genomes. Since PDF is crucial in maintaining mitochondrial function and given the atypical short distance between the end of COG8 coding sequence and the PDF initiation codon, we investigated whether the regulation of the human PDF is affected by the COG8 overlapping partner. Our data reveals that PDF has several transcription start sites, the most important of which only 18 bp from the initiation codon. Furthermore, luciferase-activation assays using differently-sized fragments defined a 97 bp minimal promoter region for human PDF, which is capable of very strong transcriptional activity. This fragment contains a potential Sp1 binding site highly conserved in mammalian species. We show that this binding site, whose mutation significantly reduces transcription activation, is a target for the Sp1 transcription factor, and possibly of other members of the Sp family. Importantly, the entire minimal promoter region is located after the end of COG8's coding region, strongly suggesting that the human PDF preserves an independent regulation from its overlapping partner.

Successful COG8 and PDF overlap is mediated by alterations in splicing and polyadenylation signals.

Although gene-free areas compose the great majority of eukaryotic genomes, a significant fraction of genes overlaps, i.e., unique nucleotide sequences are part of more than one transcription unit. In this work, the evolutionary history and origin of a same-strand gene overlap is dissected through the analysis of COG8 (component of oligomeric Golgi complex 8) and PDF (peptide deformylase). Comparative genomic surveys reveal that the relative locations of these two genes have been changing over the last 445 million years from distinct chromosomal locations in fish to overlapping in rodents and primates, indicating that the overlap between these genes precedes their divergence. The overlap between the two genes was initiated by the gain of a novel splice donor site between the COG8 stop codon and PDF initiation codon. Splicing is accomplished by the use of the PDF acceptor, leading COG8 to share the 3'end with PDF. In primates, loss of the ancestral polyadenylation signal for COG8 makes the overlap between COG8 and PDF mandatory, while in mouse and rat concurrent overlapping and non-overlapping Cog8 transcripts exist. Altogether, we demonstrate that the origin, evolution and preservation of the COG8/PDF same-strand overlap follow similar mechanistic steps as those documented for antisense overlaps where gain and/or loss of splice sites and polyadenylation signals seems to drive the process.