Personalized 3-Gene Panel for Prostate Cancer Target Therapy

Many years and billions spent for research did not yet produce an effective answer to prostate cancer (PCa). Not only each human, but even each cancer nodule in the same tumor, has unique transcriptome topology. The differences go beyond the expression level to the expression control and networking of individual genes. The unrepeatable heterogeneous transcriptomic organization among men makes the quest for universal biomarkers and “fit-for-all” treatments unrealistic. We present a bioinformatics procedure to identify each patient’s unique triplet of PCa Gene Master Regulators (GMRs) and predict consequences of their experimental manipulation. The procedure is based on the Genomic Fabric Paradigm (GFP), which characterizes each individual gene by the independent expression level, expression variability and expression coordination with each other gene. GFP can identify the GMRs whose controlled alteration would selectively kill the cancer cells with little consequence on the normal tissue. The method was applied to microarray data on surgically removed prostates from two men with metastatic PCas (each with three distinct cancer nodules), and DU145 and LNCaP PCa cell lines. The applications verified that each PCa case is unique and predicted the consequences of the GMRs’ manipulation. The predictions are theoretical and need further experimental validation.

Cooperativity and Interdependency between RNA Structure and RNA–RNA Interactions

Complex RNA–RNA interactions are increasingly known to play key roles in numerous biological processes from gene expression control to ribonucleoprotein granule formation. By contrast, the nature of these interactions and characteristics of their interfaces, especially those that involve partially or wholly structured RNAs, remain elusive. Herein, we discuss different modalities of RNA–RNA interactions with an emphasis on those that depend on secondary, tertiary, or quaternary structure. We dissect recently structurally elucidated RNA–RNA complexes including RNA triplexes, riboswitches, ribozymes, and reverse transcription complexes. These analyses highlight a reciprocal relationship that intimately links RNA structure formation with RNA–RNA interactions. The interactions not only shape and sculpt RNA structures but also are enabled and modulated by the structures they create. Understanding this two-way relationship between RNA structure and interactions provides mechanistic insights into the expanding repertoire of noncoding RNA functions, and may inform the design of novel therapeutics that target RNA structures or interactions.

Standard Intein Gene Expression Ramps (SIGER) for protein-independent expression control

Coordination of multi-gene expression is one of the key challenges of metabolic engineering for the development of cell factories. Constraints on translation initiation and early ribosome kinetics of mRNA are imposed by features at the start of the gene, referred to as the “gene ramp”, such as rare codons and mRNA secondary structures forming in relation with the 5’UTR. These features strongly influence translation yield and protein quality by regulating ribosome distribution on mRNA strands. The utilization of genetic expression sequences, such as promoters and 5’UTRs in combination with different target genes leads to a wide variety of gene ramp compositions with irregular translation rates leading to unpredictable levels of protein yield and quality. Here, we present the Standard Intein Gene Expression Ramps (SIGER) system for controlling protein expression. The SIGER system uses inteins to uncouple a characterized gene ramp from a target protein. We generated sequence-specific gene expression sequences for two inteins (DnaB and DnaX) that display defined levels of protein expression. Additionally, we used inteins that possess the ability to release the C-terminal fusion protein in vivo to avoid impairment of protein functionality by the fused intein. Overall, our results show that SIGER systems are unique tools to mitigate the undesirable effects of gene ramp variation and to control the relative ratios of enzymes involved in molecular pathways.Graphical abstract

DExCon, DExogron, LUXon: on-demand expression control of endogenous genes reveals differential dynamics of Rab11 family members

CRISPR technology has made generation of gene knockouts widely achievable in cells. However, once inactivated, their reactivation remains difficult, especially in diploid cells. Here, we present DExCon (Doxycycline-mediated endogenous gene Expression Control), DExogron (DExCon combined with auxin-mediated targeted protein degradation) and LUXon (light responsive DExCon), approaches which combine one-step CRISPR-Cas9 mediated targeted knock-in of fluorescent proteins with an advanced Tet-inducible TRE3GS promoter. These approaches combine blockade of active gene transcription with the ability to reactivate transcription on demand, including activation of silenced genes. Systematic control can be exerted using doxycycline or spatiotemporally by light, and we demonstrate functional knockout/rescue in the closely related Rab11 family of vesicle trafficking regulators. Fluorescent protein knock-in results in bright signals compatible with low-light live microscopy from monoallelic modification, the potential to simultaneously image different alleles of the same gene and bypasses the need to work with clones. Protein levels are easily tunable to correspond with endogenous expression through cell sorting (DExCon), timing of light illumination (LUXon) or by exposing cells to different levels of auxin (DExogron). Furthermore, our approach allowed us to quantify previously unforeseen differences in vesicle dynamics, expression kinetics and protein stability among highly similar endogenous Rab11 family members and their colocalization in triple knock-in cells.GRAPHICAL ABSTRACTIN BRIEFWe describe development of DExCon, LUXon and DExogron approaches, where a single CRIPR/Cas9-mediated gene editing event can block endogenous gene expression, with the ability to reactivate expression encoded such that even silent genes can be expressed. Expression can be controlled systematically using doxycycline, or spatiotemporally by light, allowing fluorescent tagging of endogenous proteins and quantification of expression kinetics, protein dynamics and stability for highly similar genes such as members of the Rab11 family.

New Putative Long Non-Coding RNAs (lncRNA) Revealed by Pan-Transcriptome of the Emerging Human Pathogenic Fungus Talaromyces Marneffei

Previous genomic/transcriptomic analyses of Talaromyces marneffei (TM) unravelled relevant pathogenicity-related elements, as well as chromosomal regions potentially involved with the production of non-coding RNAs (ncRNAs), which have been parsimoniously reported in fungi. This manuscript describes a comprehensive pan-transcriptome assembly for TM that identifies a series of previously undetected genetic elements in this emerging pathogenic fungus. Our results confirm that ~58.28% of the 9,480 genes currently annotated in the TM genome are, in fact, transcribed in vivo and that ~23.6% of them may display alternative isomorphs. Moreover, we identified 585 transcripts that do not match any gene currently mapped in the genome, represented by 90 coding transcripts and 140 ncRNAs, including 48 long non-coding RNAs (lncRNAs). Overall, we expect that the novel elements described herein may contribute to improve the currently available Talaromyces databases and foster studies aiming at characterizing lncRNA-mediated gene expression control in fungi.

Multiplexed Cell-Based Diagnostic Devices for Detection of Renal Biomarkers Using Genetic Circuits

The number of synthetic biology based solutions employed in the medical industry is growing every year. The whole cell biosensors being one of them, have been proven valuable tools for developing low-cost, portable, personalized medicine alternatives to conventional techniques. Based on this concept, we targeted one of the major health problems in the world, Chronic Kidney Disease (CKD). To do so, we developed two novel biosensors for the detection of two important renal biomarkers; urea and uric acid. Using advanced gene expression control strategies we improved the operational range and the response profiles of each biosensor to meet clinical specifications. We further engineered these systems to enable multiplexed detection as well as an AND-logic gate operating system. Finally, we tested the applicability of these systems and optimized their working dynamics inside complex medium human blood serum. This study could help the efforts to transition from labor-intensive and expensive laboratory techniques to widely available, portable, low cost diagnostic options.

Transcriptional Control of Apical-Basal Polarity Regulators

Cell polarity is essential for many functions of cells and tissues including the initial establishment and subsequent maintenance of epithelial tissues, asymmetric cell division, and morphogenetic movements. Cell polarity along the apical-basal axis is controlled by three protein complexes that interact with and co-regulate each other: The Par-, Crumbs-, and Scrib-complexes. The localization and activity of the components of these complexes is predominantly controlled by protein-protein interactions and protein phosphorylation status. Increasing evidence accumulates that, besides the regulation at the protein level, the precise expression control of polarity determinants contributes substantially to cell polarity regulation. Here we review how gene expression regulation influences processes that depend on the induction, maintenance, or abolishment of cell polarity with a special focus on epithelial to mesenchymal transition and asymmetric stem cell division. We conclude that gene expression control is an important and often neglected mechanism in the control of cell polarity.

HIF1α-AS1 is a DNA:DNA:RNA triplex-forming lncRNA interacting with the HUSH complex

DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing have been observed in vitro, but the extent to which these interactions occur in cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1α-AS1 was retrieved as a top hit. Endogenous HIF1α-AS1 reduced the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1α-AS1 was down-regulated in pulmonary hypertension and loss-of-function approaches not only resulted in gene de-repression but also enhanced angiogenic capacity. As exemplified here with HIF1α-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control.

A comprehensive map of alternative polyadenylation in African American and European American lung cancer patients

Deciphering the post-transcriptional mechanisms (PTM) regulating gene expression is critical to understand the dynamics underlying transcriptomic regulation in cancer. Alternative polyadenylation (APA)—regulation of mRNA 3′UTR length by alternating poly(A) site usage—is a key PTM mechanism whose comprehensive analysis in cancer remains an important open challenge. Here we use a method and analysis pipeline that sequences 3′end-enriched RNA directly to overcome the saturation limitation of traditional 5′–3′ based sequencing. We comprehensively map the APA landscape in lung cancer in a cohort of 98 tumor/non-involved tissues derived from European American and African American patients. We identify a global shortening of 3′UTR transcripts in lung cancer, with notable functional implications on the expression of both coding and noncoding genes. We find that APA of non-coding RNA transcripts (long non-coding RNAs and microRNAs) is a recurrent event in lung cancer and discover that the selection of alternative polyA sites is a form of non-coding RNA expression control. Our results indicate that mRNA transcripts from EAs are two times more likely than AAs to undergo APA in lung cancer. Taken together, our findings comprehensively map and identify the important functional role of alternative polyadenylation in determining transcriptomic heterogeneity in lung cancer.

Translational gene expression control in Chlamydia trachomatis.

The human pathogen Chlamydia trachomatis proceeds through a multi phenotypic developmental cycle with each cell form specialized for different roles in pathogenesis. Understanding the mechanisms regulating this complex cycle has historically been hampered by limited genetic tools. In an effort to address this issue, we developed a translational control system to regulate gene expression in Chlamydia using a synthetic riboswitch. Here we demonstrate that translational control via a riboswitch can be used in combination with a wide range of promoters in C. trachomatis. The synthetic riboswitch E, inducible with theophylline, was used to replace the ribosome binding site of the synthetic promoter T5-lac, the native chlamydial promoter of the pgp4plasmid gene and an anhydrotetracycline responsive promoter. In all cases the riboswitch inhibited translation, and high levels of protein expression was induced with theophylline. Combining the Tet transcriptional inducible promoter with the translational control of the riboswitch resulted in strong repression and allowed for the cloning and expression of the potent chlamydial regulatory protein, HctB. The ability to control the timing and strength of gene expression independently from promoter specificity is a new and important tool for studying chlamydial regulatory and virulence genes.