E-SMART: ENGINEERING OF MIRNA TARGET SITES IN CODING TRANSCRIPTS

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Applications of genome editing by programmable nucleases to the metabolic engineering of secondary metabolites

Publication . Leitão, Ana Lúcia; Costa, Marina C.; Enguita, Francisco J.

Genome engineering is a branch of modern biotechnology composed of a cohort of protocols designed to construct and modify a genotype with the main objective of giving rise to a desired phenotype. Conceptually, genome engineering is based on the so called genome editing technologies, a group of genetic techniques that allow either to delete or to insert genetic information in a particular genomic locus. Ten years ago, genome editing tools were limited to virus-driven integration and homologous DNA recombination. However, nowadays the uprising of programmable nucleases is rapidly changing this paradigm. There are two main families of modern tools for genome editing depending on the molecule that controls the specificity of the system and drives the editor machinery to its place of action. Enzymes such as Zn-finger and TALEN nucleases are protein-driven genome editors; while CRISPR system is a nucleic acid-guided editing system. Genome editing techniques are still not widely applied for the design of new compounds with pharmacological activity, but they are starting to be considered as promising tools for rational genome manipulation in biotechnology applications. In this review we will discuss the potential applications of programmable nucleases for the metabolic engineering of secondary metabolites with biological activity.

2017Journal article

Restricted access

Unzippers, resolvers and sensors: a structural and functional biochemistry tale of RNA helicases

Publication . Leitão, Ana; Costa, Marina C.; Enguita, Francisco J.

The centrality of RNA within the biological world is an irrefutable fact that currently attracts increasing attention from the scientific community. The panoply of functional RNAs requires the existence of specific biological caretakers, RNA helicases, devoted to maintain the proper folding of those molecules, resolving unstable structures. However, evolution has taken advantage of the specific position and characteristics of RNA helicases to develop new functions for these proteins, which are at the interface of the basic processes for transference of information from DNA to proteins. RNA helicases are involved in many biologically relevant processes, not only as RNA chaperones, but also as signal transducers, scaffolds of molecular complexes, and regulatory elements. Structural biology studies during the last decade, founded in X-ray crystallography, have characterized in detail several RNA-helicases. This comprehensive review summarizes the structural knowledge accumulated in the last two decades within this family of proteins, with special emphasis on the structure-function relationships of the most widely-studied families of RNA helicases: the DEAD-box, RIG-I-like and viral NS3 classes.

2015Journal article

Open access

Transcriptomic crosstalk between fungal invasive pathogens and their host cells: opportunities and challenges for next-generation sequencing methods

Publication . Enguita, Francisco J.; Costa, Marina C.; Fusco-Almeida, Ana; Mendes-Giannini, Maria; Leitão, Ana

Fungal invasive infections are an increasing health problem. The intrinsic complexity of pathogenic fungi and the unmet clinical need for new and more effective treatments requires a detailed knowledge of the infection process. During infection, fungal pathogens are able to trigger a specific transcriptional program in their host cells. The detailed knowledge of this transcriptional program will allow for a better understanding of the infection process and consequently will help in the future design of more efficient therapeutic strategies. Simultaneous transcriptomic studies of pathogen and host by high-throughput sequencing (dual RNA-seq) is an unbiased protocol to understand the intricate regulatory networks underlying the infectious process. This protocol is starting to be applied to the study of the interactions between fungal pathogens and their hosts. To date, our knowledge of the molecular basis of infection for fungal pathogens is still very limited, and the putative role of regulatory players such as non-coding RNAs or epigenetic factors remains elusive. The wider application of high-throughput transcriptomics in the near future will help to understand the fungal mechanisms for colonization and survival, as well as to characterize the molecular responses of the host cell against a fungal infection.

2016Journal article

Open access