The Emerging Role of PIWI-Interacting RNAs (piRNAs) in Gastrointestinal Cancers: An Updated Perspective

Gastrointestinal (GI) cancers produce ~3.4 million related deaths worldwide, comprising 35% of all cancer-related deaths. The high mortality among GI cancers is due to late diagnosis, the presence of metastasis and drug resistance development. Additionally, current clinical markers do not adequately guide patient management, thereby new and more reliable biomarkers and therapeutic targets are still needed for these diseases. RNA-seq technology has allowed the discovery of new types of RNA transcripts including PIWI-interacting RNAs (piRNAs), which have particular characteristics that enable these molecules to act via diverse molecular mechanisms for regulating gene expression. Cumulative evidence has described the potential role of piRNAs in the development of several tumor types as a likely explanation for certain genomic abnormalities and signaling pathways’ deregulations observed in cancer. In addition, these piRNAs might be also proposed as promising diagnostic or prognostic biomarkers or as potential therapeutic targets in malignancies. This review describes important topics about piRNAs including their molecular characteristics, biosynthesis processes, gene expression silencing mechanisms, and the manner in which these transcripts have been studied in samples and cell lines of GI cancers to elucidate their implications in these diseases. Moreover, this article discusses the potential clinical usefulness of piRNAs as biomarkers and therapeutic targets in GI cancers.

Elevation of cell-associated HIV-1 RNA transcripts in CSF CD4+ T cells, despite suppressive antiretroviral therapy, is linked to in vivo brain injury

Objective Despite effective antiretroviral therapy (ART), brain injury remains prevalent in people living with HIV-1 infection (PLHIV) possibly due to ART lacking direct inhibition of transcription with continued local production of viral transcripts and neurotoxic proteins, such as Tat, rather than cell-free whole virion toxicity. We quantified cell-associated (CA) HIV-1 RNA-transcripts in CSF and blood, in relation to proton Magnetic Resonance Spectroscopy (1H MRS) of major brain metabolites, in well characterised PLHIV. Methods RNA was extracted from cells in 16 paired samples of CSF and blood, from PLHIV on fully suppressive ART. HIV-1 CA-RNA copies were measured using the highly sensitive Double-R assay and normalized /million CD4+ T cells. 18-colour flow cytometry was used to count and analyse CD4+ T cells and monocytes in CSF and blood. The concentrations of major brain metabolites from 1H MRS in frontal white matter (FWM), posterior cingulate cortex (PCC), and caudate areas were measured. Brain injury in each voxel was defined using a composite score derived by principal component analysis. Results 14/16 CSF cell samples had quantifiable HIV-1 CA-RNA transcripts, at levels significantly higher than in their PBMCs (median 9,266 vs 185 copies /106 CD4+ T cells; p<0.0001). Higher levels of CSF transcripts were associated with greater brain injury in the FWM (Std beta=-0.73; p=0.007) and PCC (Std beta=-0.61; p=0.03). CSF cells were 91% memory T cells, equally CD4+ (median 3,605) cells and CD8+ T cells (3,632), but contained much fewer B cells (0.4 %), NK cells (2.0%) and monocytes (3.1%; 378 cells; >90% CD14+CD16+ phenotype). CXCR3+CD49d+integrin beta7-negative, CCR5+ CD4+ T cells were significantly enriched in CSF, compared with PBMC (p <0.001). Transcriptional activity in CSF cells was highly correlated with levels of transcriptional activity in CD4+ T cells in PBMC (r=0.76; p=0.002). In contrast, HIV-1 RNA in highly purified monocytes from PBMC was detected in only 6/16 samples. Conclusions Elevated HIV-1 transcripts in CSF cells were associated with in vivo brain injury, despite suppressive ART. The cellular source is most likely the predominant CXCR3+ CD49d+ integrin beta7- CCR5+ memory CD4+ T cells, not monocytes. Inhibitors of transcription to reduce local production of potentially neurotoxic proteins, should be developed.

Single-cell quantification of a broad RNA spectrum reveals unique noncoding patterns associated with cell types and states

The ability to interrogate total RNA content of single cells would enable better mapping of the transcriptional logic behind emerging cell types and states. However, current single-cell RNA-sequencing (RNA-seq) methods are unable to simultaneously monitor all forms of RNA transcripts at the single-cell level, and thus deliver only a partial snapshot of the cellular RNAome. Here we describe Smart-seq-total, a method capable of assaying a broad spectrum of coding and noncoding RNA from a single cell. Smart-seq-total does not require splitting the RNA content of a cell and allows the incorporation of unique molecular identifiers into short and long RNA molecules for absolute quantification. It outperforms current poly(A)-independent total RNA-seq protocols by capturing transcripts of a broad size range, thus enabling simultaneous analysis of protein-coding, long-noncoding, microRNA, and other noncoding RNA transcripts from single cells. We used Smart-seq-total to analyze the total RNAome of human primary fibroblasts, HEK293T, and MCF7 cells, as well as that of induced murine embryonic stem cells differentiated into embryoid bodies. By analyzing the coexpression patterns of both noncoding RNA and mRNA from the same cell, we were able to discover new roles of noncoding RNA throughout essential processes, such as cell cycle and lineage commitment during embryonic development. Moreover, we show that independent classes of short-noncoding RNA can be used to determine cell-type identity.

Influence of Escherichia coli on Expression of Selected Human Drug Addiction Genes

The impact of enteric microflora on the expression of genes associated with cocaine and amphetamine addiction was described. Human genome-wide experiments on RNA transcripts expressed in response to three selected Escherichia coli strains allowed for significant alteration (p > 0.05) of the linear regression model between HT-29 RNA transcripts associated with the KEGG pathway:hsa05030:Cocaine addiction after 3 h stimulation with intracellular pathogenic E. coli strain UM146 versus non-pathogenic E. coli Nissle 1917. Among the features influenced by the UM146 bacterial strain were visual learning, response to the presence of morphine, response to hypoxia, behavioral fear response and cognitive functions.

Developmental Aspects of SARS-CoV-2, Potential Role of Exosomes and Their Impact on the Human Transcriptome

With over 4.8 million deaths within 2 years, time is of the essence in combating COVID-19. The infection now shows devastating impacts on the younger population, who were not previously predicted to be vulnerable, such as in the older population. COVID-19-related complications have been reported in neonates whose mothers were infected with SARS-CoV-2 during pregnancy, and in children who get infected. Hence, a deeper understanding of the pathophysiology of COVID-19 during various developmental stages and placental transmission is essential. Although a connection has not yet been established between exosomal trafficking and the placental transmission of COVID-19, reports indicate that SARS-CoV-2 components may be trafficked between cells through exosomes. As the infection spreads, the transcriptome of cells is drastically perturbed, e.g., through the severe upregulation of several immune-related genes. Consequently, a major outcome of COVID-19 is an elevated immune response and the detection of viral RNA transcripts in host tissue. In this direction, this review focuses on SARS-CoV-2 virology, its in utero transmission from infected pregnant mothers to fetuses, SARS-CoV-2 and exosomal cellular trafficking, transcriptomic impacts, and RNA-mediated therapeutics against COVID-19. Future research will establish stronger connections between the above processes to develop diagnostic and therapeutic solutions towards COVID-19 and similar viral outbreaks.

Binding stoichiometry and structural model of the HIV-1 Rev/Importin beta complex

HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein Importin beta (ImpB), but how Rev associates with ImpB is poorly understood. Here we report biochemical, biophysical and structural studies of the ImpB/Rev complex. Binding and mutagenesis data reveal that ImpB associates with two Rev monomers through independent binding sites and that the N-terminal half of Rev's Arginine-Rich Motif (ARM) is a primary ImpB binding epitope. Small-angle X-ray scattering (SAXS), crosslinking mass spectrometry and compensatory mutagenesis data suggest a structural model in which one Rev monomer binds to the C-terminal half of ImpB with Rev helix alpha-2 roughly parallel to the HEAT-repeat superhelical axis while the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by ImpB and highlight an atypical binding behaviour that distinguishes Rev from canonical cellular ImpB cargos.

Preparing Planarian Cells for High-content Fluorescence Microscopy Using RNA in situ Hybridization and Immunocytochemistry

High-content fluorescence microscopy combines the efficiency of high-throughput techniques with the ability to extract quantitative information from biological systems. The planarian community has developed sensitive and robust assays for whole animals, yet cell based assays, despite their practical aspects, have not been explored to the same extent. Here we describe a modular collection of detailed protocols adapted for fixed planarian cells that enable multiplexed measurements of biomarkers in microwell plates. Methods include the detection of RNA transcripts by RNA fluorescent in situ hybridization combined with tyramide signal amplification using hapten-labeled riboprobes. In addition, immunocytochemical protocols for quantifying proliferating cells by the detection of phosphorylated histone H3 as well as 5-bromo-2'-deoxyuridine incorporation into the nuclear genome are described. The assays are compatible with planarians of virtually any size, as the tissue is disaggregated into a single cell suspension before fixation and staining. By sharing many reagents with established planarian whole mount staining protocols, preparation of samples for high-content microscopy adoption requires little additional investment. Recommendations for successful experimental workflows and common sources of errors are discussed.

A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue

Endothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.

Non-coding RNA-Encoded Peptides/Proteins in Human Cancer: The Future for Cancer Therapy

: Although non-coding RNAs (ncRNAs) were initially thought to be a class of RNA transcripts with no encoding capability, it has been established that some ncRNAs actually contain open reading frames (ORFs), which can be translated into micropeptides or microproteins. Recent studies have reported that ncRNAs-derived micropeptides/microproteins have regulatory functions on various biological and oncological processes. Some of these micropeptides/microproteins act as tumor inhibitors and some as tumor inducers. Understanding the carcinogenic role of ncRNAs-encoded micropeptides/microproteins seems to pose potential challenges to cancer research and offer promising practical perspectives on cancer treatment. In this review, we summarized the present information on the association of ncRNAs-derived micropeptides/microproteins with different types of human cancers. We also mentioned their carcinogenic mechanisms in cancer metabolism, signaling pathways, cell proliferation, angiogenesis, metastasis, and so on. Finally, we discussed the potential clinical value of these micropeptides/microproteins and their potential use in the diagnosis and treatment of cancer. This information may help discover, optimize, and develop new tools based on biological micropeptides/microproteins for the early diagnosis and development of anticancer drugs.