50 research outputs found
Regulation of lin-4 miRNA expression, organismal growth and development by a conserved RNA binding protein in C. elegans
AbstractTranscription and multiple processing steps are required to produce specific 22 nucleotide microRNAs (miRNAs) that can regulate the expression of target genes. In C. elegans, mature lin-4 miRNA accumulates at the end of the first larval stage to repress its direct targets lin-14 and lin-28, allowing the progression of several somatic cell types to later larval fates. In this study, we characterized the expression of endogenous lin-4 and found that temporally regulated independent transcripts, but not constitutive lin-4 containing RNAs derived from an overlapping gene, are processed to mature lin-4 miRNA. Through an RNAi screen, we identified a conserved RNA binding protein gene rbm-28 (R05H10.2), homologous to the human RBM28 and yeast Nop4p proteins, that is important for lin-4 expression in C. elegans. We also demonstrate that rbm-28 genetically interacts with the lin-4 developmental timing pathway and uncover a previously unrecognized role for lin-14 and lin-28 in coordinating organismal growth
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Capture of complete ciliate chromosomes in single sequencing reads reveals widespread chromosome isoforms
Background
Whole-genome shotgun sequencing, which stitches together millions of short sequencing reads into a single genome, ushered in the era of modern genomics and led to a rapid expansion of the number of genome sequences available. Nevertheless, assembly of short reads remains difficult, resulting in fragmented genome sequences. Ultimately, only a sequencing technology capable of capturing complete chromosomes in a single run could resolve all ambiguities. Even “third generation” sequencing technologies produce reads far shorter than most eukaryotic chromosomes. However, the ciliate Oxytricha trifallax has a somatic genome with thousands of chromosomes averaging only 3.2 kbp, making it an ideal candidate for exploring the benefits of sequencing whole chromosomes without assembly.
Results
We used single-molecule real-time sequencing to capture thousands of complete chromosomes in single reads and to update the published Oxytricha trifallax JRB310 genome assembly. In this version, over 50% of the completed chromosomes with two telomeres derive from single reads. The improved assembly includes over 12,000 new chromosome isoforms, and demonstrates that somatic chromosomes derive from variable rearrangements between somatic segments encoded up to 191,000 base pairs away. However, while long reads reduce the need for assembly, a hybrid approach that supplements long-read sequencing with short reads for error correction produced the most complete and accurate assembly, overall.
Conclusions
This assembly provides the first example of complete eukaryotic chromosomes captured by single sequencing reads and demonstrates that traditional approaches to genome assembly can mask considerable structural variation
Soil networks become more connected and take up more carbon as nature restoration progresses
Soil organisms have an important role in aboveground community dynamics and ecosystem functioning in terrestrial ecosystems. However, most studies have considered soil biota as a black box or focussed on specific groups, whereas little is known about entire soil networks. Here we show that during the course of nature restoration on abandoned arable land a compositional shift in soil biota, preceded by tightening of the belowground networks, corresponds with enhanced efficiency of carbon uptake. In mid- and long-term abandoned field soil, carbon uptake by fungi increases without an increase in fungal biomass or shift in bacterial-to-fungal ratio. The implication of our findings is that during nature restoration the efficiency of nutrient cycling and carbon uptake can increase by a shift in fungal composition and/or fungal activity. Therefore, we propose that relationships between soil food web structure and carbon cycling in soils need to be reconsidered
A Negative Regulatory Loop between MicroRNA and Hox Gene Controls Posterior Identities in Caenorhabditis elegans
MicroRNAs (miRNAs) have been found to regulate gene expression across eukaryotic species, but the function of most miRNA genes remains unknown. Here we describe how the analysis of the expression patterns of a well-conserved miRNA gene, mir-57, at cellular resolution for every minute during early development of Caenorhabditis elegans provided key insights in understanding its function. Remarkably, mir-57 expression shows strong positional bias but little tissue specificity, a pattern reminiscent of Hox gene function. Despite the minor defects produced by a loss of function mutation, overexpression of mir-57 causes dramatic posterior defects, which also mimic the phenotypes of mutant alleles of a posterior Hox gene, nob-1, an Abd homolog. More importantly, nob-1 expression is found in the same two posterior AB sublineages as those expressing mir-57 but with an earlier onset. Intriguingly, nob-1 functions as an activator for mir-57 expression; it is also a direct target of mir-57. In agreement with this, loss of mir-57 function partially rescues the nob-1 allele defects, indicating a negative feedback regulatory loop between the miRNA and Hox gene to provide positional cues. Given the conservation of the miRNA and Hox gene, the regulatory mechanism might be broadly used across species. The strategy used here to explore mir-57 function provides a path to dissect the regulatory relationship between genes
Identifying the research, advocacy, policy and implementation needs for the prevention and management of respiratory syncytial virus lower respiratory tract infection in low- and middle-income countries
Introduction: The high burden of respiratory syncytial virus (RSV) infection in young children disproportionately occurs in low- and middle-income countries (LMICs). The PROUD (Preventing RespiratOry syncytial virUs in unDerdeveloped countries) Taskforce of 24 RSV worldwide experts assessed key needs for RSV prevention in LMICs, including vaccine and newer preventive measures. Methods: A global, survey-based study was undertaken in 2021. An online questionnaire was developed following three meetings of the Taskforce panellists wherein factors related to RSV infection, its prevention and management were identified using iterative questioning. Each factor was scored, by non-panellists interested in RSV, on a scale of zero (very-low-relevance) to 100 (very-high-relevance) within two scenarios: (1) Current and (2) Future expectations for RSV management. Results: Ninety questionnaires were completed: 70 by respondents (71.4% physicians; 27.1% researchers/scientists) from 16 LMICs and 20 from nine high-income (HI) countries (90.0% physicians; 5.0% researchers/scientists), as a reference group. Within LMICs, RSV awareness was perceived to be low, and management was not prioritised. Of the 100 factors scored, those related to improved diagnosis particularly access to affordable point-of-care diagnostics, disease burden data generation, clinical and general education, prompt access to new interventions, and engagement with policymakers/payers were identified of paramount importance. There was a strong need for clinical education and local data generation in the lowest economies, whereas upper-middle income countries were more closely aligned with HI countries in terms of current RSV service provision. Conclusion: Seven key actions for improving RSV prevention and management in LMICs are proposed
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Cytosine methylation and hydroxymethylation mark DNA for elimination in Oxytricha trifallax
Background: Cytosine methylation of DNA is conserved across eukaryotes and plays important functional roles
regulating gene expression during differentiation and development in animals, plants and fungi.
Hydroxymethylation was recently identified as another epigenetic modification marking genes important for
pluripotency in embryonic stem cells.
Results: Here we describe de novo cytosine methylation and hydroxymethylation in the ciliate Oxytricha trifallax.
These DNA modifications occur only during nuclear development and programmed genome rearrangement.
We detect methylcytosine and hydroxymethylcytosine directly by high-resolution nano-flow UPLC mass
spectrometry, and indirectly by immunofluorescence, methyl-DNA immunoprecipitation and bisulfite sequencing.
We describe these modifications in three classes of eliminated DNA: germline-limited transposons and satellite
repeats, aberrant DNA rearrangements, and DNA from the parental genome undergoing degradation. Methylation
and hydroxymethylation generally occur on the same sequence elements, modifying cytosines in all sequence
contexts. We show that the DNA methyltransferase-inhibiting drugs azacitidine and decitabine induce
demethylation of both somatic and germline sequence elements during genome rearrangements, with
consequent elevated levels of germline-limited repetitive elements in exconjugant cells.
Conclusions: These data strongly support a functional link between cytosine DNA methylation/hydroxymethylation
and DNA elimination. We identify a motif strongly enriched in methylated/hydroxymethylated regions, and we
propose that this motif recruits DNA modification machinery to specific chromosomes in the parental
macronucleus. No recognizable methyltransferase enzyme has yet been described in O. trifallax, raising the
possibility that it might employ a novel cytosine methylation machinery to mark DNA sequences for elimination
during genome rearrangements
Piwi-Interacting RNAs Protect DNA against Loss during Oxytricha Genome Rearrangement
SummaryGenome duality in ciliated protozoa offers a unique system to showcase their epigenome as a model of inheritance. In Oxytricha, the somatic genome is responsible for vegetative growth, whereas the germline contributes DNA to the next sexual generation. Somatic nuclear development removes all transposons and other so-called “junk” DNA, which comprise ∼95% of the germline. We demonstrate that Piwi-interacting small RNAs (piRNAs) from the maternal nucleus can specify genomic regions for retention in this process. Oxytricha piRNAs map primarily to the somatic genome, representing the ∼5% of the germline that is retained. Furthermore, injection of synthetic piRNAs corresponding to normally deleted regions leads to their retention in later generations. Our findings highlight small RNAs as powerful transgenerational carriers of epigenetic information for genome programming
Programmed Genome Processing in Ciliates
The ciliates are a group of protists distinguished by the hair-like cilia on their cell surfaces. Ciliates also possess two types of nuclei, a germline micronucleus and a somatic macronucleus. The micronuclear genome contains segmented genes divided by spacer sequences of DNA that are removed to generate the macronuclear genome during development. For some species, certain micronuclear gene segments can be reordered and/or inverted with respect to their final gene sequence in the macronucleus. This chapter explores the similarities of and differences between micronuclear genomes and the processes of macronuclear development across different ciliate species