Decoding of translation-regulating entities reveals heterogeneous translation deficiency patterns in cellular senescence.

Cellular senescence constitutes a generally irreversible proliferation barrier, accompanied by macromolecular damage and metabolic rewiring. Several senescence types have been identified based on the initiating stimulus, such as replicative (RS), stress-induced (SIS) and oncogene-induced senescence (OIS). These senescence subtypes are heterogeneous and often develop subset-specific phenotypes. Reduced protein synthesis is considered a senescence hallmark, but whether this trait pertains to various senescence subtypes and if distinct molecular mechanisms are involved remain largely unknown. Here, we analyze large published or experimentally produced RNA-seq and Ribo-seq datasets to determine whether major translation-regulating entities such as ribosome stalling, the presence of uORFs/dORFs and IRES elements may differentially contribute to translation deficiency in senescence subsets. We show that translation-regulating mechanisms may not be directly relevant to RS, however uORFs are significantly enriched in SIS. Interestingly, ribosome stalling, uORF/dORF patterns and IRES elements comprise predominant mechanisms upon OIS, strongly correlating with Notch pathway activation. Our study provides for the first time evidence that major translation dysregulation mechanisms/patterns occur during cellular senescence, but at different rates depending on the stimulus type. The degree at which those mechanisms accumulate directly correlates with translation deficiency levels. Our thorough analysis contributes to elucidating crucial and so far unknown differences in the translation machinery between senescence subsets.

Cellular senescence and cardiovascular diseases: moving to the "heart" of the problem.

Cardiovascular diseases (CVDs) constitute the prime cause of global mortality, with an immense impact on patient quality of life and disability. Clinical evidence has revealed a strong connection between cellular senescence and worse cardiac outcomes in the majority of CVDs concerning both ischemic and nonischemic cardiomyopathies. Cellular senescence is characterized by cell cycle arrest accompanied by alterations in several metabolic pathways, resulting in morphological and functional changes. Metabolic rewiring of senescent cells results in marked paracrine activity, through a unique secretome, often exerting deleterious effects on neighboring cells. Here, we recapitulate the hallmarks and key molecular pathways involved in cellular senescence in the cardiac context and summarize the different roles of senescence in the majority of CVDs. In the last few years, the possibility of eliminating senescent cells in various pathological conditions has been increasingly explored, giving rise to the field of senotherapeutics. Therefore, we additionally attempt to clarify the current state of this field with a focus on cardiac senescence and discuss the potential of implementing senolytics as a treatment option in heart disease.

Postgenomic Bioinformatic Analysis of Nucleic Acid Sequences Expressed in the Salivary Gland Epithelial Cells of Primary Sjögren's Syndrome Patients in Search of Microorganisms and Endogenous Retroviruses.

Several previous studies from our laboratory have indicated that the salivary gland epithelia of primary Sjögren's syndrome (SS) patients are not only the target of autoimmune immune responses, but also key instigators of the chronic salivary gland inflammatory infiltrates of patients. In particular, the comparative analysis of salivary gland tissue specimens and of in-vitro cultured non-neoplastic salivary gland epithelial cell lines (SGEC, of ductal type) from SS-patients and non-SS disease-controls, have unequivocally highlighted the presence of intrinsic activation in the ductal epithelia of SS-patients and of aberrant expression of inflammagenic molecules thereof, that correlate with the severity of local histopathologic changes, as well as of systemic manifestations of the disease. In the same context, we have recently shown that the ductal epithelia of SS-patients manifest cell-autonomous activation of the AIM2 inflammasome owing to the presence of aberrant cytoplasmic accumulations of damaged DNA. These findings not only provide a mechanistic explanation for the intrinsic activation and inflammatory status of SS ductal epithelia, but may also point towards the putative instigating role of an exogenous or endogenous agent (i.e., a micro-organism or an endogenous retrovirus, respectively). On this basis and to further explore the nature of epithelial cell-intrinsic activation in SS, the present proposal aims to investigate the expression of endogenous retroviral and/or non-human nucleic acid sequences of microbial origin in the ductal salivary gland epithelia of SS-patients, using metagenomic analysis of high throughput DNA and RNA genome sequencing data, which will be obtained from SGEC lines derived from SS-patients and disease-controls.

HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins.

The biological role of RNA-binding proteins in the secretory pathway is not well established. Here, we describe that human HDLBP/Vigilin directly interacts with more than 80% of ER-localized mRNAs. PAR-CLIP analysis reveals that these transcripts represent high affinity HDLBP substrates and are specifically bound in their coding sequences (CDS), in contrast to CDS/3'UTR-bound cytosolic mRNAs. HDLBP crosslinks strongly to long CU-rich motifs, which frequently reside in CDS of ER-localized mRNAs and result in high affinity multivalent interactions. In addition to HDLBP-ncRNA interactome, quantification of HDLBP-proximal proteome confirms association with components of the translational apparatus and the signal recognition particle. Absence of HDLBP results in decreased translation efficiency of HDLBP target mRNAs, impaired protein synthesis and secretion in model cell lines, as well as decreased tumor growth in a lung cancer mouse model. These results highlight a general function for HDLBP in the translation of ER-localized mRNAs and its relevance for tumor progression.

Pulmonary infection by SARS-CoV-2 induces senescence accompanied by an inflammatory phenotype in severe COVID-19: possible implications for viral mutagenesis.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the respiratory system can progress to a multisystemic disease with aberrant inflammatory response. Cellular senescence promotes chronic inflammation, named senescence-associated secretory phenotype (SASP). We investigated whether coronavirus disease 2019 (COVID-19) is associated with cellular senescence and SASP. METHODS: Autopsy lung tissue samples from 11 COVID-19 patients and 43 age-matched non-COVID-19 controls with similar comorbidities were analysed by immunohistochemistry for SARS-CoV-2, markers of senescence and key SASP cytokines. Virally induced senescence was functionally recapitulated in vitro, by infecting epithelial Vero-E6 cells and a three-dimensional alveosphere system of alveolar type 2 (AT2) cells with SARS-CoV-2 strains isolated from COVID-19 patients. RESULTS: SARS-CoV-2 was detected by immunocytochemistry and electron microscopy predominantly in AT2 cells. Infected AT2 cells expressed angiotensin-converting enzyme 2 and exhibited increased senescence (p16INK4A and SenTraGor positivity) and interleukin (IL)-1ß and IL-6 expression. In vitro, infection of Vero-E6 cells with SARS-CoV-2 induced senescence (SenTraGor), DNA damage (γ-H2AX) and increased cytokine (IL-1ß, IL-6, CXCL8) and apolipoprotein B mRNA-editing (APOBEC) enzyme expression. Next-generation sequencing analysis of progenies obtained from infected/senescent Vero-E6 cells demonstrated APOBEC-mediated SARS-CoV-2 mutations. Dissemination of the SARS-CoV-2-infection and senescence was confirmed in extrapulmonary sites (kidney and liver) of a COVID-19 patient. CONCLUSIONS: We demonstrate that in severe COVID-19, AT2 cells infected by SARS-CoV-2 exhibit senescence and a proinflammatory phenotype. In vitro, SARS-CoV-2 infection induces senescence and inflammation. Importantly, infected senescent cells may act as a source of SARS-CoV-2 mutagenesis mediated by APOBEC enzymes. Therefore, SARS-CoV-2-induced senescence may be an important molecular mechanism of severe COVID-19, disease persistence and mutagenesis.

The Role of Circular RNAs in DNA Damage Response and Repair.

Circular RNAs (circRNA) comprise a distinct class of non-coding RNAs that are abundantly expressed in the cell. CircRNAs have the capacity to regulate gene expression by interacting with regulatory proteins and/or other classes of RNAs. While a vast number of circRNAs have been discovered, the majority still remains poorly characterized. Particularly, there is no detailed information on the identity and functional role of circRNAs that are transcribed from genes encoding components of the DNA damage response and repair (DDRR) network. In this article, we not only review the available published information on DDRR-related circRNAs, but also conduct a bioinformatic analysis on data obtained from public repositories to uncover deposited, yet uncharacterized circRNAs derived from components of the DDRR network. Finally, we interrogate for potential targets that are regulated by this class of molecules and look into potential functional implications.

Machine Learning Approaches on High Throughput NGS Data to Unveil Mechanisms of Function in Biology and Disease.

In this review, the fundamental basis of machine learning (ML) and data mining (DM) are summarized together with the techniques for distilling knowledge from state-of-the-art omics experiments. This includes an introduction to the basic mathematical principles of unsupervised/supervised learning methods, dimensionality reduction techniques, deep neural networks architectures and the applications of these in bioinformatics. Several case studies under evaluation mainly involve next generation sequencing (NGS) experiments, like deciphering gene expression from total and single cell (scRNA-seq) analysis; for the latter, a description of all recent artificial intelligence (AI) methods for the investigation of cell sub-types, biomarkers and imputation techniques are described. Other areas of interest where various ML schemes have been investigated are for providing information regarding transcription factors (TF) binding sites, chromatin organization patterns and RNA binding proteins (RBPs), while analyses on RNA sequence and structure as well as 3D dimensional protein structure predictions with the use of ML are described. Furthermore, we summarize the recent methods of using ML in clinical oncology, when taking into consideration the current omics data with pharmacogenomics to determine personalized treatments. With this review we wish to provide the scientific community with a thorough investigation of main novel ML applications which take into consideration the latest achievements in genomics, thus, unraveling the fundamental mechanisms of biology towards the understanding and cure of diseases.

Mechanism of Virus Attenuation by Codon Pair Deoptimization.

Codon pair deoptimization is an efficient virus attenuation strategy, but the mechanism that leads to attenuation is unknown. The strategy involves synthetic recoding of viral genomes that alters the positions of synonymous codons, thereby increasing the number of suboptimal codon pairs and CpG dinucleotides in recoded genomes. Here we identify the molecular mechanism of codon pair deoptimization-based attenuation by studying recoded influenza A viruses. We show that suboptimal codon pairs cause attenuation, whereas the increase of CpG dinucleotides has no effect. Furthermore, we show that suboptimal codon pairs reduce both mRNA stability and translation efficiency of codon pair-deoptimized genes. Consequently, reduced protein production directly causes virus attenuation. Our study provides evidence that suboptimal codon pairs are major determinants of mRNA stability. Additionally, it demonstrates that codon pair bias can be used to increase mRNA stability and protein production of synthetic genes in many areas of biotechnology.

DDX54 regulates transcriptome dynamics during DNA damage response.

The cellular response to genotoxic stress is mediated by a well-characterized network of DNA surveillance pathways. The contribution of post-transcriptional gene regulatory networks to the DNA damage response (DDR) has not been extensively studied. Here, we systematically identified RNA-binding proteins differentially interacting with polyadenylated transcripts upon exposure of human breast carcinoma cells to ionizing radiation (IR). Interestingly, more than 260 proteins, including many nucleolar proteins, showed increased binding to poly(A)+ RNA in IR-exposed cells. The functional analysis of DDX54, a candidate genotoxic stress responsive RNA helicase, revealed that this protein is an immediate-to-early DDR regulator required for the splicing efficacy of its target IR-induced pre-mRNAs. Upon IR exposure, DDX54 acts by increased interaction with a well-defined class of pre-mRNAs that harbor introns with weak acceptor splice sites, as well as by protein-protein contacts within components of U2 snRNP and spliceosomal B complex, resulting in lower intron retention and higher processing rates of its target transcripts. Because DDX54 promotes survival after exposure to IR, its expression and/or mutation rate may impact DDR-related pathologies. Our work indicates the relevance of many uncharacterized RBPs potentially involved in the DDR.