Biology subjects

Nascimento, L.

Publications and source records attributed to Nascimento, L..

3 recordsLinked to original sources

bioRxiv2025

Adaptively integrated sequencing and assembly of near-complete genomes

Advances in long-read sequencing (LRS) and assembly algorithms have made it possible to create highly complete genome assemblies for humans, animals and plants. However, ongoing development is needed to improve accessibility, affordability, and assembly quality and completeness. Cornetto is a new strategy in which we use programmable selective nanopore sequencing to focus LRS data production onto the unsolved regions of a nascent assembly. This improves assembly quality and streamlines the process, both for humans and non-human vertebrates. Cornetto enables us to generate highly complete diploid human genome assemblies using only nanopore LRS data, surpassing the quality of previous efforts at a fraction of the cost. Cornetto enables genome assembly from challenging sample types like human saliva. Finally, we obtain accurate assemblies for clinically-relevant repetitive loci at the extremes of the genome, demonstrating valid approaches for genetic diagnosis in facioscapulohumeral muscular dystrophy (FSHD) and MUC1-autosomal dominant tubulointerstitial kidney disease (MUC1-ADTKD).

genomics↗

bioRxiv2020

The trypanosome Variant Surface Glycoprotein mRNA is stabilized by an essential unconventional RNA-binding protein

Salivarian trypanosomes cause human sleeping sickness and economically important livestock diseases. The "bloodstream forms", which replicate extracellularly in the blood and tissue fluids of mammals, are coated by a monolayer of Variant Surface Glycoprotein (VSG). Switching of the expressed VSG gene is central to parasite pathogenicity because it enables the parasites to evade adaptive immunity via antigenic variation. Adequate levels of VSG expression - 10% of total protein and 7% of mRNA - are attained through very active RNA polymerase I transcription, efficient mRNA processing (trans splicing of a capped leader and polyadenylation), and high mRNA stability. We here show how VSG mRNA stability is maintained. Purification of the VSG mRNA with associated proteins specifically selected CFB2, an F-box mRNA-binding protein that lacks known RNA-binding domains. CFB2 binds to a stabilizing complex (MKT1-PBP1-XAC1-LSM12) that recruits poly(A) binding protein and a specialized cap-binding translation initiation complex, EIF4E6-EIF4G5. The interaction of CFB2 with MKT1 is essential for CFB2s expression-promoting activity, while the F-box auto-regulates CFB2 abundance via interaction with SKP1, a component of the ubiquitination machinery. The results of reporter experiments indicate that CFB2 acts via conserved sequences in the VSG mRNA 3-untranslated region. Depletion of CFB2 leads to highly specific loss of VSG mRNA. VSG expression is essential not only for antigenic variation but also for trypanosome cell division. Correspondingly, depletion of CFB2 causes cell cycle arrest, dramatic morphological abnormalities and trypanosome death.

molecular biology↗

bioRxiv2020

Essential PBP1-associated proteins of Trypanosoma brucei

Control of gene expression in kinetoplastids depends heavily on RNA-binding proteins that influence mRNA decay and translation. We previously showed that MKT1 interacts with PBP1, which in turn recruits LSM12 and poly(A) binding protein. MKT1 is recruited to mRNA by sequence-specific RNA-binding proteins, resulting in stabilisation of mRNA. We here show that PBP1, LSM12 and an additional 117-residue protein, XAC1 (Tb927.7.2780), are present in complexes that contain either MKT1 or MKT1L (Tb927.10.1490). All five proteins are present predominantly in the complexes, and there was evidence for a minor subset of complexes that contained both MKT1 and MKT1L. MKT1 appeared to be associated with many mRNAs, with the exception of those encoding ribosomal proteins. XAC1-containing complexes reproducibly contained RNA-binding proteins that were previously found associated with MKT1. In addition, however, XAC1- or MKT1-containing complexes specifically recruit one of the six translation initiation complexes, EIF4E6-EIF4G5; and yeast 2-hybrid assay results indicated that MKT1 interacts with EIF4G5. The C-terminus of MKT1L resembles MKT1: it contains MKT1 domains and a PIN domain that is probably not active as an endonuclease. MKT1L, however, also has an N-terminal extension with regions of low-complexity. Although MKT1L depletion inhibited cell proliferation, we found no evidence for specific interactions with RNA-binding proteins or mRNA. Deletion of the N-terminal extension, however, enabled MKT1L to interact with EIF4E6. We speculate that MKT1L may either enhance or inhibit the functions of MKT1-containing complexes.

molecular biology↗