Biology subjects

Bastos, M. S.

Publications and source records attributed to Bastos, M. S..

4 recordsLinked to original sources

bioRxiv2024

Genome-wide mapping of RPA1 and RAD9 reveals the management of polycistronic transcription, replication initiation, and responses to replication stress in Leishmania

When exposed single-stranded DNA accumulates at stalled or collapsed replication forks, the replication stress response is triggered to prevent genome instability. Leishmania are parasitic eukaryotes where gene expression is universally polycistronic and whose plastic genomes facilitate rapid adaptations in response to stress, with evidence implicating intrinsic replication stress as a source. Little is known about the Leishmania replication stress response. In this study, we reveal the global dynamics of the replication stress response in L. major promastigotes by performing ChIP-seq on three key replication stress response proteins, {gamma}H2A, RPA1 and RAD9, in the absence and presence of replication stress. We show that common hotspots of replication stress correlate with DNA replication initiation and transcription termination in Leishmania. When DNA replication is stalled, replication stress response factors accumulate at early S-phase origins, with a signal pattern reminiscent of bidirectional replication fork progression. Under conditions of chronic replication stress, increased accumulation of replication stress response factors emerges at wider sites of transcription initiation, suggesting Leishmania may possess compensatory strategies to limit the effects of replication stress and ensure DNA replication can complete under these conditions. In contrast, chronic replication stress enhances RSR factor accumulation at transcription termination sites, highlighting these regions as key replication stress hotspots in Leishmania. Lastly, variations in RPA dynamics in ATR-deficient cells uncover crucial roles of this protein kinase in managing polycistronic transcription and DNA replication, particularly under replication stress, in Leishmania. SummaryStrict controls operate to precisely copy an organisms DNA. However, cells need ways to rapidly adapt and respond to stimuli. In some cases, these beneficial adaptations come from problems during replication. Leishmania parasites cause serious neglected infections in humans and animals across the worlds tropics and sub-tropics. Remarkably, recent evidence suggests that Leishmania DNA experiences enhanced stress during replication that can drive its ability to rapidly adapt in response to stress. How Leishmania respond to DNA replication stress is still poorly understood. Here, using a genome-wide approach to map the locations of key proteins that manage DNA replication stress and maintain genome integrity, we show hotspots of DNA replication stress coincide with start sites of DNA replication and regions of transcription termination.

molecular biology↗

bioRxiv2024

Cap-independent translation directs stress-induced differentiation of the protozoan parasite Toxoplasma gondii

Translational control mechanisms modulate microbial latency of eukaryotic pathogens, enabling them to evade immunity and drug treatments. The protozoan parasite Toxoplasma gondii persists in hosts by differentiating from proliferative tachyzoites to latent bradyzoites, which are housed inside tissue cysts. Transcriptional changes facilitating bradyzoite conversion are mediated by a Myb domain transcription factor called BFD1, whose mRNA is present in tachyzoites but not translated into protein until stress is applied to induce differentiation. We addressed the mechanisms by which translational control drives BFD1 synthesis in response to stress-induced parasite differentiation. Using biochemical and molecular approaches, we show that the 5-leader of BFD1 mRNA is sufficient for preferential translation upon stress. The translational control of BFD1 mRNA is maintained when ribosome assembly near its 5-cap is impaired by insertion of a 5-proximal stem-loop and upon knockdown of the Toxoplasma cap-binding protein, eIF4E1. Moreover, we show that a trans-acting RNA-binding protein called BFD2/ROCY1 is necessary for cap-independent translation of BFD1 through its binding to the 5-leader. Translation of BFD2 mRNA is also suggested to be preferentially induced under stress, but by a cap-dependent mechanism. These results show that translational control and differentiation in Toxoplasma proceed through cap-independent mechanisms in addition to canonical cap-dependent translation. Our identification of cap-independent translation in protozoa underscores the antiquity of this mode of gene regulation in cellular evolution and its central role in stress-induced life-cycle events.

microbiology↗

bioRxiv2023

mRNA cap-binding protein eIF4E1 is a novel regulator of Toxoplasma gondii latency

The protozoan parasite Toxoplasma gondii causes serious opportunistic disease due to its ability to persist in patients as latent tissue cysts. The molecular mechanisms coordinating conversion between proliferative parasites (tachyzoites) and dormant cysts (bradyzoites) are not fully understood. We previously showed that phosphorylation of eIF2 accompanies bradyzoite formation, suggesting that this clinically relevant process involves regulation of mRNA translation. In this study, we investigated the composition and role of eIF4F multi-subunit complexes in translational control. Using CLIPseq, we find that the cap-binding subunit, eIF4E1, localizes to the 5-end of all tachyzoite mRNAs, many of which show evidence of stemming from heterogenous transcriptional start sites. We further show that eIF4E1 operates as the predominant cap-binding protein in two distinct eIF4F complexes. Using genetic and pharmacological approaches, we found that eIF4E1 deficiency triggers efficient spontaneous formation of bradyzoites without stress induction. Consistent with this result, we also show that stress-induced bradyzoites exhibit reduced eIF4E1 expression. Overall, our findings establish a novel role for eIF4F in translational control required for parasite latency and microbial persistence. SignificanceToxoplasma gondii is an opportunistic pathogen of importance to global human and animal health. There are currently no chemotherapies targeting the encysted form of the parasite. Consequently, a better understanding of the mechanisms controlling encystation are required. Here we show that the mRNA cap-binding protein, eIF4E1, is involved in regulating the encystation process. Encysted parasites reduce eIF4E1 levels and depletion of eIF4E1 decreases the translation of ribosome-associated machinery and drives Toxoplasma encystation. Together, these data reveal a new layer of mRNA translational control that regulates parasite encystation and latency.

microbiology↗

bioRxiv2021

m6A RNA methylation facilitates pre-mRNA 3-end formation and is essential for viability of Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that can cause serious opportunistic disease in the immunocompromised or through congenital infection. To progress through its life cycle, Toxoplasma relies on multiple layers of gene regulation that includes an array of transcription and epigenetic factors. Over the last decade, the modification of mRNA has emerged as another important layer of gene regulation called epitranscriptomics. Here, we report that epitranscriptomics machinery exists in Toxoplasma, namely the methylation of adenosines (m6A) in mRNA transcripts. We identified novel components of the m6A methyltransferase complex and determined the distribution of m6A marks within the parasite transcriptome. m6A mapping revealed the modification to be preferentially located near the 3-boundary of mRNAs within the consensus sequence, YGCAUGCR. Knockdown of the m6A writer enzyme METTL3 resulted in diminished m6A marks, loss of a target transcript, and a complete arrest of parasite replication. Furthermore, we examined the two proteins in Toxoplasma that possess YTH domains, which bind m6A marks, and showed them to be integral members of the cleavage and polyadenylation machinery that catalyzes the 3-end processing of pre-mRNAs. Together, these findings establish that the m6A epitranscriptome is essential for parasite viability by contributing to the processing of mRNA 3-ends. Author SummaryToxoplasma gondii is a parasite of medical importance that causes disease upon immuno-suppression. Uncovering essential pathways that the parasite uses for its basic biological processes may reveal opportunities for new anti-parasitic drug therapies. Here, we describe the machinery that Toxoplasma uses to modify specific adenosine residues within its messenger RNAs (mRNA) by N6-adenosine methylation (m6A). We discovered that m6A mRNA methylation is prevalent in multiple stages of the parasite life cycle and is required for parasite replication. We also establish that m6A plays a major role in the proper maturation of mRNA. Two proteins that bind m6A modifications on mRNA associate with factors responsible for the cleavage and final processing steps of mRNA maturation. Since all of the machinery is conserved from plants to Toxoplasma and other related parasites, we propose that this system operates similarly in these organisms.

microbiology↗