Mechanisms of robustness in gene regulatory networks involved in neural development.

The functions of living organisms are affected by different kinds of perturbation, both internal and external, which in many cases have functional effects and phenotypic impact. The effects of these perturbations become particularly relevant for multicellular organisms with complex body patterns and cell type heterogeneity, where transcriptional programs controlled by gene regulatory networks determine, for example, the cell fate during embryonic development. Therefore, an essential aspect of development in these organisms is the ability to maintain the functionality of their genetic developmental programs even in the presence of genetic variation, changing environmental conditions and biochemical noise, a property commonly termed robustness.

A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2.

Early detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proven crucial during the efforts to mitigate the effects of the COVID-19 pandemic. Several diagnostic methods have emerged in the past few months, each with different shortcomings and limitations. The current gold standard, RT-qPCR using fluorescent probes, relies on demanding equipment requirements plus the high costs of the probes and specific reaction mixes.

Ret Receptor Has Distinct Alterations and Functions in Breast Cancer.

Ret receptor tyrosine kinase is a proto-oncogene that participates in development of various cancers. Several independent studies have recently identified Ret as a key player in breast cancer. Although Ret overexpression and function have been under investigation, mainly in estrogen receptor positive breast cancer, a more comprehensive analysis of the impact of recurring Ret alterations in breast cancer is needed.

Accurate genome-wide predictions of spatio-temporal gene expression during embryonic development.

Comprehensive information on the timing and location of gene expression is fundamental to our understanding of embryonic development and tissue formation. While high-throughput in situ hybridization projects provide invaluable information about developmental gene expression patterns for model organisms like Drosophila, the output of these experiments is primarily qualitative, and a high proportion of protein coding genes and most non-coding genes lack any annotation. Accurate data-centric predictions of spatio-temporal gene expression will therefore complement current in situ hybridization efforts.

Non-coding RNA Expression, Function, and Variation during Drosophila Embryogenesis.

Long non-coding RNAs (lncRNAs) can often function in the regulation of gene expression during development; however, their generality as essential regulators in developmental processes and organismal phenotypes remains unclear. Here, we performed a tailored investigation of lncRNA expression and function during Drosophila embryogenesis, interrogating multiple stages, tissue specificity, nuclear localization, and genetic backgrounds. Our results almost double the number of annotated lncRNAs expressed at these embryonic stages.

The degree of enhancer or promoter activity is reflected by the levels and directionality of eRNA transcription.

Gene expression is regulated by promoters, which initiate transcription, and enhancers, which control their temporal and spatial activity. However, the discovery that mammalian enhancers also initiate transcription questions the inherent differences between enhancers and promoters. Here, we investigate the transcriptional properties of enhancers during embryogenesis using characterized developmental enhancers.

Promoter shape varies across populations and affects promoter evolution and expression noise.

Animal promoters initiate transcription either at precise positions (narrow promoters) or dispersed regions (broad promoters), a distinction referred to as promoter shape. Although highly conserved, the functional properties of promoters with different shapes and the genetic basis of their evolution remain unclear. Here we used natural genetic variation across a panel of 81 Drosophila lines to measure changes in transcriptional start site (TSS) usage, identifying thousands of genetic variants affecting transcript levels (strength) or the distribution of TSSs within a promoter (shape).

Alternative Splicing of G9a Regulates Neuronal Differentiation.

Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10) through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain.

Regulation of alternative splicing through coupling with transcription and chromatin structure.

Alternative precursor messenger RNA (pre-mRNA) splicing plays a pivotal role in the flow of genetic information from DNA to proteins by expanding the coding capacity of genomes. Regulation of alternative splicing is as important as regulation of transcription to determine cell- and tissue-specific features, normal cell functioning, and responses of eukaryotic cells to external cues. Its importance is confirmed by the evolutionary conservation and diversification of alternative splicing and the fact that its deregulation causes hereditary disease and cancer.

Coupling between transcription and alternative splicing.

The scenario of alternative splicing regulation is far more complex than the classical picture of a pre-mRNA being processed post-transcriptionally in more than one way. Introns are efficiently removed while transcripts are still being synthesized, supporting the idea of a co-transcriptional regulation of alternative splicing. Evidence of a functional coupling between splicing and transcription has recently emerged as it was observed that properties of one process may affect the outcome of the other.

Intragenic epigenetic changes modulate NCAM alternative splicing in neuronal differentiation.

Alternative splicing contributes to cell type-specific transcriptomes. Here, we show that changes in intragenic chromatin marks affect NCAM (neural cell adhesion molecule) exon 18 (E18) alternative splicing during neuronal differentiation. An increase in the repressive marks H3K9me2 and H3K27me3 along the gene body correlated with inhibition of polymerase II elongation in the E18 region, but without significantly affecting total mRNA levels.

Alternative splicing: a pivotal step between eukaryotic transcription and translation.

Alternative splicing was discovered simultaneously with splicing over three decades ago. Since then, an enormous body of evidence has demonstrated the prevalence of alternative splicing in multicellular eukaryotes, its key roles in determining tissue- and species-specific differentiation patterns, the multiple post- and co-transcriptional regulatory mechanisms that control it, and its causal role in hereditary disease and cancer. The emerging evidence places alternative splicing in a central position in the flow of eukaryotic genetic information, between transcription and translation, in that it can respond not only to various signalling pathways that target the splicing machinery but also to transcription factors and chromatin structure.

Perturbation of chromatin structure globally affects localization and recruitment of splicing factors.

Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics.

Connections between chromatin signatures and splicing.

Splicing and alternative splicing are involved in the expression of most human genes, playing key roles in differentiation, cell cycle progression, and development. Misregulation of splicing is frequently associated to disease, which imposes a better understanding of the mechanisms underlying splicing regulation. Accumulated evidence suggests that multiple trans-acting factors and cis-regulatory elements act together to determine tissue-specific splicing patterns.

Transcriptional elongation and alternative splicing.

Alternative splicing has emerged as a key contributor to proteome diversity, highlighting the importance of understanding its regulation. In recent years it became apparent that splicing is predominantly cotranscriptional, allowing for crosstalk between these two nuclear processes. We discuss some of the links between transcription and splicing, with special emphasis on the role played by transcription elongation in the regulation of alternative splicing events and in particular the kinetic model of alternative splicing regulation.

Epigenetics in alternative pre-mRNA splicing.

Alternative splicing plays critical roles in differentiation, development, and disease and is a major source for protein diversity in higher eukaryotes. Analysis of alternative splicing regulation has traditionally focused on RNA sequence elements and their associated splicing factors, but recent provocative studies point to a key function of chromatin structure and histone modifications in alternative splicing regulation. These insights suggest that epigenetic regulation determines not only what parts of the genome are expressed but also how they are spliced.

RNA Polymerase II Elongation at the Crossroads of Transcription and Alternative Splicing.

The elongation phase of transcription lies at the core of several simultaneous and coupled events leading to alternative splicing regulation. Although underestimated in the past, it is at this phase of the transcription cycle where complexes affecting the transcription machinery itself, chromatin structure, posttranscriptional gene regulation and pre-mRNA processing converge to regulate each other or simply to consolidate higher-order complexes and functions. This paper focuses on the multiple processes that take place during transcription elongation which ultimately regulate the outcome of alternative splicing decisions.

The serine/arginine-rich protein SF2/ASF regulates protein sumoylation.

Protein modification by conjugation of small ubiquitin-related modifier (SUMO) is involved in diverse biological functions, such as transcription regulation, subcellular partitioning, stress response, DNA damage repair, and chromatin remodeling. Here, we show that the serine/arginine-rich protein SF2/ASF, a factor involved in splicing regulation and other RNA metabolism-related processes, is a regulator of the sumoylation pathway. The overexpression of this protein stimulates, but its knockdown inhibits SUMO conjugation.

Intragenic chromatin modifications: a new layer in alternative splicing regulation.

The multiple steps that contribute to gene expression in the metazoan nucleus are highly integrated and regulated. Most of pre-mRNA processing is believed to occur co-transcriptionally and choices regarding alternative processing reactions are influenced by transcription. Several articles published in the last year unveiled a connection between chromatin structure and the splicing process, strengthening a view in which the dynamic of intragenic chromatin modifications has an important role regulating alternative splicing (AS) choices.

Playing inside the genes: Intragenic histone acetylation after membrane depolarization of neural cells opens a path for alternative splicing regulation.

Regulation of alternative splicing is coupled to transcription quality, the polymerase elongation rate being an important factor in modulating splicing choices. In a recently published work, we provide evidence that intragenic histone acetylation patterns can be affected by neural cell excitation in order to regulate alternative splicing of the neural cell adhesion molecule (NCAM) mRNA. This example illustrates how an extracellular stimulus can influence transcription-coupled alternative splicing, strengthening the link between chromatin structure, transcriptional elongation and mRNA processing.

Neuronal cell depolarization induces intragenic chromatin modifications affecting NCAM alternative splicing.

In search for physiological pathways affecting alternative splicing through its kinetic coupling with transcription, we found that membrane depolarization of neuronal cells triggers the skipping of exon 18 from the neural cell adhesion molecule (NCAM) mRNA, independently of the calcium/calmodulin protein kinase IV pathway. We show that this exon responds to RNA polymerase II elongation, because its inclusion is increased by a slow polymerase II mutant. Depolarization affects the chromatin template in a specific way, by causing H3K9 hyper-acetylation restricted to an internal region of the NCAM gene surrounding the alternative exon.