Biology subjects

Abascal-Palacios, G.

Publications and source records attributed to Abascal-Palacios, G..

5 recordsLinked to original sources

bioRxiv2026

Structural characterization of human endogenous retrovirus integration and strand transfer inhibition

Human endogenous retroviruses (hERVs) are remnants of ancestral retroviral infections that have shaped the human genome through their capacity to mobilize and retrotranspose. Among these, hERV-K (HML-2) remains the most recently active family and its dysregulation is strongly associated with diverse cancers and neurodegenerative pathologies, yet the structural basis of its integration remains poorly understood. Here, we combine activity assays with high-resolution cryo-electron microscopy to resolve the hERV-K integration machinery in three distinct states: asymmetric target-DNA engagement, strand transfer, and pharmacological inhibition. Our structures reveal a compact architecture defined by a unique organization of the outer integrase domains, which distinguishes hERV-K from other known retroviral intasomes. Biochemical validation confirms the catalytic competence of this compact tetrameric assembly, which relies on specialized polar motifs to optimize synaptic stability while retaining sensitivity to competitive antagonism by strand transfer inhibitors. Notably, beyond canonical restriction by Raltegravir, we discovered that the drug binding stabilizes an unanticipated, "closed" conformation not observed in previously characterized intasomes. Together, these findings elucidate the molecular mechanism of endogenous retroviral integration and provide a structural framework for rational therapeutic targeting of hERV-K-driven diseases.

biophysics↗

bioRxiv2023

A synthetic CRISPR-Cas nuclease with expanded enzymatic activities

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonucleases have revolutionized biotechnology for their potential as programmable genome editors. Yet, most natural nucleases and their variants have limitations. Here, we report a fully synthetic CRISPR-associated (Cas) nuclease (-synCas) designed by Ancestral Sequence Reconstruction (ASR) that displays a set of robust and distinct targeting properties, not found in any other known CRISPR-Cas Class 2 system. We show that -synCas is a PAMless nuclease able to catalyse RNA-guided, specific cleavage of dsDNA, ssDNA and ssRNA. The synthetic enzyme is also capable of sequence-nonspecific degradation of dsDNA, ssDNA and ssRNA following activation by complementary dsDNA, ssDNA and ssRNA targets. Furthermore, -synCas exhibits a robust genome editing activity in human cells and bacteria. Cryo-electron microscopy structures of -synCas ternary and quaternary complexes provide a framework to understand the structural basis for its expanded enzymatic activities. The capability for programmable multimodal targeting of virtually any nucleic acid sequence distinguishes -synCas as a promising new tool to extend current CRISPR-based technologies.

molecular biology↗

bioRxiv2021

The human RNA polymerase I structure reveals an HMG-like transcription factor docking domain specific to metazoans

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP-fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This dock II domain resembles a truncated HMG-box incapable of DNA-binding which may serve as a downstream-transcription factor binding platform in metazoans. Biochemical analysis and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG-box domain containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.

biochemistry↗

bioRxiv2021

Structural basis of Ty3 retrotransposon integration at RNA Polymerase III-transcribed genes

Retrotransposons are endogenous elements that have the ability to mobilise their DNA and integrate at different locations in the host genome. In budding yeast, the Ty3 retrotransposon integrates with an exquisite specificity in a narrow window upstream of RNA Polymerase III-transcribed genes, such as the genes of transfer RNAs, representing a paradigm for specific targeted integration. Here we present the cryo-EM reconstruction at 4.0 [A]-resolution of an active Ty3 strand-transfer complex (Ty3 intasome) caught in the act of integrating onto a specific tRNA gene bound to the RNA Polymerase III general transcription factor TFIIIB, which is required for Ty3 specific targeting. The structure unravels the molecular mechanisms underlying Ty3 integration specificity at RNA Polymerase III-transcribed genes and sheds light into the architecture of a retrotransposon integration machinery during the process of strand transfer at a genomic locus. The Ty3 intasome establishes contacts with a region of the TATA-binding protein (TBP), a subunit of TFIIIB, which is blocked by the ubiquitous transcription regulator negative cofactor 2 (NC2) in RNA Pol II-transcribed genes. A previously unrecognised chromodomain of the Ty3 integrase mediates non- canonical interactions with TFIIIB and the tRNA gene itself, defining with extreme precision the position of the integration site. Surprisingly, Ty3 retrotransposon tethering to TFIIIB topologically resembles LEDGF/p75 transcription factor targeting by HIV retrovirus, highlighting mechanisms of convergent evolution by unrelated mobile elements and host organisms. The Ty3 intasome-TFIIIB-tRNA promoter complex presented here represents a detailed molecular snapshot of a general transcription factors co-option by a mobile element, resulting in harmless integration into the host genome.

biophysics↗

bioRxiv2020

Structure of human RNA Polymerase III

ABSTRACTIn eukaryotes, RNA Polymerase (Pol) III is the enzyme specialised for the transcription of the entire pool of tRNAs and several other short, essential, untranslated RNAs. Pol III is a critical determinant of cellular growth and lifespan across the eukaryotic kingdom. Upregulation of Pol III transcription is often observed in cancer cells and causative Pol III mutations have been described in patients affected by severe neurodevelopmental disorders and hypersensitivity to viral infection.Harnessing CRISPR-Cas9 genome editing in HeLa cells, we isolated endogenous human Pol III and obtained a cryo-EM reconstruction at 4.0 Å. The structure of human Pol III allowed us to map the reported genetic mutations and rationalise them. Mutations causing neurodevelopmental defects cluster in hotspots that affect the stability and/or biogenesis of Pol III, thereby resulting in loss-of-function of the enzyme. Mutations affecting viral sensing are located in the periphery of the enzyme in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing activity.Furthermore, integrating x-ray crystallography and SAXS data, we describe the structure of the RPC5 C-terminal extension, which is absent in lower eukaryotes and not visible in our EM map. Surprisingly, experiments in living cells highlight a role for the RPC5 C-terminal extension in the correct assembly and stability of the human Pol III enzyme, thus suggesting an added layer of regulation during the biogenesis of Pol III in higher eukaryotes.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗