RESUMEN
Small RNA pathways, including RNA interference (RNAi), play crucial roles in regulation of gene expression. Initially considered to be cytoplasmic, these processes have later been demonstrated to associate with membranes. For example, maturation of late endosomes/multivesicular bodies (MVBs) is required for efficient RNAi, whereas fusion of MVBs to lysosomes appears to reduce silencing efficiency. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane fusion and are thus at the core of membrane trafficking. In spite of this, no SNARE has previously been reported to affect RNAi. Here, we demonstrate that in Caenorhabditis elegans, loss of the conserved SNARE SEC-22 results in enhanced RNAi upon ingestion of double-stranded RNA. Furthermore, SEC-22 overexpression inhibits RNAi in wild-type animals. We find that overexpression of SEC-22 in the target tissue (body wall muscle) strongly suppresses the sec-22(-) enhanced RNAi phenotype, supporting a primary role for SEC-22 in import of RNAi silencing signals or cell autonomous RNAi. A functional mCherry::SEC-22 protein localizes primarily to late endosomes/MVBs and these compartments are enlarged in animals lacking sec-22 SEC-22 interacts with late endosome-associated RNA transport protein SID-5 in a yeast two-hybrid assay and functions in a sid-5-dependent manner. Taken together, our data indicate that SEC-22 reduces RNAi efficiency by affecting late endosome/MVB function, for example, by promoting fusion between late endosomes/MVBs and lysosomes. To our knowledge, this is the first report of a SNARE with a function in small RNA-mediated gene silencing.
Asunto(s)
Caenorhabditis elegans/genética , Endosomas/metabolismo , Proteínas Tirosina Quinasas/genética , Interferencia de ARN , ARN Bicatenario/genética , Proteínas SNARE/genética , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Genes Reporteros , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Lisosomas/metabolismo , Fusión de Membrana , Proteínas Tirosina Quinasas/metabolismo , ARN Bicatenario/metabolismo , ARN de Helminto/genética , ARN de Helminto/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas SNARE/metabolismo , Proteína Fluorescente RojaRESUMEN
Evolutionary theory of ageing maintains that increased allocation to early-life reproduction results in reduced somatic maintenance, which is predicted to compromise longevity and late-life reproduction. This prediction has been challenged by the discovery of long-lived mutants with no loss of fecundity. The first such long-lived mutant was found in the nematode worm Caenorhabditis elegans Specifically, partial loss-of-function mutation in the age-1 gene, involved in the nutrient-sensing insulin/insulin-like growth factor signalling pathway, confers longevity, as well as increased resistance to pathogens and to temperature stress without appreciable fitness detriment. Here, we show that the long-lived age-1(hx546) mutant has reduced fecundity and offspring production in early-life, but increased fecundity, hatching success, and offspring production in late-life compared with wild-type worms under standard conditions. However, reduced early-life performance of long-lived mutant animals was not fully compensated by improved performance in late-life and resulted in reduced individual fitness. These results suggest that the age-1(hx546) allele has opposing effects on early-life versus late-life fitness in accordance with antagonistic pleiotropy (AP) and disposable soma theories of ageing. These findings support the theoretical conjecture that experimental studies based on standing genetic variation underestimate the importance of AP in the evolution of ageing.
Asunto(s)
Caenorhabditis elegans/fisiología , Aptitud Genética , Pleiotropía Genética , Longevidad , Reproducción , Envejecimiento , Alelos , Animales , Caenorhabditis elegans/genética , FertilidadRESUMEN
Ribonucleotide reductases (RNRs) catalyze the only pathway for de novo synthesis of deoxyribonucleotides needed for DNA replication and repair. The vast majority of eukaryotes encodes only a class I RNR, but interestingly some eukaryotes, including the social amoeba Dictyostelium discoideum, encode both a class I and a class II RNR. The amino acid sequence of the D. discoideum class I RNR is similar to other eukaryotic RNRs, whereas that of its class II RNR is most similar to the monomeric class II RNRs found in Lactobacillus spp. and a few other bacteria. Here we report the first study of RNRs in a eukaryotic organism that encodes class I and class II RNRs. Both classes of RNR genes were expressed in D. discoideum cells, although the class I transcripts were more abundant and strongly enriched during mid-development compared with the class II transcript. The quaternary structure, allosteric regulation, and properties of the diiron-oxo/radical cofactor of D. discoideum class I RNR are similar to those of the mammalian RNRs. Inhibition of D. discoideum class I RNR by hydroxyurea resulted in a 90% reduction in spore formation and decreased the germination viability of the surviving spores by 75%. Class II RNR could not compensate for class I inhibition during development, and an excess of vitamin B12 coenzyme, which is essential for class II activity, did not improve spore formation. We suggest that class I is the principal RNR during D. discoideum development and growth and is important for spore formation, possibly by providing dNTPs for mitochondrial replication.
Asunto(s)
Dictyostelium/enzimología , Proteínas Protozoarias/metabolismo , Ribonucleótido Reductasas/metabolismo , Regulación Alostérica , Complejos de Coordinación/química , Citidina Difosfato/química , Dictyostelium/genética , Dictyostelium/fisiología , Inhibidores Enzimáticos/farmacología , Radicales Libres/química , Expresión Génica , Regulación Enzimológica de la Expresión Génica , Guanosina Difosfato/química , Hierro/química , Cinética , Filogenia , Proteínas Protozoarias/genética , Ribonucleótido Reductasas/antagonistas & inhibidores , Ribonucleótido Reductasas/química , Ribonucleótido Reductasas/genética , Espectrofotometría Ultravioleta , Esporas Protozoarias/enzimología , Esporas Protozoarias/genética , Tirosina/químicaRESUMEN
Germline regulates the expression of life-history traits and mediates the trade-off between reproduction and somatic maintenance. However, germline maintenance in itself can be costly, and the costs can vary between the sexes depending on the number of gametes produced across the lifetime. We tested this directly by germline ablation using glp-1 RNA interference (RNAi) in a dioecious nematode Caenorhabditis remanei. Germline removal strongly increased heat-shock resistance in both sexes, thus confirming the role of the germline in regulating somatic maintenance. However, germline removal resulted in increased lifespan only in males. High costs of mating strongly reduced lifespan in both sexes and obliterated the survival benefit of germline-less males even though neither sex produced any offspring. Furthermore, germline removal reduced male growth before maturation but not in adulthood, while female growth rate was reduced both before and especially after maturation. Thus, germline removal improves male lifespan without major growth costs, while germline-less females grow slower and do not live longer than reproductively functional counterparts in the absence of environmental stress. Overall, these results suggest that germline maintenance is costlier for males than for females in C. remanei.
RESUMEN
Anthelmintic resistance in equine parasite Parascaris univalens, compromises ivermectin (IVM) effectiveness and necessitates an in-depth understanding of its resistance mechanisms. Most research, primarily focused on holistic gene expression analyses, may overlook vital tissue-specific responses and often limit the scope of novel genes. This study leveraged gene co-expression network analysis to elucidate tissue-specific transcriptional responses and to identify core genes implicated in the IVM response in P. univalens. Adult worms (n = 28) were exposed to 10-11 M and 10-9 M IVM in vitro for 24 hours. RNA-sequencing examined transcriptional changes in the anterior end and intestine. Differential expression analysis revealed pronounced tissue differences, with the intestine exhibiting substantially more IVM-induced transcriptional activity. Gene co-expression network analysis identified seven modules significantly associated with the response to IVM. Within these, 219 core genes were detected, largely expressed in the intestinal tissue and spanning diverse biological processes with unspecific patterns. After 10-11 M IVM, intestinal tissue core genes showed transcriptional suppression, cell cycle inhibition, and ribosomal alterations. Interestingly, genes PgR028_g047 (sorb-1), PgB01_g200 (gmap-1) and PgR046_g017 (col-37 & col-102) switched from downregulation at 10-11 M to upregulation at 10-9 M IVM. The 10-9 M concentration induced expression of cuticle and membrane integrity core genes in the intestinal tissue. No clear core gene patterns were visible in the anterior end after 10-11 M IVM. However, after 10-9 M IVM, the anterior end mostly displayed downregulation, indicating disrupted transcriptional regulation. One interesting finding was the non-modular calcium-signaling gene, PgR047_g066 (gegf-1), which uniquely connected 71 genes across four modules. These genes were enriched for transmembrane signaling activity, suggesting that PgR047_g066 (gegf-1) could have a key signaling role. By unveiling tissue-specific expression patterns and highlighting biological processes through unbiased core gene detection, this study reveals intricate IVM responses in P. univalens. These findings suggest alternative drug uptake of IVM and can guide functional validations to further IVM resistance mechanism understanding.
Asunto(s)
Antihelmínticos , Ascaridoidea , Caballos/genética , Animales , Ivermectina/farmacología , Antihelmínticos/farmacología , Regulación de la Expresión Génica , Perfilación de la Expresión Génica , Ascaridoidea/genética , Resistencia a Medicamentos/genéticaRESUMEN
Parasitic nematodes pose a significant threat to human and animal health, as well as cause economic losses in the agricultural sector. The use of anthelmintic drugs, such as Ivermectin (IVM), to control these parasites has led to widespread drug resistance. Identifying genetic markers of resistance in parasitic nematodes can be challenging, but the free-living nematode Caenorhabditis elegans provides a suitable model. In this study, we aimed to analyze the transcriptomes of adult C. elegans worms of the N2 strain exposed to the anthelmintic drug Ivermectin (IVM), and compare them to those of the resistant strain DA1316 and the recently identified Abamectin Quantitative Trait Loci (QTL) on chromosome V. We exposed pools of 300 adult N2 worms to IVM (10-7 and 10-8 M) for 4 hours at 20°C, extracted total RNA and sequenced it on the Illumina NovaSeq6000 platform. Differentially expressed genes (DEGs) were determined using an in-house pipeline. The DEGs were compared to genes from a previous microarray study on IVM-resistant C. elegans and Abamectin-QTL. Our results revealed 615 DEGs (183 up-regulated and 432 down-regulated genes) from diverse gene families in the N2 C. elegans strain. Of these DEGs, 31 overlapped with genes from IVM-exposed adult worms of the DA1316 strain. We identified 19 genes, including the folate transporter (folt-2) and the transmembrane transporter (T22F3.11), which exhibited an opposite expression in N2 and the DA1316 strain and were deemed potential candidates. Additionally, we compiled a list of potential candidates for further research including T-type calcium channel (cca-1), potassium chloride cotransporter (kcc-2), as well as other genes such as glutamate-gated channel (glc-1) that mapped to the Abamectin-QTL.
Asunto(s)
Antihelmínticos , Ivermectina , Animales , Humanos , Ivermectina/farmacología , Ivermectina/metabolismo , Caenorhabditis elegans/metabolismo , Transcriptoma , Sitios de Carácter Cuantitativo , Antihelmínticos/farmacología , Resistencia a Medicamentos/genéticaRESUMEN
BACKGROUND: The nematode Parascaris univalens is one of the most prevalent parasitic pathogens infecting horses but anthelmintic resistance undermines treatment approaches. The molecular mechanisms underlying drug activity and resistance remain poorly understood in this parasite since experimental in vitro models are lacking. The aim of this study was to evaluate the use of Caenorhabditis elegans as a model for P. univalens drug metabolism/resistance studies by a comparative gene expression approach after in vitro exposure to the anthelmintic drug ivermectin (IVM). METHODS: Twelve adult P. univalens worms in groups of three were exposed to ivermectin (IVM, 10-13 M, 10-11 M, 10-9 M) or left unexposed for 24 h at 37 °C, and total RNA, extracted from the anterior end of the worms, was sequenced using Illumina NovaSeq. Differentially expressed genes (DEGs) involved in metabolism, transportation, or gene expression with annotated Caernorhabditis elegans orthologues were identified as candidate genes to be involved in IVM metabolism/resistance. Similarly, groups of 300 adult C. elegans worms were exposed to IVM (10-9 M, 10-8 M and 10-7 M) or left unexposed for 4 h at 20 °C. Quantitative RT-PCR of RNA extracted from the C. elegans worm pools was used to compare against the expression of selected P. univalens candidate genes after drug treatment. RESULTS: After IVM exposure, 1085 DEGs were found in adult P. univalens worms but the relative gene expression changes were small and large variabilities were found between different worms. Fifteen of the DEGs were chosen for further characterization in C. elegans after comparative bioinformatics analyses. Candidate genes, including the putative drug target lgc-37, responded to IVM in P. univalens, but marginal to no responses were observed in C. elegans despite dose-dependent behavioral effects observed in C. elegans after IVM exposure. Thus, the overlap in IVM-induced gene expression in this small set of genes was minor in adult worms of the two nematode species. CONCLUSION: This is the first time to our knowledge that a comparative gene expression approach has evaluated C. elegans as a model to understand IVM metabolism/resistance in P. univalens. Genes in P. univalens adults that responded to IVM treatment were identified. However, identifying conserved genes in P. univalens and C. elegans involved in IVM metabolism/resistance by comparing gene expression of candidate genes proved challenging. The approach appears promising but was limited by the number of genes studied (n = 15). Future studies comparing a larger number of genes between the two species may result in identification of additional candidate genes involved in drug metabolism and/or resistance.
Asunto(s)
Antihelmínticos , Ascaridoidea , Animales , Antihelmínticos/uso terapéutico , Caenorhabditis elegans , Resistencia a Medicamentos/genética , Expresión Génica , Caballos , Ivermectina/uso terapéutico , ARN/metabolismoRESUMEN
Small RNAs play crucial roles in regulation of gene expression in many eukaryotes. Here, we report the cloning and characterization of 18-26 nt RNAs in the social amoeba Dictyostelium discoideum. This survey uncovered developmentally regulated microRNA candidates whose biogenesis, at least in one case, is dependent on a Dicer homolog, DrnB. Furthermore, we identified a large number of 21 nt RNAs originating from the DIRS-1 retrotransposon, clusters of which have been suggested to constitute centromeres. Small RNAs from another retrotransposon, Skipper, were significantly up-regulated in strains depleted of the second Dicer-like protein, DrnA, and a putative RNA-dependent RNA polymerase, RrpC. In contrast, the expression of DIRS-1 small RNAs was not altered in any of the analyzed strains. This suggests the presence of multiple RNAi pathways in D. discoideum. In addition, we isolated several small RNAs with antisense complementarity to mRNAs. Three of these mRNAs are developmentally regulated. Interestingly, all three corresponding genes express longer antisense RNAs from which the small RNAs may originate. In at least one case, the longer antisense RNA is complementary to the spliced but not the unspliced pre-mRNA, indicating synthesis by an RNA-dependent RNA polymerase.
Asunto(s)
Dictyostelium/genética , Interferencia de ARN , ARN Protozoario/metabolismo , ARN no Traducido/metabolismo , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Regulación de la Expresión Génica , Biblioteca de Genes , MicroARNs/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Protozoario/química , ARN no Traducido/química , ARN Polimerasa Dependiente del ARN/metabolismo , RetroelementosRESUMEN
Classical theory maintains that ageing evolves via energy trade-offs between reproduction and survival leading to accumulation of unrepaired cellular damage with age. In contrast, the emerging new theory postulates that ageing evolves because of deleterious late-life hyper-function of reproduction-promoting genes leading to excessive biosynthesis in late-life. The hyper-function theory uniquely predicts that optimizing nutrient-sensing molecular signaling in adulthood can simultaneously postpone ageing and increase Darwinian fitness. Here, we show that reducing evolutionarily conserved insulin/IGF-1 nutrient-sensing signaling via daf-2 RNA interference (RNAi) fulfils this prediction in Caenorhabditis elegans nematodes. Long-lived daf-2 RNAi parents showed normal fecundity as self-fertilizing hermaphrodites and improved late-life reproduction when mated to males. Remarkably, the offspring of daf-2 RNAi parents had higher Darwinian fitness across three different genotypes. Thus, reduced nutrient-sensing signaling in adulthood improves both parental longevity and offspring fitness supporting the emerging view that suboptimal gene expression in late-life lies at the heart of ageing.
RESUMEN
We have identified a putative RNA helicase from Dictyostelium that is closely related to drh-1, the 'dicer-related-helicase' from Caenorhabditis elegans and that also has significant similarity to proteins from vertebrates and plants. Green fluorescent protein (GFP)-tagged HelF protein was localized in speckles in the nucleus. Disruption of the helF gene resulted in a mutant morphology in late development. When transformed with RNAi constructs, HelF- cells displayed enhanced RNA interference on four tested genes. One gene that could not be knocked-down in the wild-type background was efficiently silenced in the mutant. Furthermore, the efficiency of silencing in the wild-type was dramatically improved when helF was disrupted in a secondary transformation. Silencing efficiency depended on transcription levels of hairpin RNA and the threshold was dramatically reduced in HelF- cells. However, the amount of siRNA did not depend on hairpin transcription. HelF is thus a natural nuclear suppressor of RNA interference. In contrast, no improvement of gene silencing was observed when mutant cells were challenged with corresponding antisense constructs. This indicates that RNAi and antisense have distinct requirements even though they may share parts of their pathways.
Asunto(s)
Dictyostelium/enzimología , Dictyostelium/genética , Proteínas Nucleares/fisiología , ARN Helicasas/fisiología , Interferencia de ARN , Secuencia de Aminoácidos , Animales , Proteínas de Caenorhabditis elegans/química , Línea Celular , Núcleo Celular/enzimología , ARN Helicasas DEAD-box , Dictyostelium/crecimiento & desarrollo , Eliminación de Gen , Silenciador del Gen , Locomoción , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , ARN Helicasas/química , ARN Helicasas/genética , ARN sin Sentido/genética , ARN Bicatenario/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The quest for non-coding RNAs (ncRNAs) in the last few years has revealed a surprisingly large number of small RNAs belonging to previously known as well as entirely novel classes. Computational and experimental approaches have uncovered new ncRNAs in all kingdoms of life. In this work, we used a shotgun cloning approach to construct full-length cDNA libraries of small RNAs from the eukaryotic model organism Dictyostelium discoideum. Interestingly, two entirely novel classes of RNAs were identified of which one is developmentally regulated. The RNAs within each class share conserved 5'- and 3'-termini that can potentially form stem structures. RNAs of both classes show predominantly cytoplasmic localization. In addition, based on conserved structure and/or sequence motifs, several of the identified ncRNAs could be divided into classes known from other organisms, e.g. 18 small nucleolar RNA candidates (17 box C/D, of which a few are developmentally regulated, and one box H/ACA). Two ncRNAs showed a high degree of similarity to the small nuclear U2 RNA and signal recognition particle RNA (SRP RNA), respectively. Furthermore, the majority of the regions upstream of the sequences encoding the isolated RNAs share conserved motifs that may constitute new promoter elements.
Asunto(s)
Dictyostelium/crecimiento & desarrollo , Dictyostelium/genética , ARN no Traducido/genética , Animales , Secuencia de Bases , Dictyostelium/metabolismo , Regulación del Desarrollo de la Expresión Génica , Biblioteca de Genes , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Nuclear Pequeño/química , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , ARN no Traducido/análisis , ARN no Traducido/metabolismoRESUMEN
Proteoglycans are proteins that carry sulfated glycosaminoglycans (GAGs). They help form and maintain morphogen gradients, guiding cell migration and differentiation during animal development. While no sulfated GAGs have been found in marine sponges, chondroitin sulfate (CS) and heparan sulfate (HS) have been identified in Cnidarians, Lophotrocozoans and Ecdysozoans. The general view that nematodes such as Caenorhabditis elegans, which belong to Ecdysozoa, produce HS but only chondroitin without sulfation has therefore been puzzling. We have analyzed GAGs in C. elegans using reversed-phase ion-pairing HPLC, mass spectrometry and immunohistochemistry. Our analyses included wild type C. elegans but also a mutant lacking two HS sulfotransferases (hst-6 hst-2), as we suspected that the altered HS structure could boost CS sulfation. We could indeed detect sulfated CS in both wild type and mutant nematodes. While 4-O-sulfation of galactosamine dominated, we also detected 6-O-sulfated galactosamine residues. Finally, we identified the product of the gene C41C4.1 as a C. elegans CS-sulfotransferase and renamed it chst-1 (CarboHydrate SulfoTransferase) based on loss of CS-4-O-sulfation in a C41C4.1 mutant and in vitro sulfotransferase activity of recombinant C41C4.1 protein. We conclude that C. elegans indeed manufactures CS, making this widely used nematode an interesting model for developmental studies involving CS.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Sulfatos de Condroitina/biosíntesis , Mutación , Sulfotransferasas/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Sulfatos de Condroitina/genética , Espectrometría de Masas , Sulfotransferasas/genéticaRESUMEN
In the nematode C. elegans, RNAi silencing signals are efficiently taken up from the environment and transported between cells and tissues. Previous studies implicating endosomal proteins in systemic RNAi lack conclusive evidence. Here, we report the identification and characterization of SID-5, a C. elegans endosome-associated protein that is required for efficient systemic RNAi in response to both ingested and expressed double-stranded RNA (dsRNA). SID-5 is detected in cytoplasmic foci that partially colocalize with GFP fusions of late endosomal proteins RAB-7 and LMP-1. Furthermore, knockdown of various endosomal proteins similarly relocalizes both SID-5 and LMP-1::GFP. Consistent with a non-cell-autonomous function, intestine-specific SID-5 expression restored body wall muscle (bwm) target gene silencing in response to ingested dsRNA. Finally, we show that sid-5 is required for the previously described sid-1-independent transport of ingested RNAi triggers across the intestine. Together, these data demonstrate that an endosome-associated protein, SID-5, promotes the transport of RNAi silencing signals between cells. Furthermore, SID-5 acts differently than the previously described SID-1, SID-2, and SID-3 proteins, thus expanding the systemic RNAi pathway.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Glicoproteínas de Membrana/metabolismo , Interferencia de ARN , Proteínas de Unión al GTP rab/metabolismo , Animales , Transporte Biológico , Proteínas de Caenorhabditis elegans/genética , ADN/metabolismo , Endosomas/genética , Endosomas/metabolismo , Proteínas Fluorescentes Verdes/genética , Mucosa Intestinal/metabolismo , Glicoproteínas de Membrana/genética , ARN Bicatenario/metabolismo , ARN Citoplasmático Pequeño , Eliminación de Secuencia , Proteínas de Unión a GTP rab7RESUMEN
Genome data are increasingly important in the computational identification of novel regulatory non-coding RNAs (ncRNAs). However, most ncRNA gene-finders are either specialized to well-characterized ncRNA gene families or require comparisons of closely related genomes. We developed a method for de novo screening for ncRNA genes with a nucleotide composition that stands out against the background genome based on a partial sum process. We compared the performance when assuming independent and first-order Markov-dependent nucleotides, respectively, and used Karlin-Altschul and Karlin-Dembo statistics to evaluate the significance of hits. We hypothesized that a first-order Markov-dependent process might have better power to detect ncRNA genes since nearest-neighbor models have been shown to be successful in predicting RNA structures. A model based on a first-order partial sum process (analyzing overlapping dinucleotides) had better sensitivity and specificity than a zeroth-order model when applied to the AT-rich genome of the amoeba Dictyostelium discoideum. In this genome, we detected 94% of previously known ncRNA genes (at this sensitivity, the false positive rate was estimated to be 25% in a simulated background). The predictions were further refined by clustering candidate genes according to sequence similarity and/or searching for an ncRNA-associated upstream element. We experimentally verified six out of 10 tested ncRNA gene predictions. We conclude that higher-order models, in combination with other information, are useful for identification of novel ncRNA gene families in single-genome analysis of D. discoideum. Our generalizable approach extends the range of genomic data that can be searched for novel ncRNA genes using well-grounded statistical methods.
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Dictyostelium/genética , Genómica/métodos , ARN no Traducido/genética , Adenina/análisis , Animales , Composición de Base , Secuencia de Bases , Secuencia Conservada , Genes Protozoarios , Genoma de Protozoos , Cadenas de Markov , Datos de Secuencia Molecular , Familia de Multigenes , Nucleótidos/análisis , ARN no Traducido/química , ARN no Traducido/metabolismo , Timina/análisisRESUMEN
The traditional view of RNA being merely an intermediate in the transfer of genetic information, as mRNA, spliceosomal RNA, tRNA, and rRNA, has become outdated. The recent discovery of numerous regulatory RNAs with a plethora of functions in biological processes has truly revolutionized our understanding of gene regulation. Tiny RNAs such as microRNAs and small interfering RNAs play vital roles at different levels of gene control. Small nucleolar RNAs are much more abundant than previously recognized, and new functions beyond processing and modification of rRNA have recently emerged. Longer non-coding RNAs (ncRNAs) can also have important regulatory roles in the cell, e.g., antisense RNAs that control their target mRNAs. The majority of these important findings arose from analyses in various model organisms. In this review, we focus on ncRNAs in the social amoeba Dictyostelium discoideum. This important genetically tractable model organism has recently received renewed attention in terms of discovery, regulation and functional studies of ncRNAs. Old and recent findings are discussed and put in context of what we today know about ncRNAs in other organisms.
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Dictyostelium/genética , ARN Protozoario/genética , ARN no Traducido/genética , Animales , Biología Computacional/métodos , Biología Computacional/tendencias , Dictyostelium/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Procesamiento Postranscripcional del ARN , ARN Protozoario/aislamiento & purificación , ARN Protozoario/metabolismo , ARN no Traducido/aislamiento & purificación , ARN no Traducido/metabolismoRESUMEN
Most eukaryotic mRNAs depend upon precise removal of introns by the spliceosome, a complex of RNAs and proteins. Splicing of pre-mRNA is known to take place in Dictyostelium discoideum, and we previously isolated the U2 spliceosomal RNA experimentally. In this study, we identified the remaining major spliceosomal RNAs in Dictyostelium by a bioinformatical approach. Expression was verified from 17 small nuclear RNA (snRNA) genes. All these genes are preceded by a putative noncoding RNA gene promoter. Immunoprecipitation showed that snRNAs U1, U2, U4, and U5, but not U6, carry the conserved trimethylated 5' cap structure. A number of divergent U2 species are expressed in Dictyostelium. These RNAs carry the U2 RNA hallmark sequence and structure motifs but have an additional predicted stem-loop structure at the 5' end. Surprisingly, and in contrast to the other spliceosomal RNAs in this study, the new U2 variants were enriched in the cytoplasm and were developmentally regulated. Furthermore, all of the snRNAs could also be detected as polyadenylated species, and polyadenylated U1 RNA was demonstrated to be located in the cytoplasm.