TbAGO1, an Argonaute protein required for RNA interference, is involved in mitosis and chromosome segregation in Trypanosoma brucei

BACKGROUND: RNA silencing processes are widespread in almost all eukaryotic organisms. They have various functions including genome protection, and the control of gene expression, development and heterochromatin formation. RNA interference (RNAi) is the post-transcriptional destruction of RNA, which is mediated by a ribonucleoprotein complex that contains, among several components, RNA helicases and Argonaute proteins. RNAi is functional in trypanosomes, protozoan parasites that separated very early from the main eukaryotic lineage and exhibit several intriguing features in terms of the control of gene expression. In this report, we investigated the functions of RNAi in Trypanosoma brucei. RESULTS: By searching through genome databases, novel Argonaute-like proteins were identified in several protozoa that belong to the kinetoplastid order, a group of organisms that diverged early from the main eukaryotic lineage. T. brucei possesses two Argonaute-like genes termed TbAGO1 and TbPWI1. Dual transient transfection assays suggest that TbAGO1, but not TbPWI1, is involved in RNAi. The entire coding region of TbAGO1 was deleted by double gene knockout. TbAGO1-/- cells turned out to be completely resistant to RNAi generated either by transfected double-stranded RNA or by expression of an inverted repeat. TbAGO1-/- cells were viable but showed a dramatically reduced growth rate. This was probably due to defects in mitosis and abnormal chromosome segregation as revealed by in situ analysis. The RNAi and growth phenotypes were complemented by the inducible expression of a GFP::TbAGO1 fusion protein that revealed the cytoplasmic location of the protein. CONCLUSIONS: The requirement of TbAGO1 for RNAi in trypanosomes demonstrates the evolutionary ancient involvement of Argonaute proteins in RNAi silencing processes. RNAi-deficient TbAGO1-/- cells showed numerous defects in chromosome segregation and mitotic spindle assembly. We propose a working hypothesis in which RNAi would be involved in heterochromatin formation at the centromere and therefore in chromosome segregation

The Schizosaccharomyces pombe JmjC-protein, Msc1, prevents H2A.Z localization in centromeric and subtelomeric chromatin domains

Eukaryotic genomes are repetitively packaged into chromatin by nucleosomes, however they are regulated by the differences between nucleosomes, which establish various chromatin states. Local chromatin cues direct the inheritance and propagation of chromatin status via self-reinforcing epigenetic mechanisms. Replication-independent histone exchange could potentially perturb chromatin status if histone exchange chaperones, such as Swr1C, loaded histone variants into wrong sites. Here we show that in Schizosaccharomyces pombe, like Saccharomyces cerevisiae, Swr1C is required for loading H2A.Z into specific sites, including the promoters of lowly expressed genes. However S. pombe Swr1C has an extra subunit, Msc1, which is a JumonjiC-domain protein of the Lid/Jarid1 family. Deletion of Msc1 did not disrupt the S. pombe Swr1C or its ability to bind and load H2A.Z into euchromatin, however H2A.Z was ectopically found in the inner centromere and in subtelomeric chromatin. Normally this subtelomeric region not only lacks H2A.Z but also shows uniformly lower levels of H3K4me2, H4K5, and K12 acetylation than euchromatin and disproportionately contains the most lowly expressed genes during vegetative growth, including many meiotic-specific genes. Genes within and adjacent to subtelomeric chromatin become overexpressed in the absence of either Msc1, Swr1, or paradoxically H2A.Z itself. We also show that H2A.Z is N-terminally acetylated before, and lysine acetylated after, loading into chromatin and that it physically associates with the Nap1 histone chaperone. However, we find a negative correlation between the genomic distributions of H2A.Z and Nap1/Hrp1/Hrp3, suggesting that the Nap1 chaperones remove H2A.Z from chromatin. These data describe H2A.Z action in S. pombe and identify a new mode of chromatin surveillance and maintenance based on negative regulation of histone variant misincorporation.publishedVersio

SWI/SNF-like chromatin remodeling factor Fun30 supports point centromere function in S. cerevisiae

Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4, coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture

Harnessing the power of resistant starch: a narrative review of its health impact and processing challenges

Starch is a primary energy storage for plants, making it an essential component of many plant-based foods consumed today. Resistant starch (RS) refers to those starch fractions that escape digestion in the small intestine and reach the colon where they are fermented by the microflora. RS has been repeatedly reported as having benefits on health, but ensuring that its content remains in food processing may be challenging. The present work focuses on the impact RS on health and explores the different processes that may influence its presence in foods, thus potentially interfering with these effects. Clinical evidence published from 2010 to 2023 and studying the effect of RS on health parameters in adult populations, were identified, using PUBMED/Medline and Cochrane databases. The search focused as well on observational studies related to the effect of food processes on RS content. While processes such as milling, fermentation, cooking and heating seem to have a deleterious influence on RS content, other processes, such as cooling, cooking time, storage time, or water content, may positively impact its presence. Regarding the influence on health parameters, there is a body of evidence suggesting an overall significant beneficial effect of RS, especially type 1 and 2, on several health parameters such as glycemic response, insulin resistance index, bowel function or inflammatory markers. Effects are more substantiated in individuals suffering from metabolic diseases. The effects of RS may however be exerted differently depending on the type. A better understanding of the influence of food processes on RS can guide the development of dietary intake recommendations and contribute to the development of food products rich in RS

Régulations génétique et moléculaire par ARN interférence chez Trypanosoma brucei

RNA interference (RNAi) is a phenomenon discovered in 1998 in which the presence of double-stranded RNA (dsRNA) in a cell leads to degradation of RNA of homologous sequence. RNAi is mediated by a ribonucleoprotein complex that contains short double stranded RNA and a member of a the Argonaute protein family. This thesis is focusing on RNAi in the protist Trypanosoma brucei. We first defined the degree of specificity and efficiency of RNAi generated after expression of dsRNA, parameters that were instrumental in the design of a software allowing selection of dsRNA for silencing experiments. Next, we searched for candidate genes coding for proteins potentially involved in RNAi. The best candidate is TbAGO1, a member of the Argonaute protein family characterised by an extra domain possibly involved in RNA binding. This gene is essential for RNAi. Its deletion leads to significant mitosis defects, that we established were due to defects in spindle formation and chromosome migration. A second phenotype in the absence of RNAi is the overexpression of retroposon (retrotransposons without LTR) transcripts, without a concomitant increase in retroposition. Both phenotypes are independent. We next demonstrated that presence of dsRNA leads to destruction of homologous RNA in the cytoplasm but could also induce transcriptional silencing of the corresponding gene. This type of mechanism could control expression of retroposons but also of genes in which these retro-elements are inserted.L'ARN interférence (ARNi) est un phénomène découvert en 1998 par lequel la présence d'ARN double brin au sein d'une cellule entraîne la dégradation d'ARN de séquence homologue. L'ARNi est effectué par un complexe ribonucléoprotéique contenant des petits ARN double brin et au moins une protéine de la famille Argonaute. Cette thèse a été consacrée à l'étude de l'ARNi chez le protozoaire Trypanosoma brucei. Nous avons d'abord défini les conditions d'utilisation de l'ARNi au niveau de la spécificité et de l'efficacité, paramètres qui ont servi à l'élaboration d'un logiciel permettant la sélection de l'ARN double brin pour les études fonctionnelles. Ensuite, nous avons recherché plusieurs gènes candidats codant pour des protéines participant à l'ARNi. Le meilleur d'entre eux, TbAGO1, appartient à la famille Argonaute et se caractérise par la présence d'un domaine supplémentaire, capable de lier les ARN. Il est essentiel pour l'ARNi chez le trypanosome. Sa délétion produit des défauts significatifs lors de la mitose et nous avons établi que l'ARNi contribue à la formation du fuseau mitotique et à la ségrégation des chromosomes. Un second phénotype observé en l'absence d'ARNi est la surexpression des ARN de deux types de rétroposons (rétrotransposons sans LTR), sans toutefois augmentation de leur activité de rétroposition. Les deux phénotypes sont indépendants l'un de l'autre. Nous avons ensuite démontré que la présence d'ARN double brin entraîne la destruction d'ARN cible de séquence homologue dans le cytoplasme mais peut aussi conduire à une extinction de la transcription du gène correspondant. Ce type de mécanisme pourrait non seulement contrôler l'expression des ARN des rétroposons, mais aussi celle des gènes dans lesquels ils sont insérés

Régulations génétique et moléculaire par ARN interférence chez Trypanosoma brucei

L'ARN interférence (ARNi) est un phénomène découvert en 1998 par lequel la présence d'ARN double brin au sein d'une cellule entraîne la dégradation d'ARN de séquence homologue. L'ARNi est effectué par un complexe ribonucléoprotéique contenant des petits ARN double brin et au moins une protéine de la famille Argonaute. Cette thèse a été consacrée à l'étude de l'ARNi chez le protozoaire Trypanosoma brucei. Nous avons d'abord défini les conditions d'utilisation de l'ARNi au niveau de la spécificité et de l'efficacité, paramètres qui ont servi à l'élaboration d'un logiciel permettant la sélection de l'ARN double brin pour les études fonctionnelles. Ensuite, nous avons recherché plusieurs gènes candidats codant pour des protéines participant à l'ARNi. Le meilleur d'entre eux, TbAGO1, appartient à la famille Argonaute et se caractérise par la présence d'un domaine supplémentaire, capable de lier les ARN. Il est essentiel pour l'ARNi chez le trypanosome. Sa délétion produit des défauts significatifs lors de la mitose et nous avons établi que l'ARNi contribue à la formation du fuseau mitotique et à la ségrégation des chromosomes. Un second phénotype observé en l'absence d'ARNi est la surexpression des ARN de deux types de rétroposons (rétrotransposons sans LTR), sans toutefois augmentation de leur activité de rétroposition. Les deux phénotypes sont indépendants l'un de l'autre. Nous avons ensuite démontré que la présence d'ARN double brin entraîne la destruction d'ARN cible de séquence homologue dans le cytoplasme mais peut aussi conduire à une extinction de la transcription du gène correspondant. Ce type de mécanisme pourrait non seulement contrôler l'expression des ARN des rétroposons, mais aussi celle des gènes dans lesquels ils sont insérésRNA interference (RNAi) is a phenomenon discovered in 1998 in which the presence of double-stranded RNA (dsRNA) in a cell leads to degradation of RNA of homologous sequence. RNAi is mediated by a ribonucleoprotein complex that contains short double stranded RNA and a member of a the Argonaute protein family. This thesis is focusing on RNAi in the protist Trypanosoma brucei. We first defined the degree of specificity and efficiency of RNAi generated after expression of dsRNA, parameters that were instrumental in the design of a software allowing selection of dsRNA for silencing experiments. Next, we searched for candidate genes coding for proteins potentially involved in RNAi. The best candidate is TbAGO1, a member of the Argonaute protein family characterised by an extra domain possibly involved in RNA binding. This gene is essential for RNAi. Its deletion leads to significant mitosis defects, that we established were due to defects in spindle formation and chromosome migration. A second phenotype in the absence of RNAi is the overexpression of retroposon (retrotransposons without LTR) transcripts, without a concomitant increase in retroposition. Both phenotypes are independent. We next demonstrated that presence of dsRNA leads to destruction of homologous RNA in the cytoplasm but could also induce transcriptional silencing of the corresponding gene. This type of mechanism could control expression of retroposons but also of genes in which these retro-elements are insertedPARIS-Museum Hist.Naturelle (751052304) / SudocSudocFranceF

Régulations génétique et moléculaire par ARN interférence chez Trypanosoma brucei

L'ARN interférence (ARNi) est un phénomène découvert en 1998 par lequel la présence d'ARN double brin au sein d'une cellule entraîne la dégradation d'ARN de séquence homologue. L'ARNi est effectué par un complexe ribonucléoprotéique contenant des petits ARN double brin et au moins une protéine de la famille Argonaute. Cette thèse a été consacrée à l'étude de l'ARNi chez le protozoaire Trypanosoma brucei. Nous avons d'abord défini les conditions d'utilisation de l'ARNi au niveau de la spécificité et de l'efficacité, paramètres qui ont servi à l'élaboration d'un logiciel permettant la sélection de l'ARN double brin pour les études fonctionnelles. Ensuite, nous avons recherché plusieurs gènes candidats codant pour des protéines participant à l'ARNi. Le meilleur d'entre eux, TbAGO1, appartient à la famille Argonaute et se caractérise par la présence d'un domaine supplémentaire, capable de lier les ARN. Il est essentiel pour l'ARNi chez le trypanosome. Sa délétion produit des défauts significatifs lors de la mitose et nous avons établi que l'ARNi contribue à la formation du fuseau mitotique et à la ségrégation des chromosomes. Un second phénotype observé en l'absence d'ARNi est la surexpression des ARN de deux types de rétroposons (rétrotransposons sans LTR), sans toutefois augmentation de leur activité de rétroposition. Les deux phénotypes sont indépendants l'un de l'autre. Nous avons ensuite démontré que la présence d'ARN double brin entraîne la destruction d'ARN cible de séquence homologue dans le cytoplasme mais peut aussi conduire à une extinction de la transcription du gène correspondant. Ce type de mécanisme pourrait non seulement contrôler l'expression des ARN des rétroposons, mais aussi celle des gènes dans lesquels ils sont insérésRNA interference (RNAi) is a phenomenon discovered in 1998 in which the presence of double-stranded RNA (dsRNA) in a cell leads to degradation of RNA of homologous sequence. RNAi is mediated by a ribonucleoprotein complex that contains short double stranded RNA and a member of a the Argonaute protein family. This thesis is focusing on RNAi in the protist Trypanosoma brucei. We first defined the degree of specificity and efficiency of RNAi generated after expression of dsRNA, parameters that were instrumental in the design of a software allowing selection of dsRNA for silencing experiments. Next, we searched for candidate genes coding for proteins potentially involved in RNAi. The best candidate is TbAGO1, a member of the Argonaute protein family characterised by an extra domain possibly involved in RNA binding. This gene is essential for RNAi. Its deletion leads to significant mitosis defects, that we established were due to defects in spindle formation and chromosome migration. A second phenotype in the absence of RNAi is the overexpression of retroposon (retrotransposons without LTR) transcripts, without a concomitant increase in retroposition. Both phenotypes are independent. We next demonstrated that presence of dsRNA leads to destruction of homologous RNA in the cytoplasm but could also induce transcriptional silencing of the corresponding gene. This type of mechanism could control expression of retroposons but also of genes in which these retro-elements are insertedPARIS-Museum Hist.Naturelle (751052304) / SudocSudocFranceF

Functional complementation of RNA interference mutants in trypanosomes.

BACKGROUND: In many eukaryotic cells, double-stranded RNA (dsRNA) triggers RNA interference (RNAi), the specific degradation of RNA of homologous sequence. RNAi is now a major tool for reverse-genetics projects, including large-scale high-throughput screens. Recent reports have questioned the specificity of RNAi, raising problems in interpretation of RNAi-based experiments. RESULTS: Using the protozoan Trypanosoma brucei as a model, we designed a functional complementation assay to ascertain that phenotypic effect(s) observed upon RNAi were due to specific silencing of the targeted gene. This was applied to a cytoskeletal gene encoding the paraflagellar rod protein 2 (TbPFR2), whose product is essential for flagellar motility. We demonstrate the complementation of TbPFR2, silenced via dsRNA targeting its UTRs, through the expression of a tagged RNAi-resistant TbPFR2 encoding a protein that could be immunolocalized in the flagellum. Next, we performed a functional complementation of TbPFR2, silenced via dsRNA targeting its coding sequence, through heterologous expression of the TbPFR2 orthologue gene from Trypanosoma cruzi: the flagellum regained its motility. CONCLUSIONS: This work shows that functional complementation experiments can be readily performed in order to ascertain that phenotypic effects observed upon RNAi experiments are indeed due to the specific silencing of the targetted gene. Further, the results described here are of particular interest when reverse genetics studies cannot be easily achieved in organisms not amenable to RNAi. In addition, our strategy should constitute a firm basis to elaborate functional-dissection studies of genes from other organisms

Use of bacterial magnetosomes in the magnetic hyperthermia treatment of tumours: A review

International audienceWe review the most recent and significant results published in the field of magnetotactic bacteria (MTB), in particular data relating to the use of bacterial magnetosomes in magnetic hyperthermia for the treatment of tumours. We review different methods for cultivating MTB and preparing suspensions of bacterial magnetosomes. As well as the production of magnetosomes, we also review key data on the toxicity of the magnetosomes as well as their heating and anti-tumour efficiencies. The toxicity and efficiency of magnetosomes needs to be understood and the risk–benefit ratio with which to evaluate their use in the magnetic hyperthermia treatment of tumours needs to be measured

Functional complementation of RNA interference mutants in trypanosomes-0

<p><b>Copyright information:</b></p><p>Taken from "Functional complementation of RNA interference mutants in trypanosomes"</p><p>BMC Biotechnology 2005;5():6-6.</p><p>Published online 9 Feb 2005</p><p>PMCID:PMC549545.</p><p>Copyright © 2005 Rusconi et al; licensee BioMed Central Ltd.</p> four copies of coding sequence and specific UTRs. Regions targeted by RNAi are highlighted. The coding sequence was targeted using CDS dsRNA and the UTRs were targeted all together with a set of dsRNA homologous to the 3' UTR, the intergenic UTR (igUTR) and the 5' UTR (UTRs MIX dsRNAs). (B) Not-to-scale representation of the constructs used for the transfection of WT cells (pTbPFR2TAG430 and pTbPFR2TAG430-ΔHLA; integration in the rDNA spacer) or cells (pPCGFP and pPCTcPFR2; integration in the tubulin intergenic region). Large boxes represent protein coding sequences (black boxes: proteins of interest; grey boxes: antibiotic-resistance activities). Each plasmid was linearized with the indicated restriction enzyme prior to transfection into the cells. : 3' UTR of the aldolase gene; : 5' or 3' UTR of the actin gene; : EP procyclin intergenic region; : tubulin intergenic region