The role of deadenylation in the degradation of unstable mRNAs in trypanosomes

Removal of the poly(A) tail is the first step in the degradation of many eukaryotic mRNAs. In metazoans and yeast, the Ccr4/Caf1/Not complex has the predominant deadenylase activity, while the Pan2/Pan3 complex may trim poly(A) tails to the correct size, or initiate deadenylation. In trypanosomes, turnover of several constitutively-expressed or long-lived mRNAs is not affected by depletion of the 5′–3′ exoribonuclease XRNA, but is almost completely inhibited by depletion of the deadenylase CAF1. In contrast, two highly unstable mRNAs, encoding EP procyclin and a phosphoglycerate kinase, PGKB, accumulate when XRNA levels are reduced. We here show that degradation of EP mRNA was partially inhibited after CAF1 depletion. RNAi-targeting trypanosome PAN2 had a mild effect on global deadenylation, and on degradation of a few mRNAs including EP. By amplifying and sequencing degradation intermediates, we demonstrated that a reduction in XRNA had no effect on degradation of a stable mRNA encoding a ribosomal protein, but caused accumulation of EP mRNA fragments that had lost substantial portions of the 5′ and 3′ ends. The results support a model in which trypanosome mRNAs can be degraded by at least two different, partially independent, cytoplasmic degradation pathways attacking both ends of the mRNA

Management of cytoskeleton architecture by molecular chaperones and immunophilins

Cytoskeletal structure is continually remodeled to accommodate normal cell growth and to respond to pathophysiological cues. As a consequence, several cytoskeleton-interacting proteins become involved in a variety of cellular processes such as cell growth and division, cell movement, vesicle transportation, cellular organelle location and function, localization and distribution of membrane receptors, and cell-cell communication. Molecular chaperones and immunophilins are counted among the most important proteins that interact closely with the cytoskeleton network, in particular with microtubules and microtubule-associated factors. In several situations, heat-shock proteins and immunophilins work together as a functionally active heterocomplex, although both types of proteins also show independent actions. In circumstances where homeostasis is affected by environmental stresses or due to genetic alterations, chaperone proteins help to stabilize the system. Molecular chaperones facilitate the assembly, disassembly and/or folding/refolding of cytoskeletal proteins, so they prevent aberrant protein aggregation. Nonetheless, the roles of heat-shock proteins and immunophilins are not only limited to solve abnormal situations, but they also have an active participation during the normal differentiation process of the cell and are key factors for many structural and functional rearrangements during this course of action. Cytoskeleton modifications leading to altered localization of nuclear factors may result in loss- or gain-of-function of such factors, which affects the cell cycle and cell development. Therefore, cytoskeletal components are attractive therapeutic targets, particularly microtubules, to prevent pathological situations such as rapidly dividing tumor cells or to favor the process of cell differentiation in other cases. In this review we will address some classical and novel aspects of key regulatory functions of heat-shock proteins and immunophilins as housekeeping factors of the cytoskeletal network.Fil: Quintá, Héctor Ramiro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Galigniana, Natalia Maricel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Erlejman, Alejandra Giselle. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Lagadari, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Piwien Pilipuk, Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Galigniana, Mario Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

The FIP-1 like polyadenylation factor in trypanosomes and the structural basis for its interaction with CPSF30

In trypanosomes transcription is polycistronic and individual mRNAs are generated by a trans-splicing/polyadenylation coupled reaction. We identified a divergent trypanosome FIP1-like, a factor required for mRNA 3′ end formation from yeasts to human. Here we showed that it is a nuclear protein with a speckled distribution essential for trypanosome viability. A strong interaction was found between TcFIP1-like and TcCPSF30, a component of the polyadenylation complex. We determined the specific amino acids in each protein involved in the interaction. Significant differences were found between the trypanosome interaction surface and its human counterpart. Although CPSF30/FIP1 interaction is known in other organisms, this is the first report mapping the interaction surface at the amino acid level.Fil: Bercovich, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Levin, Mariano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Vazquez, Martin Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentin

Sunflower pan-genome analysis shows that hybridization altered gene content and disease resistance

Domesticated plants and animals often display dramatic responses to selection, but the origins of the genetic diversity underlying these responses remain poorly understood. Despite domestication and improvement bottlenecks, the cultivated sunflower remains highly variable genetically, possibly due to hybridization with wild relatives. To characterize genetic diversity in the sunflower and to quantify contributions from wild relatives, we sequenced 287 cultivated lines, 17 Native American landraces and 189 wild accessions representing 11 compatible wild species. Cultivar sequences failing to map to the sunflower reference were assembled de novo for each genotype to determine the gene repertoire, or ‘pan-genome’, of the cultivated sunflower. Assembled genes were then compared to the wild species to estimate origins. Results indicate that the cultivated sunflower pan-genome comprises 61,205 genes, of which 27% vary across genotypes. Approximately 10% of the cultivated sunflower pan-genome is derived through introgression from wild sunflower species, and 1.5% of genes originated solely through introgression. Gene ontology functional analyses further indicate that genes associated with biotic resistance are over-represented among introgressed regions, an observation consistent with breeding records. Analyses of allelic variation associated with downy mildew resistance provide an example in which such introgressions have contributed to resistance to a globally challenging disease

Identification of core components of the exon junction complex in trypanosomes

In animal cells, the exon junction complex (EJC) is deposited onto mRNAs during the second step of splicing, 20-24 nt upstream of the exon-exon junction. The EJC core contains four proteins: Mago, Y14, eIF4AIII and Btz. In trypanosomes, cis-splicing is very rare but all mRNAs are subject to 5′ trans-splicing of a 39-nt RNA sequence. Here we show that trypanosomes have a conserved Mago and a divergent Y14 protein, but we were unable to identify a Btz orthologue. We demonstrate that Mago and Y14 form a stable heterodimer using yeast two hybrid analyses. We also show that this complex co-purifies in vivo in trypanosomes with a protein containing an NTF2 domain, typically involved in mRNA transport.Fil: Bercovich, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Levin, Mariano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Clayton, Christine. Heidelberg Universität; AlemaniaFil: Vazquez, Martin Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentin

The activation of the decapping enzyme DCP2 by DCP1 occurs on the EDC4 scaffold and involves a conserved loop in DCP1

The removal of the 5′-cap structure by the decapping enzyme DCP2 and its coactivator DCP1 shuts down translation and exposes the mRNA to 5′-to-3′ exonucleolytic degradation by XRN1. Although yeast DCP1 and DCP2 directly interact, an additional factor, EDC4, promotes DCP1–DCP2 association in metazoan. Here, we elucidate how the human proteins interact to assemble an active decapping complex and how decapped mRNAs are handed over to XRN1. We show that EDC4 serves as a scaffold for complex assembly, providing binding sites for DCP1, DCP2 and XRN1. DCP2 and XRN1 bind simultaneously to the EDC4 C-terminal domain through short linear motifs (SLiMs). Additionally, DCP1 and DCP2 form direct but weak interactions that are facilitated by EDC4. Mutational and functional studies indicate that the docking of DCP1 and DCP2 on the EDC4 scaffold is a critical step for mRNA decapping in vivo. They also revealed a crucial role for a conserved asparagine–arginine containing loop (the NR-loop) in the DCP1 EVH1 domain in DCP2 activation. Our data indicate that DCP2 activation by DCP1 occurs preferentially on the EDC4 scaffold, which may serve to couple DCP2 activation by DCP1 with 5′-to-3′ mRNA degradation by XRN1 in human cells

Analysis of a nuclear localization signal in the p14 splicing factor in Trypanosoma cruzi

There are only a few reported nuclear localization signals (NLS) in trypanosomes despite intensive research on nuclear metabolic processes such as mRNA processing and transcription during the recent past. Moreover, there are only two reports for a monopartite (La protein) and bipartite (H2B histone, ESAG8) NLS in Trypanosoma brucei. We decided to investigate a NLS in Trypanosoma cruzi by selecting p14, a small RNA recognition motif (RRM) containing protein involved in the splicing process in the nucleus. Its small size (117 amino acids), and an optimized streamlined workflow for analysis in T. cruzi, allowed us to define a region of basic amino acids (RRKRRR) located at the C-terminus that is necessary for nuclear localization. However, the NLS for p14 appeared to be more complex since the signature RRKRRR alone is necessary but not sufficient to direct heterologous proteins, such as GFP, to the nucleus. Since p14 interacts strongly with splicing factor SF3b155, a much larger protein, we designed a p14 variant unable to interact with it. The results allowed us to discard the notion that p14 is entering the nucleus, or is retained within, as the sole consequence of being part of a larger complex. Extensive mapping showed that all of the information for nuclear import resides within the small p14 protein in a bipartite NLS composed of the signature RRKRRR and a region of the RRM domain. Thus, NLS definition in T. cruzi is more complex than previously described.Fil: Westergaard, Gaston G.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Bercovich, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Max Planck for developmental Biology; AlemaniaFil: Reinert, Marina Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Vazquez, Martin Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentin

Mapping of the protein-binding interface between splicing factors SF3b155 and p14 of Trypanosoma cruzi

SF3b155 and p14 are essential components of spliceosome core that recognize the branch point adenosine, a critical step in splicing in eukaryotes. Trypanosomes are unusual since every transcribed gene is processed by trans-splicing instead of cis-splicing. Thus, the trans-spliceosome emerges as an interesting anti-parasitic drug target since this process is not present in mammalian hosts. Here, we present the orthologues of these proteins in Trypanosoma cruzi that interact strongly with each other. To define similarities and differences with the human pair, we performed a detailed alanine scan analysis that allowed us to identify the regions and the critical amino acids of T. cruzi SF3b155 involved in interaction with p14. We demonstrate that the T. cruzi SF3b155 interface is larger and contains more complex elements than its human counterpart. Additionally, our results provide the first insights into the core of the putative mRNA processing complex of trypanosomes.Fil: Avila, Maria Lara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Bercovich, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Westergaard, Gaston. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Levin, Mariano Jorge. Institut Cochin; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vazquez, Martin Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentin

Trypanosoma cruzi: Analysis of the complete PUF RNA-binding protein family

The members of the PUF family of RNA-binding proteins regulate the fate of mRNAs by binding to their 3′UTR sequence elements in eukaryotes. In trypanosomes, for which gene expression is polycistronic and controlled almost exclusively by post-transcriptional processes, PUF proteins could play a crucial role. We report here the complete analysis of the PUF protein family of Trypanosoma cruzi composed of 10 members. In silico analysis predicts the existence of at least three major groups within the T. cruzi family, based on their putative binding specificity. Using yeast three hybrid assays, we tested some of these predictions for TcPUF1, TcPUF3, TcPUF5, and TcPUF8 as representatives of these groups. Data mining of the T. cruzi genome led us to describe putative binding targets for the TcPUFs of the most conserved group, TcPUF1 and TcPUF2. The targets include genes for mitochondrial proteins and protein kinases. Finally, immunolocalization experiments showed that TcPUF1 is localized in multiple discrete foci in the cytoplasm supporting its proposed function.Fil: Caro, Florence. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Bercovich, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Atorrasagasti, María Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Levin, Mariano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Vazquez, Martin Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentin

Neonatal herpes simplex encephalitis: Two twins, two cases Encefalitis herpética neonatal: Dos gemelas, dos casos

Herpes simplex encephalitis is an infrequent infection with high mortality and morbidity. Antiviral therapies decrease mortality but long term sequelae are still high, so early diagnosis is important for opportune treatment. We present a pair of twins with central nervous system herpes simplex infection during the first month of life. Both twins presented non-specific symptoms and consulted with 48 hours apart needing intensive care admission, the first one for noninvasive mechanical ventilation and the second for hemodynamic support. Diagnosis was made by cerebrospinal fluid PCR, in the first twin at day 9 of disease and in the second at admission. Both twins were treated with acyclovir, but only the second one at the beginning of her illness. Initial study with electroencephalogram and magnetic resonance was normal and cerebrospinal fluid on day 18 of treatment was negative for herpes simplex virus DNA detection in both patients