RESUMEN
The vault ribonucleoprotein (RNP), comprising vault RNA (vtRNA) and telomerase-associated protein 1 (TEP1), is found in many eukaryotes. However, previous studies of vtRNAs, for example in mammalian cells, have failed to reach a definitive conclusion about their function. vtRNAs are related to Y RNAs, which are complexed with Ro protein and influence Ro's function in noncoding RNA (ncRNA) quality control and processing. In Trypanosoma brucei, the small noncoding TBsRNA-10 was first described in a survey of the ncRNA repertoire in this organism. Here, we report that TBsRNA-10 in T. brucei is a vtRNA, based on its association with TEP1 and sequence similarity to those of other known and predicted vtRNAs. We observed that like vtRNAs in other species, TBsRNA-10 is transcribed by RNA polymerase III, which in trypanosomes also generates the spliceosomal U-rich small nuclear RNAs. In T. brucei, spliced leader (SL)-mediated trans-splicing of pre-mRNAs is an obligatory step in gene expression, and we found here that T. brucei's vtRNA is highly enriched in a non-nucleolar locus in the cell nucleus implicated in SL RNP biogenesis. Using a newly developed permeabilized cell system for the bloodstream form of T. brucei, we show that down-regulated vtRNA levels impair trans-spliced mRNA production, consistent with a role of vtRNA in trypanosome mRNA metabolism. Our results suggest a common theme for the functions of vtRNAs and Y RNAs. We conclude that by complexing with their protein-binding partners TEP1 and Ro, respectively, these two RNA species modulate the metabolism of various RNA classes.
Asunto(s)
Proteínas Protozoarias/genética , ARN Protozoario/genética , Trans-Empalme/genética , Trypanosoma brucei brucei/genética , Partículas Ribonucleoproteicas en Bóveda/genética , Emparejamiento Base/genética , Secuencia de Bases , Nucléolo Celular/metabolismo , Secuencia Conservada/genética , ADN Polimerasa III/metabolismo , Proteínas Protozoarias/metabolismo , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/química , Transcripción GenéticaRESUMEN
The infectious metacyclic forms of Trypanosoma brucei result from a complex development in the tsetse fly vector. When they infect mammals, they cause African sleeping sickness in humans. Due to scarcity of biological material and difficulties of the tsetse fly as an experimental system, very limited information is available concerning the gene expression profile of metacyclic forms. We used an in vitro system based on expressing the RNA binding protein 6 to obtain infectious metacyclics and determined their protein and mRNA repertoires by mass-spectrometry (MS) based proteomics and mRNA sequencing (RNA-Seq) in comparison to non-infectious procyclic trypanosomes. We showed that metacyclics are quiescent cells, and propose this influences the choice of a monocistronic variant surface glycoprotein expression site. Metacyclics have a largely bloodstream-form type transcriptome, and thus are programmed to translate a bloodstream-form type proteome upon entry into the mammalian host and resumption of cell division. Genes encoding cell surface components showed the largest changes between procyclics and metacyclics, observed at both the transcript and protein levels. Genes encoding metabolic enzymes exhibited expression in metacyclics with features of both procyclic and bloodstream forms, suggesting that this intermediate-type metabolism is dictated by the availability of nutrients in the tsetse fly vector.
Asunto(s)
Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Animales , Enfermedades Transmisibles , Humanos , Mamíferos , Espectrometría de Masas , Glicoproteínas de Membrana/metabolismo , Proteoma , Proteómica , ARN Mensajero , Transcriptoma , Tripanosomiasis Africana/microbiología , Moscas Tse-Tse/parasitologíaRESUMEN
Parasitic protozoa of the flagellate order Kinetoplastida represent one of the deepest branches of the eukaryotic tree. Among this group of organisms, the mechanism of RNA interference (RNAi) has been investigated in Trypanosoma brucei and to a lesser degree in Leishmania (Viannia) spp. The pathway is triggered by long double-stranded RNA (dsRNA) and in T. brucei requires a set of five core genes, including a single Argonaute (AGO) protein, T. brucei AGO1 (TbAGO1). The five genes are conserved in Leishmania (Viannia) spp. but are absent in other major kinetoplastid species, such as Trypanosoma cruzi and Leishmania major. In T. brucei small interfering RNAs (siRNAs) are methylated at the 3' end, whereas Leishmania (Viannia) sp. siRNAs are not. Here we report that T. brucei HEN1, an ortholog of the metazoan HEN1 2'-O-methyltransferases, is required for methylation of siRNAs. Loss of TbHEN1 causes a reduction in the length of siRNAs. The shorter siRNAs in hen1(-/-) parasites are single stranded and associated with TbAGO1, and a subset carry a nontemplated uridine at the 3' end. These findings support a model wherein TbHEN1 methylates siRNA 3' ends after they are loaded into TbAGO1 and this methylation protects siRNAs from uridylation and 3' trimming. Moreover, expression of TbHEN1 in Leishmania (Viannia) panamensis did not result in siRNA 3' end methylation, further emphasizing mechanistic differences in the trypanosome and Leishmania RNAi mechanisms.
Asunto(s)
Metiltransferasas/metabolismo , Proteínas Protozoarias/metabolismo , Procesamiento Postranscripcional del ARN , ARN Protozoario/metabolismo , ARN Interferente Pequeño/metabolismo , Trypanosoma brucei brucei/metabolismo , Secuencia de Aminoácidos , Leishmania/genética , Leishmania/metabolismo , Metiltransferasas/química , Metiltransferasas/genética , Datos de Secuencia Molecular , Mutación , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/enzimologíaRESUMEN
Among trypanosomatid protozoa the mechanism of RNA interference (RNAi) has been investigated in Trypanosoma brucei and to a lesser extent in Leishmania braziliensis. Although these two parasitic organisms belong to the same family, they are evolutionarily distantly related raising questions about the conservation of the RNAi pathway. Here we carried out an in-depth analysis of small interfering RNAs (siRNAs) associated with L. braziliensis Argonaute1 (LbrAGO1). In contrast to T. brucei, Leishmania siRNAs are sensitive to 3' end oxidation, indicating the absence of blocking groups, and the Leishmania genome does not code for a HEN1 RNA 2'-O-methyltransferase, which modifies small RNA 3' ends. Consistent with this observation, ~20% of siRNA 3' ends carry non-templated uridines. Thus siRNA biogenesis, and most likely their metabolism, is different in these organisms. Similarly to T. brucei, putative mobile elements and repeats constitute the major Leishmania siRNA-producing loci and AGO1 ablation leads to accumulation of long transcripts derived from putative mobile elements. However, contrary to T. brucei, no siRNAs were detected from other genomic regions with the potential to form double-stranded RNA, namely sites of convergent transcription and inverted repeats. Thus, our results indicate that organism-specific diversification has occurred in the RNAi pathway during evolution of the trypanosomatid lineage.
Asunto(s)
Variación Genética , Leishmania braziliensis/genética , ARN Interferente Pequeño/genética , Proteínas Argonautas/genética , Regulación de la Expresión Génica , ARN Interferente Pequeño/química , Trypanosoma brucei brucei/genéticaRESUMEN
The introduction ten years ago of RNA interference (RNAi) as a tool for molecular exploration in Trypanosoma brucei has led to a surge in our understanding of the pathogenesis and biology of this human parasite. In particular, a genome-wide RNAi screen has recently been combined with next-generation Illumina sequencing to expose catalogues of genes associated with loss of fitness in distinct developmental stages. At present, this technology is restricted to RNAi-positive protozoan parasites, which excludes T. cruzi, Leishmania major, and Plasmodium falciparum. Therefore, elucidating the mechanism of RNAi and identifying the essential components of the pathway is fundamental for improving RNAi efficiency in T. brucei and for transferring the RNAi tool to RNAi-deficient pathogens. Here we used comparative genomics of RNAi-positive and -negative trypanosomatid protozoans to identify the repertoire of factors in T. brucei. In addition to the previously characterized Argonaute 1 (AGO1) protein and the cytoplasmic and nuclear Dicers, TbDCL1 and TbDCL2, respectively, we identified the RNA Interference Factors 4 and 5 (TbRIF4 and TbRIF5). TbRIF4 is a 3'-5' exonuclease of the DnaQ superfamily and plays a critical role in the conversion of duplex siRNAs to the single-stranded form, thus generating a TbAGO1-siRNA complex required for target-specific cleavage. TbRIF5 is essential for cytoplasmic RNAi and appears to act as a TbDCL1 cofactor. The availability of the core RNAi machinery in T. brucei provides a platform to gain mechanistic insights in this ancient eukaryote and to identify the minimal set of components required to reconstitute RNAi in RNAi-deficient parasites.
Asunto(s)
Proteínas Argonautas/genética , Exonucleasas/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Trypanosoma brucei brucei/genética , Secuencia de Bases , Técnicas de Inactivación de Genes , Genómica , Análisis de Secuencia de ARNRESUMEN
BACKGROUND: At the core of the RNA interference (RNAi) pathway in Trypanosoma brucei is a single Argonaute protein, TbAGO1, with an established role in controlling retroposon and repeat transcripts. Recent evidence from higher eukaryotes suggests that a variety of genomic sequences with the potential to produce double-stranded RNA are sources for small interfering RNAs (siRNAs). RESULTS: To test whether such endogenous siRNAs are present in T. brucei and to probe the individual role of the two Dicer-like enzymes, we affinity purified TbAGO1 from wild-type procyclic trypanosomes, as well as from cells deficient in the cytoplasmic (TbDCL1) or nuclear (TbDCL2) Dicer, and subjected the bound RNAs to Illumina high-throughput sequencing. In wild-type cells the majority of reads originated from two classes of retroposons. We also considerably expanded the repertoire of trypanosome siRNAs to encompass a family of 147-bp satellite-like repeats, many of the regions where RNA polymerase II transcription converges, large inverted repeats and two pseudogenes. Production of these newly described siRNAs is strictly dependent on the nuclear DCL2. Notably, our data indicate that putative centromeric regions, excluding the CIR147 repeats, are not a significant source for endogenous siRNAs. CONCLUSIONS: Our data suggest that endogenous RNAi targets may be as evolutionarily old as the mechanism itself.
Asunto(s)
ARN Interferente Pequeño/metabolismo , Trypanosoma brucei brucei/genética , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sitios Genéticos , Secuencias Invertidas Repetidas , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Interferencia de ARN , Retroelementos , Ribonucleasa III/genética , Ribonucleasa III/metabolismoRESUMEN
The genome of Trypanosoma brucei, the causative agent of African trypanosomiasis, was published five years ago, yet identification of all genes and their transcripts remains to be accomplished. Annotation is challenged by the organization of genes transcribed by RNA polymerase II (Pol II) into long unidirectional gene clusters with no knowledge of how transcription is initiated. Here we report a single-nucleotide resolution genomic map of the T. brucei transcriptome, adding 1,114 new transcripts, including 103 non-coding RNAs, confirming and correcting many of the annotated features and revealing an extensive heterogeneity of 5' and 3' ends. Some of the new transcripts encode polypeptides that are either conserved in T. cruzi and Leishmania major or were previously detected in mass spectrometry analyses. High-throughput RNA sequencing (RNA-Seq) was sensitive enough to detect transcripts at putative Pol II transcription initiation sites. Our results, as well as recent data from the literature, indicate that transcription initiation is not solely restricted to regions at the beginning of gene clusters, but may occur at internal sites. We also provide evidence that transcription at all putative initiation sites in T. brucei is bidirectional, a recently recognized fundamental property of eukaryotic promoters. Our results have implications for gene expression patterns in other important human pathogens with similar genome organization (Trypanosoma cruzi, Leishmania sp.) and revealed heterogeneity in pre-mRNA processing that could potentially contribute to the survival and success of the parasite population in the insect vector and the mammalian host.
Asunto(s)
Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Precursores del ARN/genética , ARN Bacteriano/genética , Transcripción Genética , Trypanosoma brucei brucei/patogenicidad , Tripanosomiasis Africana/genética , Secuencia de Bases , Genoma Bacteriano , Humanos , Datos de Secuencia Molecular , ARN Polimerasa II/genética , Homología de Secuencia de Ácido Nucleico , Sitio de Iniciación de la Transcripción , Trypanosoma brucei brucei/genética , Tripanosomiasis Africana/microbiologíaRESUMEN
RNA interference (RNAi) pathways are widespread in metaozoans but the genes required show variable occurrence or activity in eukaryotic microbes, including many pathogens. While some Leishmania lack RNAi activity and Argonaute or Dicer genes, we show that Leishmania braziliensis and other species within the Leishmania subgenus Viannia elaborate active RNAi machinery. Strong attenuation of expression from a variety of reporter and endogenous genes was seen. As expected, RNAi knockdowns of the sole Argonaute gene implicated this protein in RNAi. The potential for functional genetics was established by testing RNAi knockdown lines lacking the paraflagellar rod, a key component of the parasite flagellum. This sets the stage for the systematic manipulation of gene expression through RNAi in these predominantly diploid asexual organisms, and may also allow selective RNAi-based chemotherapy. Functional evolutionary surveys of RNAi genes established that RNAi activity was lost after the separation of the Leishmania subgenus Viannia from the remaining Leishmania species, a divergence associated with profound changes in the parasite infectious cycle and virulence. The genus Leishmania therefore offers an accessible system for testing hypothesis about forces that may select for the loss of RNAi during evolution, such as invasion by viruses, changes in genome plasticity mediated by transposable elements and gene amplification (including those mediating drug resistance), and/or alterations in parasite virulence.
Asunto(s)
Interferencia de ARN , Transducción de Señal/genética , Trypanosomatina/genética , Evolución Molecular , Genes Protozoarios , Especiación Genética , Inestabilidad Genómica/genética , Inestabilidad Genómica/fisiología , Leishmania braziliensis/genética , Leishmania braziliensis/metabolismo , Fenotipo , Filogenia , Interferencia de ARN/fisiología , ARN Interferente Pequeño/farmacología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Trypanosomatina/inmunología , Virus/genéticaRESUMEN
Expression of dsRNA complementary to small nucleolar RNAs (snoRNAs) in Trypanosoma brucei results in snoRNA silencing, termed snoRNAi. Here, we demonstrate that snoRNAi requires the nuclear TbDCL2 protein, but not TbDCL1, which is involved in RNA interference (RNAi) in the cytoplasm. snoRNAi depends on Argonaute1 (Slicer), and on TbDCL2, suggesting that snoRNA dicing and slicing takes place in the nucleus, and further suggesting that AGO1 is active in nuclear silencing. snoRNAi was next utilized to elucidate the function of an abundant snoRNA, TB11Cs2C2 (92 nt), present in a cluster together with the spliced leader associated RNA (SLA1) and snR30, which are both H/ACA RNAs with special nuclear functions. Using AMT-UV cross-linking and RNaseH cleavage, we provide evidence for the interaction of TB11Cs2C2 with the small rRNAs, srRNA-2 and srRNA-6, which are part of the large subunit (LSU) rRNA. snoRNAi of TB11Cs2C2 resulted in defects in generating srRNA-2 and LSUß rRNA. This is the first snoRNA described so far to engage in trypanosome-specific processing events.
Asunto(s)
Interferencia de ARN , ARN Nucleolar Pequeño/fisiología , Trypanosoma brucei brucei/genética , Emparejamiento Base , Núcleo Celular/enzimología , Endorribonucleasas/fisiología , Proteínas Protozoarias/fisiología , Procesamiento Postranscripcional del ARN , ARN Ribosómico/química , ARN Ribosómico/metabolismo , ARN Nucleolar Pequeño/antagonistas & inhibidores , ARN Nucleolar Pequeño/química , Ribonucleasa III/fisiología , Trypanosoma brucei brucei/enzimologíaRESUMEN
Trypanosoma brucei is one of the most ancient eukaryotes where RNA interference (RNAi) is operational and is the only single-cell pathogen where RNAi has been extensively studied and used as a tool for functional analyses. Here, we report that the T. brucei RNAi pathway, although relying on a single Argonaute protein (AGO1), is initiated by the activities of two distinct Dicer-like enzymes. Both TbDCL1, a mostly cytoplasmic protein, and the previously undescribed nuclear enzyme TbDCL2 contribute to the biogenesis of siRNAs from retroposons. However, TbDCL2 has a predominant role in generating siRNAs from chromosomal internal repeat transcripts that accumulate at the nucleolus in RNAi-deficient cells and in initiating the endogenous RNAi response against retroposons and repeats alike. Moreover, siRNAs generated by both TbDCL1 and TbDCL2 carry a 5'-monophosphate and a blocked 3' terminus, suggesting that 3' end modification is an ancient trait of siRNAs. We thus propose a model whereby TbDCL2 fuels the T. brucei nuclear RNAi pathway and TbDCL1 patrols the cytoplasm, posttranscriptionally silencing potentially harmful nucleic acid parasites that may access the cytoplasm. Nevertheless, we also provide evidence for cross-talk between the two Dicer-like enzymes, because TbDCL2 is implicated in the generation of 35- to 65-nucleotide intermediate transcripts that appear to be substrates for TbDCL1. Our finding that dcl2KO cells are more sensitive to RNAi triggers than wild-type cells has significant implications for reverse genetic analyses in this important human pathogen.
Asunto(s)
Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Interferencia de ARN , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Trypanosoma brucei rhodesiense/genética , Trypanosoma brucei rhodesiense/metabolismo , Animales , Humanos , ARN Protozoario/genética , ARN Interferente Pequeño/genética , Retroelementos/genética , Transcripción Genética , Trypanosoma brucei rhodesiense/patogenicidadRESUMEN
OBJECTIVE: To determine the accuracy of two dismensional sonography and color doppler in diagnosing placenta previa accreta in patients with previous cesarean section. METHODS: Forty-one patients with previous cesarean sections were confirmed to have partial or total placenta previa in the current pregnancy and were given ultrasound examinations after the 28th week of gestation. Specific ultrasound features of the placenta and its interphase with the uterus and the bladder for placenta accreta were checked by two-dimensional ultrasonography and color Doppler. All the patients were traced until delivery. The golden standard in diagnosis was the intraoperative finding and the pathologic exam. RESULTS: Twenty-two patients had ultrasonographic evidence of placenta previa, 20 of which were later confirmed placenta previa accreta intraoperatively. Nineteen patients had no ultrasound evidence of placenta previa, and 1 of which was later confirmed placenta previa accreta. The sensitivity and specificity of antenatal ultrasound diagnosis of placenta previa accreta were 95.24% and 94.74% respectively. The most prominent feature to suggest placenta accreta in twodismensional sonography was the presence of multiple lakes that represented dilated vessels extending from the placenta through the myometrium. The most prominent color Doppler feature was the presence of interphase hypervascularity with abnormal vessels linking the placenta to the bladder, and the rate was 95.24%. CONCLUSION: Placenta previa accreta can be diagnosed made with a thorough two dimensional ultrasonographic and color Doppler examination in patients with previous cesarean scar and placenta previa.
Asunto(s)
Cesárea , Placenta Accreta/diagnóstico por imagen , Placenta Previa/diagnóstico por imagen , Ultrasonografía Doppler en Color , Ultrasonografía/métodos , Adulto , Cicatriz/complicaciones , Femenino , Humanos , Embarazo , Útero/patología , Adulto JovenRESUMEN
Trypanosoma brucei is a protozoan parasite that causes important human and livestock diseases in sub-Saharan Africa. By overexpressing a single RNA-binding protein, RBP6, in non-infectious procyclics trypanosomes, we previously recapitulated in vitro the events occurring in the tsetse fly vector, namely the development of epimastigotes and infectious, quiescent metacyclic parasites. To identify genes involved in this developmental progression, we individually targeted 86 transcripts by RNAi in the RBP6 overexpression cell line and assessed the loss-of-function phenotypes on repositioning the kinetoplast, an organelle that contains the mitochondrial genome, the expression of BARP or brucei alanine rich protein, a marker for epimastigotes, and metacyclic variant surface glycoprotein. This screen identified 22 genes that positively or negatively regulate the stepwise progression towards infectivity at different stages. Two previously uncharacterized putative nucleic acid binding proteins emerged as potent regulators, namely the cold shock domain-containing proteins CSD1 and CSD2. RNA-Seq data from a selected group of cell lines further revealed that the components of gene expression regulatory networks identified in this study affected the abundance of a subset of transcripts in very similar fashion. Finally, our data suggest a considerable overlap between the genes that regulate the formation of stumpy bloodstream form trypanosomes and the genes that govern the development of metacyclic form parasites.
Asunto(s)
Progresión de la Enfermedad , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/patogenicidad , Tripanosomiasis Africana/patología , Tripanosomiasis Africana/parasitología , Línea Celular , Regulación hacia Abajo/genética , Perfilación de la Expresión Génica , Polirribosomas/metabolismo , Biosíntesis de Proteínas , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , RNA-Seq , Tripanosomiasis Africana/genética , Regulación hacia Arriba/genéticaRESUMEN
Argonaute proteins (AGOs) are central to RNA interference (RNAi) and related silencing pathways. At the core of the RNAi pathway in the ancient parasitic eukaryote Trypanosoma brucei is a single Argonaute protein, TbAGO1, with an established role in the destruction of potentially harmful retroposon transcripts. One notable feature of TbAGO1 is that a fraction sediments with polyribosomes, and this association is facilitated by an arginine/glycine-rich domain (RGG domain) at the N terminus of the protein. Here we report that reducing the size of the RGG domain and, in particular, mutating all arginine residues severely reduced the association of TbAGO1 with polyribosomes and RNAi-induced cleavage of mRNA. However, these mutations did not change the cellular localization of Argonaute and did not affect the accumulation of single-stranded siRNAs, an essential step in the activation of the RNA-induced silencing complex. We further show that mRNA on polyribosomes can be targeted for degradation, although this alliance is not a pre-requisite. Finally, sequestering tubulin mRNAs from translation with antisense morpholino oligonucleotides reduced the RNAi response indicating that mRNAs not engaged in translation may be less accessible to the RNAi machinery. We conclude that the association of the RNAi machinery and target mRNA on polyribosomes promotes an efficient RNAi response. This mechanism may represent an ancient adaptation to ensure that retroposon transcripts are efficiently destroyed, if they become associated with the translational apparatus.
Asunto(s)
Polirribosomas/metabolismo , Proteínas Protozoarias/metabolismo , Interferencia de ARN/fisiología , ARN Mensajero/metabolismo , ARN Protozoario/metabolismo , Proteínas de Unión al ARN/metabolismo , Trypanosoma brucei brucei/metabolismo , Proteínas Argonautas , Polirribosomas/genética , Estructura Terciaria de Proteína/fisiología , Proteínas Protozoarias/genética , ARN Mensajero/genética , ARN Protozoario/genética , Proteínas de Unión al ARN/genética , Retroelementos/fisiología , Trypanosoma brucei brucei/genéticaRESUMEN
The mortality of pregnant women with pulmonary arterial hypertension (PAH) remains high. The aim of this study was to evaluate and analyze perinatal and postpartum outcomes in patients with PAH.A total of 79 pregnant patients with PAH who underwent abortion or parturition were reviewed retrospectively. Preoperative characteristics, anesthesia method, intensive care management, PAH-specific therapy, and maternal and neonatal outcomes were analyzed in this case series study.This study was a retrospective analysis of 79 pregnant women with PAH. We collected data on maternal, obstetrical, and neonatal outcomes. The mean age of the parturient women with mild and severe PAH was 26.6â±â5.7 and 26.0â±â4.9 years, respectively, and the mean systolic pulmonary arterial pressure of the 2 groups was 43.8â±â4.2 mmHg and 76.7â±â15.6 mmHg, respectively. Of the 79 patients, 43 (54.4%) had severe PAH and 36 (45.6%) had mild PAH. The gestational weeks were significantly shorter and the rate of fetal death was higher in the severe PAH group than in the mild PAH group (36.0 vs 37.3 weeks and 6/24 vs 1/30, respectively; Pâ<â.05). Fifty-seven patients received PAH-specific therapy during pregnancy, including sildenafil, iloprost, and treprostinil. Overall, 22 PAH patients underwent therapeutic abortion and 57 continued their pregnancy. A total of 9 women, all of whom had severe PAH, died within 3 months of labor, giving a mortality rate of 15.8% (9/57). Of the 57 parturients, 21 (35.6%) gave birth prematurely and 36 (64.4%) delivered at term. Overall, 55 (96.5%) patients delivered by cesarean section and 2 (3.5%) delivered vaginally. There were 7 fetal deaths - 6 in the severe PAH group and one in the mild PAH group (6/24 vs 1/30).Although the mortality rate of this group of women with PAH was lower than that previously reported, patients with PAH should still be advised against pregnancy.
Asunto(s)
Complicaciones Cardiovasculares del Embarazo/epidemiología , Resultado del Embarazo/epidemiología , Hipertensión Arterial Pulmonar/epidemiología , Aborto Inducido , Adulto , Anestesia/métodos , Antihipertensivos/uso terapéutico , Cesárea/estadística & datos numéricos , Cuidados Críticos/métodos , Femenino , Muerte Fetal , Edad Gestacional , Humanos , Embarazo , Complicaciones Cardiovasculares del Embarazo/tratamiento farmacológico , Complicaciones Cardiovasculares del Embarazo/mortalidad , Hipertensión Arterial Pulmonar/tratamiento farmacológico , Hipertensión Arterial Pulmonar/mortalidad , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
We previously established an in vitro differentiation system based on the inducible expression of the RNA binding protein 6 (RBP6), which initiated differentiation of Trypanosoma brucei non-infectious procyclics to infectious metacyclics (MFs). However, further differentiation to bloodstream forms (BFs) required infection of mice. Here we report the serendipitous isolation of a single point mutation in RBP6 (Q109K), whose expression not only generated MFs, but purified MFs continued the developmental cycle in vitro to BFs expressing variant surface glycoprotein-2 (VSG-2), formerly known as VSG 221. This transition occurred over a period of 11 days and by RNA-Seq, VSG-2 was first measureable on day 1, whereas metacyclic VSGs were detected up to 8 days. We further showed that inducible expression of mutant RBP6 appeared to skip the intermediate epimastigote stage and we highlight the potential involvement of RBP33 in the establishment of metacyclics and in particular in the generation of metacyclics uncharacteristically arrested at the G2/M checkpoint.
Asunto(s)
Proteínas Mutantes/genética , Mutación Puntual , Proteínas Protozoarias/genética , Proteínas de Unión al ARN/genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/genética , Ciclo Celular , Perfilación de la Expresión Génica , Proteínas Mutantes/metabolismo , Mutación Missense , Proteínas Protozoarias/metabolismo , Proteínas de Unión al ARN/metabolismo , Análisis de Secuencia de ARN , Factores de Tiempo , Trypanosoma brucei brucei/química , Glicoproteínas Variantes de Superficie de Trypanosoma/análisisRESUMEN
We used an in vitro system based on the inducible expression of the RNA binding protein 6 (RBP6) to monitor transcriptome changes during the differentiation of Trypanosoma brucei from non-infectious procyclics to infectious metacyclics and from metacyclics to bloodstream forms. This data file describes the bioinformatics analysis of 20 distinct RNA-Seq samples, with four biological replicates each, highlighting differential transcript abundance. Additional functional annotation analysis using Gene Ontology is also presented. Complete raw data files were deposited at the NCBI Sequence Read Archive - SRA at http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi with accession numbers: SRP153824, SRP153562, and SRP152737.
RESUMEN
Members of the Argonaute protein family have been linked through a combination of genetic and biochemical studies to RNA interference (RNAi) and related phenomena. Here, we describe the characterization of the first Argonaute protein (AGO1) in Trypanosoma brucei, the earliest divergent eukaryote where RNAi has been described so far. AGO1 is predominantly cytoplasmic and is found in a ribonucleoprotein particle with small interfering RNAs (siRNAs), and this particle is present in a soluble form, as well as associated with polyribosomes. A genetic knockout of AGO1 leads to a loss of RNAi, and concomitantly, endogenous retroposon-derived siRNAs as well as siRNAs derived from transgenic double-stranded RNA are reduced to almost undetectable levels. Furthermore, AGO1 deficiency leads to an increase in retroposon transcript abundance via mechanisms operating at the transcriptional level and at the RNA stability level. Our results suggest that AGO1 function is required for production and/or stabilization of siRNAs and provide the first evidence for an Argonaute protein being involved in the regulation of retroposon transcript levels.