Baltica: integrated splice junction usage analysis

Alternative splicing is a tightly regulated co- and post-transcriptional process contributing to the transcriptome diversity observed in eukaryotes. Several methods for detecting differential junction usage (DJU) from RNA sequencing (RNA-seq) datasets exist. Yet, efforts to integrate the results from DJU methods are lacking. Here, we present Baltica, a framework that provides workflows for quality control, de novo transcriptome assembly with StringTie2, and currently 4 DJU methods: rMATS, JunctionSeq, Majiq, and LeafCutter. Baltica puts the results from different DJU methods into context by integrating the results at the junction level. We present Baltica using 2 datasets, one containing known artificial transcripts (SIRVs) and the second dataset of paired Illumina and Oxford Nanopore Technologies RNA-seq. The data integration allows the user to compare the performance of the tools and reveals that JunctionSeq outperforms the other methods, in terms of F1 score, for both datasets. Finally, we demonstrate for the first time that meta-classifiers trained on scores of multiple methods outperform classifiers trained on scores of a single method, emphasizing the application of our data integration approach for differential splicing identification. Baltica is available at https://github.com/dieterich-lab/Baltica under MIT license.

Conservation of A-to-I RNA editing in bowhead whale and pig

RNA editing is a post-transcriptional process in which nucleotide changes are introduced into an RNA sequence, many of which can contribute to proteomic sequence variation. The most common type of RNA editing, contributing to nearly 99% of all editing events in RNA, is A-to-I (adenosine-to-inosine) editing mediated by double-stranded RNA-specific adenosine deaminase (ADAR) enzymes. A-to-I editing at ‘recoding’ sites results in non-synonymous substitutions in protein-coding sequences. Here, we present studies of the conservation of A-to-I editing in selected mRNAs between pigs, bowhead whales, humans and two shark species. All examined mRNAs–NEIL1, COG3, GRIA2, FLNA, FLNB, IGFBP7, AZIN1, BLCAP, GLI1, SON, HTR2C and ADAR2 –showed conservation of A-to-I editing of recoding sites. In addition, novel editing sites were identified in NEIL1 and GLI1 in bowhead whales. The A-to-I editing site of human NEIL1 in position 242 was conserved in the bowhead and porcine homologues. A novel editing site was discovered in Tyr244. Differential editing was detected at the two adenosines in the NEIL1 242 codon in both pig and bowhead NEIL1 mRNAs in various tissues and organs. No conservation of editing of KCNB1 and EEF1A mRNAs was seen in bowhead whales. In silico analyses revealed conservation of five adenosines in ADAR2, some of which are subject to A-to-I editing in bowheads and pigs, and conservation of a regulatory sequence in GRIA2 mRNA that is responsible for recognition of the ADAR editing enzyme.

Suppression of CPSF6 Enhances Apoptosis Through Alternative Polyadenylation-Mediated Shortening of the VHL 3′UTR in Gastric Cancer Cells

Alternative polyadenylation (APA) is an important RNA post-transcriptional process, which can generate diverse mRNA isoforms. Increasing evidence shows that APA is involved in cell self-renewal, development, immunity, and cancer. CPSF6 is one of the core proteins of CFIm complex and can modulate the APA process. Although it has been reported to play oncogenic roles in cancer, the underlying mechanisms remain unclear. The aim of the present study was to characterize CPSF6 in human gastric cancer (GC). We observed that CPSF6 was upregulated in GC. Knockdown of CPSF6 inhibited proliferation and enhanced apoptosis of GC cells both in vitro and in vivo. Global APA site profiling analysis revealed that knockdown of CPSF6 induced widespread 3′UTR shortening of genes in GC cells, including VHL. We also found CPSF6 negatively regulated the expression of VHL through APA and VHL short-3′UTR isoform enhanced apoptosis and inhibited cell growth in GC cells. Our data suggested that CPSF6-induced cell proliferation and inhibition of apoptosis were mediated by the preferential usage of poly(A) in VHL. Our data provide insights into the function of CPSF6 and may imply potential therapeutic targets against GC.

Comparative Analysis of Transcriptional Regulation Patterns: Understanding the Gene Expression Profile in Nucleocytoviricota

The nucleocytoplasmic large DNA viruses (NCLDV) possess unique characteristics that have drawn the attention of the scientific community, and they are now classified in the phylum Nucleocytoviricota. They are characterized by sharing many genes and have their own transcriptional apparatus, which provides certain independence from their host’s machinery. Thus, the presence of a robust transcriptional apparatus has raised much discussion about the evolutionary aspects of these viruses and their genomes. Understanding the transcriptional process in NCLDV would provide information regarding their evolutionary history and a better comprehension of the biology of these viruses and their interaction with hosts. In this work, we reviewed NCLDV transcription and performed a comparative functional analysis of the groups of genes expressed at different times of infection of representatives of six different viral families of giant viruses. With this analysis, it was possible to observe a temporal profile of their gene expression and set of genes activated in specific phases throughout the multiplication cycle as a common characteristic of this group. Due to the lack of information regarding the transcriptional regulation process of this group of pathogens, we sought to provide information that contributes to and opens up the field for transcriptional studies of other viruses belonging to Nucleocytoviricota.

KSR1-and ERK-dependent translational regulation of the epithelial-to-mesenchymal transition

The epithelial-to-mesenchymal transition (EMT) is considered a transcriptional process that induces a switch in cells from a polarized state to a migratory phenotype. Here we show that KSR1 and ERK promote EMT-like phenotype through the preferential translation of Epithelial-Stromal Interaction 1 (EPSTI1), which is required to induce the switch from E- to N-cadherin and coordinate migratory and invasive behavior. EPSTI1 is overexpressed in human colorectal cancer (CRC) cells. Disruption of KSR1 or EPSTI1 significantly impairs cell migration and invasion in vitro, and reverses EMT-like phenotype, in part, by decreasing the expression of N-cadherin and the transcriptional repressors of E-cadherin expression, ZEB1 and Slug. In CRC cells lacking KSR1, ectopic EPSTI1 expression restored the E- to N-cadherin switch, migration, invasion, and anchorage-independent growth. KSR1-dependent induction of EMT-like phenotype via selective translation of mRNAs reveals its underappreciated role in remodeling the translational landscape of CRC cells to promote their migratory and invasive behavior.

Internal promoters of the epothilone biosynthetic gene cluster and their activation in Myxococcus xanthus by CRISPR-dCas9

Background Multiple genes involving in a complex pathway are often clustered into a giant operon with no transcription terminator before the end, and this leads to frangibility of the transcriptional process and arduous engineering work to control the transcription of operon genes. Internal promoters might occur in operon to coordinate the transcription of individual genes, but their effects on the transcription of operon genes have been less investigated. Results Epothilones are a kind of polyketides synthesized by seven multifunctional enzymes, which are encoded by a 56-kb operon of the myxobacterium Sorangium cellulosum. In this study, we determined that the epothilone operon contained multiple internal promoters. These promoters were activatable by the CRISPRa technique, and the yields of epothilones were accordingly increased. However, the activation efficiencies of promoters in operon and separate forms were greatly different. Further, we found that the transcriptional levels of the epothilone genes were always increased at a greater extent than the epothilone yields, which suggested that the transcriptional activation of single genes probably had a weak effect on the final epothilone yield, and higher yield required an overall transcriptional increase of the multiple operonic genes. Finally, we combined the activation of the starting promoter PepoA together with internal promoters in different epothilone-producing strains, and obtained the highest 15-fold increase of epothilone yield in Myxococcus xanthus ZE5. Conclusions This is the first time to report the internal promoters in epothilone gene clusters in Myxococcus xanthus and the first time to assay the activation effects of these internal promoters by CRISPR-dCas9. Our results highlight that tuning internal promoter activities is critical to control the transcription of operon genes and the production efficiency of microbial secondary metabolites.

KSR1- and ERK-dependent Translational Regulation of the Epithelial-to-Mesenchymal Transition

The epithelial-to-mesenchymal transition (EMT) is considered a transcriptional process that induces a switch in cells from a polarized state to a migratory phenotype. Here we show that KSR1 and ERK promote EMT through the preferential translation of Epithelial-Stromal Interaction 1 (EPSTI1), which is required to induce the switch from E-to N-cadherin and coordinate migratory and invasive behavior. EPSTI1 is overexpressed in human colorectal cancer (CRC) cells. Disruption of KSR1 or EPSTI1 significantly impairs cell migration and invasion in vitro, and reverses EMT, in part, by decreasing the expression of N-cadherin and the transcriptional repressors of E-cadherin expression, ZEB1 and Slug. In CRC cells lacking KSR1, ectopic EPSTI1 expression restored the E-to N-cadherin switch, migration, invasion, and anchorage-independent growth. KSR1-dependent induction of EMT via selective translation of mRNAs reveals its underappreciated role in remodeling the translational landscape of CRC cells to promote their migratory and invasive behavior.

MYC as a driver of stochastic chromatin networks: implications for the fitness of cancer cells

The relationship between stochastic transcriptional bursts and dynamic 3D chromatin states is not well understood. Using an innovated, ultra-sensitive technique, we address here enigmatic features underlying the communications between MYC and its enhancers in relation to the transcriptional process. MYC thus interacts with its flanking enhancers in a mutually exclusive manner documenting that enhancer hubs impinging on MYC detected in large cell populations likely do not exist in single cells. Dynamic encounters with pathologically activated enhancers responsive to a range of environmental cues, involved <10% of active MYC alleles at any given time in colon cancer cells. Being the most central node of the chromatin network, MYC itself likely drives its communications with flanking enhancers, rather than vice versa. We submit that these features underlie an acquired ability of MYC to become dynamically activated in response to a diverse range of environmental cues encountered by the cell during the neoplastic process.

RNA editing in mesothelioma: a look forward

RNA editing is a post-transcriptional process increasing transcript diversity, thereby regulating different biological processes. We recently observed that mutations resulting from RNA editing due to hydrolytic deamination of adenosine increase during the development of mesothelioma, a rare cancer linked to chronic exposure to asbestos. This review gathers information from the published literature and public data mining to explore several aspects of RNA editing and their possible implications for cancer growth and therapy. We address possible links between RNA editing and particular types of mesothelioma genetic and epigenetic alterations and discuss the relevance of an edited substrate in the context of current chemotherapy or immunotherapy.

Engineering Transcriptional Interference through RNA Polymerase Processivity Control

ABSTRACTAntisense transcription is widespread in all kingdoms of life and has been shown to influence gene expression through transcriptional interference (TI), a phenomenon in which one transcriptional process negatively influences another in cis. The processivity, or uninterrupted transcription, of an RNA Polymerase (RNAP) is closely tied to levels of antisense transcription in bacterial genomes, but its influence on TI, while likely important, is not well-characterized. Here we show that TI can be tuned through processivity control via three distinct antitermination strategies: the antibiotic bicyclomycin, phage protein Psu, and ribosome-RNAP coupling. We apply these methods toward TI and tune ribosome-RNAP coupling to produce 38-fold gene repression due to RNAP collisions. We then couple protein roadblock and RNAP collisions to design minimal genetic NAND and NOR logic gates. Together these results show the importance of processivity control for strong TI and demonstrate the potential for TI to create sophisticated switching responses.