Biology subjects

Faria, J. R.

Publications and source records attributed to Faria, J. R..

5 recordsLinked to original sources

bioRxiv2026

CRISPR-Cas9 precision editing of kinetochore protein phosphosite codons in Leishmania mexicana

Leishmania mexicana, like other trypanosomatids, possesses a unique kinetochore--the protein complex crucial for chromosome segregation during mitosis. To investigate the functional significance of specific phosphorylation sites on essential kinetochore proteins, we adapted a selection-free precision editing strategy using CRISPR-Cas9 in Leishmania mexicana promastigotes. Our method targeted genomic DNA with 160-bp double-stranded DNA repair templates and guide RNAs to introduce targeted modifications. We focused on six phosphosites within the kinetochore proteins KKT2, KKT4, and KKT7, generating phosphodeficient, phosphomimetic, and synonymous mutants for each site. Across 18 independent transfections, we achieved a successful editing rate of 27.5% as determined by PCR screening, with 30.4% of clones confirmed as edited by Sanger sequencing. A significant portion of these edited clones (22.1%) were homozygous. Despite these precise genomic modifications, none of the phosphosite mutant clones exhibited any apparent growth defects or cell cycle dysregulation, suggesting these phosphorylation sites individually may not be critical for these processes under standard culture conditions. To facilitate higher-throughput precision editing, we developed a Python script that automates the design of the 160-bp repair templates. This script uses a FASTA file, a codon usage table, and a simple configuration file to design templates with a single nonsynonymous mutation and additional synonymous mutations for screening purposes. It also generates a corresponding synonymous-only repair template and primers for both screening and repair template generation, offering a "ready-to-go" approach. While designed for Leishmania, this powerful tool is adaptable for use with other kinetoplastids.

microbiology↗

bioRxiv2025

Genome-wide mapping of DNA G-quadruplexes in Trypanosoma brucei chromatin reveals enrichment in coding regions

G-quadruplexes (G4s) are non-canonical DNA structures formed in guanine-rich sequences that are proposed to act as regulatory elements in trypanosomatid parasites, including Trypanosoma brucei, the causative agent of African sleeping sickness. However, their functional roles remain poorly understood, largely due to limited knowledge of their genomic distribution. Herein, we performed computational analyses across 63 trypanosomatid species uncovering high degree of variability in G4-prevalence and species-specific patterns. We generated the first genome-wide map of G4s in T. brucei using G4 chromatin immunoprecipitation followed by sequencing (G4 ChIP-Seq), which revealed a striking enrichment of G4s within coding DNA sequences (CDSs). This pattern diverges markedly from in silico predictions and previous genome-wide G4 mapping studies in humans, suggesting that G4s may play unique roles exclusive to trypanosome biology. To investigate their functional relevance, we profiled the transcriptome of T. brucei upon treatment with the G4-stabilising ligand PhenDC3. We observed that PhenDC3 exerts targeted gene expression perturbation of genes bearing G4s, particularly those located within coding CDSs, where G4s are mostly enriched. Altogether, our findings highlight a distinctive role for G4s in the regulation of gene expression in T. brucei and support their potential as therapeutic targets in the treatment of African sleeping sickness. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/666098v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1a2e0b5org.highwire.dtl.DTLVardef@4eba13org.highwire.dtl.DTLVardef@9a46fdorg.highwire.dtl.DTLVardef@aabc90_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Specialised RNA decay fine-tunes monogenic antigen expression in African trypanosomes

Antigenic variation is a sophisticated immune evasion strategy employed by many pathogens. Trypanosoma brucei expresses a single Variant-Surface-Glycoprotein (VSG) from a large genetic repertoire, which they periodically switch throughout an infection. Co-transcribed with the active-VSG within a specialised nuclear body are expression-site-associated-genes (ESAGs), involved in important host-parasite interactions, including protecting the parasite from human serum lytic effects, modulating the hosts innate immune response and uptake of essential nutrients. Despite expression within the same polycistron, there is a significant differential expression between ESAGs and VSGs (>140-fold), however, the regulatory mechanism has remained elusive for decades. Here, using a combination of genetic tools, super resolution microscopy, proteomics and transcriptomics analyses, we identified three novel proteins, which are recruited in a hierarchical manner, forming discreet sub-nuclear condensates that are developmentally regulated and negatively regulate ESAG transcripts. Among them, Expression-Site-Body-specific-protein-2 (ESB2) contains a nuclease domain that shares structural similarity to the endonuclease domain found in SMG6, a critical component of nonsense mediated decay in mammals. Mutation of key residues required for the nuclease activity impaired ESB2 localisation and function. Overall, our findings reveal a novel mechanism of post-transcriptional regulation and shed light on how specialised RNA decay can regulate expression of specific genes.

molecular biology↗

bioRxiv2025

A non-coding role for trypanosome VSG transcripts in allelic exclusion

Bloodstream-form African trypanosomes display mono-telomeric expression of a Variant Surface Glycoprotein (VSG) gene in an inter-chromosomally bridged transcription and splicing compartment, such that the dominant gene produces 10,000 times more transcript than excluded VSG genes. Antigenic variation, whereby parasites switch to express other VSGs, then underpins a robust host immune evasion strategy. Specific chromatin and RNA-associated factors are required to maintain VSG exclusion, but our understanding of the mechanisms involved remains incomplete. Here we show that the VSG transcript impacts allelic competition. We induced either specific translation blockade by recruiting MS2 coat protein to the active VSG 5-untranslated region, or VSG transcript depletion using RNA interference. Neither perturbation substantially compromised exclusion of native VSGs, as determined by transcriptomic analyses. In contrast, exclusion of a VSG transgene was compromised when the native transcript was transiently depleted. Notably, while both perturbations blocked cytokinesis, an additional round of DNA replication and mitosis was observed when the transcript, known to be stabilized by a bloodstream-form specific cyclin-like F-box protein, was translationally blocked. We conclude that the VSG transcript is a bi-functional coding and non-coding RNA that participates in allelic competition to establish exclusion. Significance statementAllelic exclusion mechanisms underpin immune evasion in parasites and olfaction in mammals but the mechanisms responsible remain mysterious. VSG exclusion factors have been identified in trypanosomes, while RNA has been implicated in olfactory receptor exclusion, and in var gene exclusion in the parasites that cause malaria. The current study demonstrates a role for RNA in VSG exclusion in trypanosomes.

molecular biology↗

bioRxiv2025

Precision-edited histone tails disrupt polycistronic gene expression controls in trypanosomes

Transcription of protein coding genes in trypanosomatids is atypical and almost exclusively polycistronic. In Trypanosoma brucei, approximately 150 polycistrons, and 8000 genes, are constitutively transcribed by RNA polymerase II. RNA polymerase II promoters are unconventional and characterised by regions of chromatin enriched for histones with specific patterns of post-translational modification on their highly divergent N-terminal tails. To investigate the roles of histone tail-residues in gene expression control in T. brucei, we engineered strains exclusively expressing novel mutant histones. We used an inducible CRISPR-Cas9 system to delete >40 native copies of histone H4, complementing the tandem arrays with a single ectopic H4 gene. The resulting histoneH4 strains were validated using whole-genome sequencing and transcriptome analysis. We then performed saturation mutagenesis of six histone H4 N-terminal tail lysine (K) residues and used multiplex amplicon-seq to profile the relative fitness of 384 distinct precision edited mutants. H4K10 mutations were not tolerated, but we could derive a panel of nineteen strains exclusively expressing novel H4K4 or H4K14 mutants. Both proteomic and transcriptomic analysis of H4K4Q mutants revealed significantly reduced expression of genes adjacent to RNA polymerase II promoters, where the glutamine (Q) mutation mimics an abnormally high level of acetylation. Thus, we present direct evidence for polycistronic expression control by histone H4 N-terminal tails in trypanosomes.

molecular biology↗