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[This corrects the article DOI: 10.3389/fcell.2021.713415.].
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The Leishmania developmental cycle comprises three main life forms in two hosts, indicating that the parasite is continually challenged due to drastic environmental changes. The disruption of this cycle is critical for discovering new therapies to eradicate leishmaniasis, a neglected disease that affects millions worldwide. Telomeres, the physical ends of chromosomes, maintain genome stability and cell proliferation and are potential antiparasitic drug targets. Therefore, understanding how telomere length is regulated during parasite development is vital. Here, we show that telomeres form clusters spread in the nucleoplasm of the three parasite life forms. We also observed that amastigotes telomeres are shorter than metacyclic and procyclic promastigotes and that in parasites with continuous in vitro passages, telomere length increases over time. These observed differences in telomere length among parasite's life stages were not due to lack/inhibition of telomerase since enzyme activity was detected in all parasite life stages, although the catalysis was temperature-dependent. These data led us to test if, similar to other eukaryotes, parasite telomere length maintenance could be regulated by Hsp83, the ortholog of Hsp90 in trypanosomatids, and Leishmania (LHsp90). Parasites were then treated with the Hsp90 inhibitor 17AAG. The results showed that 17AAG disturbed parasite growth, induced accumulation into G2/M phases, and telomere shortening in a time-dependent manner. It has also inhibited procyclic promastigote's telomerase activity. Besides, LHsp90 interacts with the telomerase TERT component as shown by immunoprecipitation, strongly suggesting a new role for LHsp90 as a parasite telomerase component involved in controlling telomere length maintenance and parasite life span.
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Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for the stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach. However, it can generate unintended transgenic integrations, while Cas9 prolonged-expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated ones, especially plant cells, is still challenging. In a protoplast system, previous studies claimed that such pitfalls would be averted by delivering pre-assembled ribonucleoprotein complexes (RNPs) composed of purified recombinant Cas9 enzyme and in vitro transcribed guide RNA (gRNA) molecules. We, therefore, aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with polyethylene glycol (PEG) 4000. We performed an effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene, and by applying two different exposure times to RNPs. Using a low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between the exposure time and mutation frequency. The mutation frequency was gRNA sequence- and exposure time-dependent. In the present study, we demonstrated that the suitability of RNP transfection was proven as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for the selection of gRNA suitable for the editing of the gene of interest will be highly useful for genome editing in maize, since the genome size and GC-content are large and high in the maize genome, respectively. Nevertheless, the large amplitude of mutations at the target site require scrutiny when checking mutations at off-target sites and potential safety concerns.
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Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Polietilenoglicóis/química , Ribonucleoproteínas/genética , Zea mays/genética , Edição de Genes/métodos , Genoma de Planta/genética , Células Vegetais/fisiologia , Protoplastos/fisiologia , RNA Guia de Cinetoplastídeos/genética , Zea mays/fisiologiaRESUMO
The Fragile Mental Retardation 1 gene (FMR1), at Xq27.3, encodes the fragile mental retardation protein (FMRP), and displays in its 5'-untranslated region a series of polymorphic CGG triplet repeats that may undergo dynamic mutation. Fragile X syndrome (FXS) is the leading cause of inherited intellectual disability among men, and is most frequently due to FMR1 full mutation and consequent transcription repression. FMR1 premutations may associate with at least two other clinical conditions, named fragile X-associated primary ovarian insufficiency (FXPOI) and tremor and ataxia syndrome (FXTAS). While FXPOI and FXTAS appear to be mediated by FMR1 mRNA accumulation, relative reduction of FMRP, and triplet repeat translation, FXS is due to the lack of the RNA-binding protein FMRP. Besides its function as mRNA translation repressor in neuronal and stem/progenitor cells, RNA editing roles have been assigned to FMRP. In this review, we provide a brief description of FMR1 transcribed microsatellite and associated clinical disorders, and discuss FMRP molecular roles in ribonucleoprotein complex assembly and trafficking, as well as aspects of RNA homeostasis affected in FXS cells.
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Ataxia/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Homeostase/genética , Insuficiência Ovariana Primária/genética , RNA/metabolismo , Ribonucleoproteínas/genética , Tremor/genética , Ataxia/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Feminino , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Humanos , Masculino , Mutação , Insuficiência Ovariana Primária/metabolismo , RNA/genética , Edição de RNA , Ribonucleoproteínas/metabolismo , Tremor/metabolismoRESUMO
The sorting of RNAs to specific regions of the cell for local translation represents an important mechanism directing protein distribution and cell compartmentalization. While significant progress has been made in understanding the mechanisms underlying the transport and localization of mRNAs, the mechanisms governing ribosome mobilization are less well understood. Ribosomes present in the cytoplasm of multiple cell types can form ribonucleoprotein complexes that also contain myosin-Va (Myo5a), a processive, actin-dependent molecular motor. Here, we report that Myo5a can be disassociated from ribosomes when ribonucleoprotein complexes are exposed to calcium, both in vitro and in vivo. We suggest that Myo5a may act as a molecular switch able to anchor or release ribosomes from the actin cytoskeleton in response to intracellular signaling.
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Cálcio/farmacologia , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Células 3T3-L1 , Animais , Cálcio/metabolismo , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Masculino , Camundongos , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Trypanosomes regulate gene expression mostly by posttranscriptional mechanisms, including control of mRNA turnover and translation efficiency. This regulation is carried out via certain elements located at the 3'-untranslated regions of mRNAs, which are recognized by RNA-binding proteins. In trypanosomes, trans-splicing is of central importance to control mRNA maturation. We have previously shown that TcDRBD4/PTB2, a trypanosome homolog of the human polypyrimidine tract-binding protein splicing regulator, interacts with the intergenic region of one specific dicistronic transcript, referred to as TcUBP (and encoding for TcUBP1 and TcUBP2, two closely kinetoplastid-specific proteins). In this work, a survey of TcUBP RNA processing revealed certain TcDRBD4/PTB2-regulatory elements within its intercistronic region, which are likely to influence the trans-splicing rate of monocistronic-derived transcripts. Furthermore, TcDRBD4/PTB2 overexpression in epimastigote cells notably decreased both UBP1 and UBP2 protein expression. This type of posttranscriptional gene regulatory mechanism could be extended to other transcripts as well, as we identified several other RNA precursor molecules that specifically bind to TcDRBD4/PTB2. Altogether, these findings support a model in which TcDRBD4/PTB2-containing ribonucleoprotein complexes can prevent trans-splicing. This could represent another stage of gene expression regulation mediated by the masking of trans-splicing/polyadenylation signals.