RNA-Sequencing data supports the existence of novel VEGFA splicing events but not of VEGFAxxxb isoforms

AbstractVascular endothelial growth factor (VEGFA), a pivotal regulator of angiogenesis and valuable therapeutic target, is characterised by alternative splicing which generates three principal isoforms, VEGFA121, VEGFA165 and VEGFA189. A second set of anti-angiogenic isoforms termed VEGFAxxxb that utilise an alternative splice site in the final exon have been widely reported, with mRNA detection based principally upon RT-PCR assays. We sought confirmation of the existence of the VEGFAxxxb isoforms within the abundant RNA sequencing data available publicly. Whilst sequences derived specifically from each of the canonical VEGFA isoforms were present in many tissues, there were no sequences derived from VEGFAxxxb isoforms. Sequencing of approximately 50,000 RT-PCR products spanning the exon 7–8 junction in 10 tissues did not identify any VEGFAxxxb transcripts. The absence or extremely low expression of these transcripts in vivo indicates that VEGFAxxxb isoforms are unlikely to play a role in normal physiology. Our analyses also revealed multiple novel splicing events supported by more reads than previously reported for VEGFA145 and VEGFA148 isoforms, including three from novel first exons consistent with existing transcription start site data. These novel VEGFA isoforms may play significant roles in specific cell types.</jats:p

Regulation of mitochondrial biogenesis in erythropoiesis by mTORC1-mediated protein translation.

Advances in genomic profiling present new challenges of explaining how changes in DNA and RNA are translated into proteins linking genotype to phenotype. Here we compare the genome-scale proteomic and transcriptomic changes in human primary haematopoietic stem/progenitor cells and erythroid progenitors, and uncover pathways related to mitochondrial biogenesis enhanced through post-transcriptional regulation. Mitochondrial factors including TFAM and PHB2 are selectively regulated through protein translation during erythroid specification. Depletion of TFAM in erythroid cells alters intracellular metabolism, leading to elevated histone acetylation, deregulated gene expression, and defective mitochondria and erythropoiesis. Mechanistically, mTORC1 signalling is enhanced to promote translation of mitochondria-associated transcripts through TOP-like motifs. Genetic and pharmacological perturbation of mitochondria or mTORC1 specifically impairs erythropoiesis in vitro and in vivo. Our studies support a mechanism for post-transcriptional control of erythroid mitochondria and may have direct relevance to haematologic defects associated with mitochondrial diseases and ageing

Untranslated Parts of Genes Interpreted: Making Heads or Tails of High-Throughput Transcriptomic Data via Computational Methods Computational methods to discover and quantify isoforms with alternative untranslated regions

In this review we highlight the importance of defining the untranslated parts of transcripts, and present a number of computational approaches for the discovery and quantification of alternative transcription start and poly‐adenylation events in high‐throughput transcriptomic data. The fate of eukaryotic transcripts is closely linked to their untranslated regions, which are determined by the position at which transcription starts and ends at a genomic locus. Although the extent of alternative transcription starts and alternative poly‐adenylation sites has been revealed by sequencing methods focused on the ends of transcripts, the application of these methods is not yet widely adopted by the community. We suggest that computational methods applied to standard high‐throughput technologies are a useful, albeit less accurate, alternative to the expertise‐demanding 5′ and 3′ sequencing and they are the only option for analysing legacy transcriptomic data. We review these methods here, focusing on technical challenges and arguing for the need to include better normalization of the data and more appropriate statistical models of the expected variation in the signal

Untranslated parts of genes interpreted: making heads or tails of high-throughput transcriptomic data via computational methods

The fate of eukaryotic transcripts is closely linked to their untranslated regions, which are determined by where transcription starts and ends on a genomic locus. The extent of alternative transcription start and alternative poly-adenylation has been revealed by sequencing methods focused on the ends of transcripts, but the application of these methods is not yet widely adopted by the community. In this review we highlight the importance of defining the untranslated parts of transcripts and suggest that computational methods applied to standard high-throughput technologies are a useful alternative to the expertise-demanding 5’ and 3’ sequencing. We present a number of computational approaches for the discovery and quantification of alternative transcription start and poly-adenylation events, focusing on technical challenges and arguing for the need to include better normalization of the data and more appropriate statistical models of the expected variation in the signal

Identification and expression analysis of human and murine overlapping genes

Wydział BiologiiZjawisko nakładania się genów może pełnić wiele funkcji regulatorowych, a większość prowadzonych obecnie w tym temacie badań skupia się na parach genów kodujących białka, nakładających się z niekodującymi RNA. Stosunkowo mało wiadomo na temat zjawiska nakładania w przypadku dwóch genów kodujących białka. W niniejszej pracy przeanalizowano wpływ nakładania się genów kodujących białka końcami 5’ na poziom ich ekspresji. W oparciu o analizę miejsc startu transkrypcji (TSS) w 73 ludzkich i 10 mysich organach, tkankach i liniach komórkowych, zidentyfikowano 582 i 113 par genów nakładających się w przynajmniej jednej bibliotece odpowiednio u człowieka i myszy. Wykazano, że rejon nakładania jest rzadko zachowany między gatunkami i tkankami. Pokazano również, że tylko w przypadku połowy par genów, transkrypcja inicjowana była wyłącznie w rejonie nakładania. W pozostałych przypadkach inicjacja transkrypcji następowała z miejsc TSS położonych zarówno w rejonie nakładania jak i po za nim. Analizy 26 linii komórkowych gruczolakoraka płuc oparte zostały dodatkowo również o dane pochodzące z eksperymentów RNA-Seq oraz ChIP-Seq dla siedmiu typów modyfikacji histonów i aktywności polimerazy RNA II, co pozwoliło przestudiować zjawisko nakładania w kontekście interferencji transkrypcji. Kluczowe wyniki zdeponowane zostały w bazie danych, dostępnej pod adresem http://overgenedb.amu.edu.pl.Gene overlap is known to play various regulatory functions on transcriptional and post-transcriptional levels. Most of the currently held studies is focused on protein coding genes overlapping with non-protein coding counterparts. Much less is known about the role of the gene overlap in the case of two protein-coding genes. Here we have studied 5’ end protein-coding overlapping genes in human and mice genomes. We have identified 582 human and 113 mouse pairs of genes that are transcribed using overlapping promoters in at least one analyzed library. Gene pairs were identified based on the analysis of the transcription start sites (TSSs) coordinates in 73 human and 10 mouse organs, tissues and cell lines. The collected data revealed that the overlap region is rarely conserved between the studied species and tissues. The results also shown that only in the case of about 50% of overlapping genes, transcription started explicitly in the overlap regions. In remaining half, the transcription was initiated both from overlapping and non-overlapping TSSs. Analyses for 26 human lung adenocarcinoma cell lines were additionally based on RNA-Seq and ChIP-Seq data for seven histone modifications and RNA Polymerase II activity which were studied in the context of the transcriptional interference. Key results were stored in a database, accessible under http://overgenedb.amu.edu.pl

Transcription Start Site selection within a single cluster and G quadruplex structures: a novel mechanism regulating gene expression

The 5' untranslated region (UTR) in a messenger RNA (mRNA) can greatly influence translation. Depending on where the transcription starts, the 5’ UTR will contain (or not) regulatory elements that can modulate mRNA translation. Although studies have demonstrated the effect of differential transcription start sites (TSSs) usage on translation efficiency, these are mainly restricted to TSSs in different clusters many nucleotides apart. However, it is currently unknown if there is any relevance to the TSS variation within a single cluster. Here, we present our findings on single cluster TSS-mediated regulation of protein expression, using mainly AGAP2 as an example. Using 5' RLM-RACE, we have identified different TSSs usage for AGAP2 mRNA in chronic myeloid leukaemia (CML) and prostate cancer (PC) cell lines, giving rise to populations of transcripts with variable lengths of 5' UTR. The population of longer 5' UTR were relatively higher in CML cell lines (P < 0.05), and those extra nucleotides contained the consensus sequence for a G-quadruplex (G4). The G4 formation was verified by CD spectroscopy. Additionally, we developed an immunoprecipitation method termed 'GRIP' [G4 RNA Immunoprecipitation] and demonstrated the existence of these RNA secondary structures in the living cells. To study the impact of the longer 5' UTR and the G4 on translation efficiency, we cloned three 5' UTR isoforms (shorter, longer and mutated-longer version) into a bicistronic plasmid and reported a significant decrease in luciferase activity by the G4 in the longer 5' UTR (P < 0.001). This result coincides with the discrepancy noted in AGAP2 mRNA and protein levels in these cell lines. Furthermore, polysome fractionation studies also confirmed that mRNA with longer 5' UTR associated less prominently with polyribosomes (P < 0.001). Our bioinformatics pipeline has identified 4,920 transcripts in the FANTOM database that contained putative G4 sequences between the major and upstream TSSs within the same TSS cluster. By integrating the NCI-60 microarray and SWATH-MS database, we curated a list of genes that displayed discrepancies in RNA and protein expression with a significantly higher level of G4 forming TSS; and validated our findings in another gene target (HK1). This highlights that the TSS-G4 mediated mechanism is not only limited to AGAP2 expression regulation but is also implicated in controlling the expression of other genes

Untangling the contribution of untranslated regions to mRNA translation in health and disease

Protein homeostasis is essential for living organisms and a consequence of steps of the gene expression pathway such as transcription, mRNA translation as well as the degradation of mRNA and proteins. These major cellular events demand tight control, high maintenance, and require a large proportion of cellular resources. mRNA translation is regulated by a plethora of cellular pathways as well as features of the mRNA molecule itself and is implicated in malignancies such as cancer. To decipher the contributions of mRNA elements in shaping the proteome, we developed a computational approach to identify mRNA features helping to understand their role in posttranscriptional gene regulation (paper III). While using mTOR-sensitive translation as a model, we characterized an mRNA subset, which despite its transcriptional regulation, does not lead to altered protein levels. This phenomenon, termed translational offsetting, associated with distinct features in the 3’ untranslated region and mRNA stability. The initiation step of mRNA translation is widely considered as rate-limiting and is greatly influenced by features in the 5’ untranslated region. These characteristics can lead to impaired scanning and initiation and have been attributed to cellular cues such as the mTOR pathway and the integrated stress response. In paper II, we applied nano-cap analysis of gene expression (nanoCAGE) and identified 5’UTR variants containing upstream open reading frames. In combination with the development of a reporter-based high-throughput method we studied these variants in a 5’UTR-centric manner, which led to the discovery of an mRNA subset being stressresistant due to precise transcription start site positioning. In paper IV, we studied the coordination of gene expression upon depletion of the transcription factor ERα, known for its role in hormone-dependent cancers. Post-transcriptional regulation upon ERα depletion is characterized by extensive translational offsetting, which is largely assigned to features in the coding sequence of mRNAs. These mRNAs are enriched for codons requiring U34-modified tRNAs for their translation, while these modifications are regulated by ERα. A large proportion of cancer types are characterized by aberrant tumor suppressor activity such as mutations or dysregulated protein levels of p53. Its reactivation by small molecules presents a promising strategy for cancer treatment. However, the exact mode of action of such compounds remains often elusive. RITA, a small molecule initially discovered for its induction of apoptosis upon p53 reactivation, induces cell death in a predominantly p53-independent manner. We studied RITA in the context of mRNA translation and found its activity is dependent on the phosphorylation of eIF2α, a major regulator of mRNA translation (paper I)

Ciblage Tissu-Spécifique des Cascades Enzymatiques de l’Angiotensinogène dans l’Athérome Humain

Atherosclerosis remains and continues to be the leading cause of death and disability in the world. The implication of Renin-angiotensin-aldosterone system (RAAS) in the development of the disease is well experimentally and clinically documented. However, due to the complexity of the system, these studies remain dispersed and give no clear global view of the association between the system and the disease. In this regard, we studied the functional organization of a set of 37 genes encoding classical and newly discovered RAAS participants, including substrate, enzymes and receptors. This set was called extended RAAS (extRAAS). Using statistical analysis of human carotid atheroma transcriptome involving gene clustering, we revealed special features of extRAAS expression associated with atheromatous remodeling. An important feature of this pattern was the coordination of 2 clusters of genes that are known to favor atheroma formation. The first cluster constitutes genes that encode for angiotensin peptidases, including ACE, CTSG, CTSD and RNPEP. Whereas the second encode for receptors (AGTR1, MR, GR and LNPEP). We hypothesized that the local pattern of extRAAS gene expression plays a key role in the development of atherosclerosis by orienting the metabolism of active peptidesL'Athérosclérose est la principale cause de décès et d'invalidité dans le monde. L'implication du système rénine-angiotensine-aldostérone (RAAS) dans le développement de la maladie est expérimentalement et cliniquement bien documentée. Toutefois, en raison de la complexité du système, ces études ne donnent pas de vision claire sur l'association entre le système et la maladie. À cet égard, nous avons étudié l'organisation fonctionnelle d'un ensemble de 37 gènes codant pour les composants classiques et nouvellement découverts du RAAS, y compris les substrats, les enzymes et les récepteurs. Cet ensemble a été appelé RAAS étendu (extRAAS). En utilisant une analyse statistique des données du transcriptome de l'athérome carotidien humain, nous avons révélé des caractéristiques spéciales de l'expression de l'extRAAS associées au remodelage athéromateux. Une caractéristique importante de ce modèle est la coordination de 2 groupes de gènes qui sont connus pour favoriser la formation de l'athérome. Le premier groupe est constitué de gènes codant pour les peptidases de l'angiotensine, y compris ACE, CTSG, CTSD et RNPEP. Le deuxième groupe est constitué des gènes codant pour les récepteurs AGTR1, MR, GR et LNPE