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.

Cell division protein FtsK coordinates bacterial chromosome segregation and daughter cell separation in Staphylococcus aureus.

Unregulated cell cycle progression may have lethal consequences and therefore, bacteria have various mechanisms in place for the precise spatiotemporal control of cell cycle events. We have uncovered a new link between chromosome replication/segregation and splitting of the division septum. We show that the DNA translocase domain-containing divisome protein FtsK regulates cellular levels of a peptidoglycan hydrolase Sle1, which is involved in cell separation in the bacterial pathogen Staphylococcus aureus. FtsK interacts with a chaperone (trigger factor, TF) and establishes a FtsK-dependent TF concentration gradient that is higher in the septal region. Trigger factor binds Sle1 and promotes its preferential export at the septal region, while also preventing Sle1 degradation by the ClpXP proteolytic machinery. Upon conditions that lead to paused septum synthesis, such as DNA damage or impaired DNA replication/segregation, TF gradient is dissipated and Sle1 levels are reduced, thus halting premature septum splitting.

Searching for SARS-CoV-2 in Cancer Tissues: Results of an Extensive Methodologic Approach based on ACE2 and Furin Expression.

SARS-CoV-2 pandemics have been massively characterized on a global scale by the rapid generation of in-depth genomic information. The main entry gate of SARS-CoV-2 in human cells is the angiotensin-converting enzyme 2 (ACE2) receptor. The expression of this protein has been reported in several human tissues, suggesting a correlation between SARS-CoV-2 organotropism and ACE2 distribution. In this study, we selected (a series of) 90 patients who were submitted to surgery for tumor removal between the beginning of the SARS-CoV-2 pandemic and the closure of operating rooms (by the end of March 2020) in two different countries-Portugal and Brazil. We evaluated the expressions of ACE2 and furin (another important factor for virus internalization) in colon (n = 60), gastric (n = 19), and thyroid (n = 11) carcinomas. In a subseries of cases with PCR results for SARS-CoV-2 detection in the peri-operatory window (n = 18), we performed different methodological approaches for viral detections in patient tumor samples. Our results show that colon and gastric carcinomas display favorable microenvironments to SARS-CoV-2 tropism, presenting high expression levels of ACE2 and furin. From the subseries of 18 cases, 11 tested positive via PCR detection performed in tumor blocks; however, a direct association between the ACE2 expression and SARS-CoV-2 infection was not demonstrated in cancer cells using histology-based techniques, such as immunohistochemistry or in situ hybridization. This study raises the possibility of ACE2-mediated viral tropism in cancer tissues to be clarified in future studies.

Simultaneous studies of gene expression and alternative polyadenylation in primary human immune cells.

Transcription termination in eukaryotic cells involves the recognition of polyadenylation signals (PAS) that signal the site of pre-mRNA cleavage and polyadenylation. Most eukaryotic genes contain multiple PAS that are used by alternative polyadenylation (APA), a co-transcriptional process that increases transcriptomic diversity and modulates the fate of the mRNA and protein produced. However, current tools to pinpoint the relationship between mRNAs in different subcellular fractions and the gene expression outcome are lacking, particularly in primary human immune cells, which, due to their nature, are challenging to study. Here, we describe an integrative approach using subcellular fractionation and RNA isolation, chromatin-bound and nucleoplasmic RNA-Sequencing, 3' RNA-Sequencing and bioinformatics, to identify accurate APA mRNA isoforms and to quantify gene expression in primary human macrophages. Our protocol includes macrophage differentiation and polarization, co-culture with cancer cells, and gene silencing by siRNA. This method allows the simultaneous identification of macrophage APA mRNA isoforms integrated with the characterization of nuclear APA events, the identification of the molecular mechanisms involved, as well as the gene expression alterations caused by the cancer-macrophage crosstalk. With this methodology we identified macrophage APA mRNA signatures driven by the cancer cells that alter the macrophage inflammatory and transcriptomic profiles, with consequences for macrophage physiology and tumor evasion.

Absence of tmRNA Has a Protective Effect against Fluoroquinolones in Streptococcus pneumoniae.

The transfer messenger RNA (tmRNA), encoded by the ssrA gene, is a small non-coding RNA involved in trans-translation that contributes to the recycling of ribosomes stalled on aberrant mRNAs. In most bacteria, its inactivation has been related to a decreased ability to respond to and recover from a variety of stress conditions. In this report, we investigated the role of tmRNA in stress adaptation in the human pathogen Streptococcus pneumoniae. We constructed a tmRNA deletion mutant and analyzed its response to several lethal stresses. The ΔssrA strain grew slower than the wild type, indicating that, although not essential, tmRNA is important for normal pneumococcal growth. Moreover, deletion of tmRNA increased susceptibility to UV irradiation, to exogenous hydrogen peroxide and to antibiotics that inhibit protein synthesis and transcription. However, the ΔssrA strain was more resistant to fluoroquinolones, showing twofold higher MIC values and up to 1000-fold higher survival rates than the wild type. Deletion of SmpB, the other partner in trans-translation, also reduced survival to levofloxacin in a similar extent. Accumulation of intracellular reactive oxygen species associated to moxifloxacin and levofloxacin treatment was also highly reduced (∼100-fold). Nevertheless, the ΔssrA strain showed higher intracellular accumulation of ethidium bromide and levofloxacin than the wild type, suggesting that tmRNA deficiency protects pneumococcal cells from fluoroquinolone-mediated killing. In fact, analysis of chromosome integrity revealed that deletion of tmRNA prevented the fragmentation of the chromosome associated to levofloxacin treatment. Moreover, such protective effect appears to relay mainly on inhibition of protein synthesis, since a similar effect was observed with antibiotics that inhibit that process. The emergence and spread of drug-resistant pneumococci is a matter of concern and these results contribute to a better comprehension of the mechanisms underlying fluoroquinolones action.

Small regulatory RNAs in Streptococcus pneumoniae: discovery and biological functions.

Streptococcus pneumoniae is a prominent human pathogen responsible for many severe diseases and the leading cause of childhood mortality worldwide. The pneumococcus is remarkably adept at colonizing and infecting different niches in the human body, and its adaptation to dynamic host environment is a central aspect of its pathogenesis. In the last decade, increasing findings have evidenced small RNAs (sRNAs) as vital regulators in a number of important processes in bacteria. In S. pneumoniae, a small antisense RNA was first discovered in the pMV158 plasmid as a copy number regulator. More recently, genome-wide screens revealed that the pneumococcal genome also encodes multiple sRNAs, many of which have important roles in virulence while some are implicated in competence control. The knowledge of the sRNA-mediated regulation in pneumococcus remains very limited, and future research is needed for better understanding of functions and mechanisms. Here, we provide a comprehensive summary of the current knowledge on sRNAs from S. pneumoniae, focusing mainly on the trans-encoded sRNAs.