Auto Tag: G-quadruplex

April 21, 2020  |  

Transcriptional initiation of a small RNA, not R-loop stability, dictates the frequency of pilin antigenic variation in Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the sole causative agent of gonorrhea, constitutively undergoes diversification of the Type IV pilus. Gene conversion occurs between one of the several donor silent copies located in distinct loci and the recipient pilE gene, encoding the major pilin subunit of the pilus. A guanine quadruplex (G4) DNA structure and a cis-acting sRNA (G4-sRNA) are located upstream of the pilE gene and both are required for pilin antigenic variation (Av). We show that the reduced sRNA transcription lowers pilin Av frequencies. Extended transcriptional elongation is not required for Av, since limiting the transcript to 32 nt allows for normal Av frequencies. Using chromatin immunoprecipitation (ChIP) assays, we show that cellular G4s are less abundant when sRNA transcription is lower. In addition, using ChIP, we demonstrate that the G4-sRNA forms a stable RNA:DNA hybrid (R-loop) with its template strand. However, modulating R-loop levels by controlling RNase HI expression does not alter G4 abundance quantified through ChIP. Since pilin Av frequencies were not altered when modulating R-loop levels by controlling RNase HI expression, we conclude that transcription of the sRNA is necessary, but stable R-loops are not required to promote pilin Av. © 2019 John Wiley & Sons Ltd.

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April 21, 2020  |  

Rapid antigen diversification through mitotic recombination in the human malaria parasite Plasmodium falciparum.

Malaria parasites possess the remarkable ability to maintain chronic infections that fail to elicit a protective immune response, characteristics that have stymied vaccine development and cause people living in endemic regions to remain at risk of malaria despite previous exposure to the disease. These traits stem from the tremendous antigenic diversity displayed by parasites circulating in the field. For Plasmodium falciparum, the most virulent of the human malaria parasites, this diversity is exemplified by the variant gene family called var, which encodes the major surface antigen displayed on infected red blood cells (RBCs). This gene family exhibits virtually limitless diversity when var gene repertoires from different parasite isolates are compared. Previous studies indicated that this remarkable genome plasticity results from extensive ectopic recombination between var genes during mitotic replication; however, the molecular mechanisms that direct this process to antigen-encoding loci while the rest of the genome remains relatively stable were not determined. Using targeted DNA double-strand breaks (DSBs) and long-read whole-genome sequencing, we show that a single break within an antigen-encoding region of the genome can result in a cascade of recombination events leading to the generation of multiple chimeric var genes, a process that can greatly accelerate the generation of diversity within this family. We also found that recombinations did not occur randomly, but rather high-probability, specific recombination products were observed repeatedly. These results provide a molecular basis for previously described structured rearrangements that drive diversification of this highly polymorphic gene family.

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April 21, 2020  |  

Intragenomic heterogeneity of intergenic ribosomal DNA spacers in Cucurbita moschata is determined by DNA minisatellites with variable potential to form non-canonical DNA conformations.

The intergenic spacer (IGS) of rDNA is frequently built of long blocks of tandem repeats. To estimate the intragenomic variability of such knotty regions, we employed PacBio sequencing of the Cucurbita moschata genome, in which thousands of rDNA copies are distributed across a number of loci. The rRNA coding regions are highly conserved, indicating intensive interlocus homogenization and/or high selection pressure. However, the IGS exhibits high intragenomic structural diversity. Two repeated blocks, R1 (300-1250 bp) and R2 (290-643 bp), account for most of the IGS variation. They exhibit minisatellite-like features built of multiple periodically spaced short GC-rich sequence motifs with the potential to adopt non-canonical DNA conformations, G-quadruplex-folded and left-handed Z-DNA. The mutual arrangement of these motifs can be used to classify IGS variants into five structural families. Subtle polymorphisms exist within each family due to a variable number of repeats, suggesting the coexistence of an enormous number of IGS variants. The substantial length and structural heterogeneity of IGS minisatellites suggests that the tempo of their divergence exceeds the tempo of the homogenization of rDNA arrays. As frequently occurring among plants, we hypothesize that their instability may influence transcription regulation and/or destabilize rDNA units, possibly spreading them across the genome. © The Author(s) 2019. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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April 21, 2020  |  

Long-read sequencing identifies GGC repeat expansions in NOTCH2NLC associated with neuronal intranuclear inclusion disease.

Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease that is characterized by eosinophilic hyaline intranuclear inclusions in neuronal and somatic cells. The wide range of clinical manifestations in NIID makes ante-mortem diagnosis difficult1-8, but skin biopsy enables its ante-mortem diagnosis9-12. The average onset age is 59.7 years among approximately 140 NIID cases consisting of mostly sporadic and several familial cases. By linkage mapping of a large NIID family with several affected members (Family 1), we identified a 58.1 Mb linked region at 1p22.1-q21.3 with a maximum logarithm of the odds score of 4.21. By long-read sequencing, we identified a GGC repeat expansion in the 5′ region of NOTCH2NLC (Notch 2 N-terminal like C) in all affected family members. Furthermore, we found similar expansions in 8 unrelated families with NIID and 40 sporadic NIID cases. We observed abnormal anti-sense transcripts in fibroblasts specifically from patients but not unaffected individuals. This work shows that repeat expansion in human-specific NOTCH2NLC, a gene that evolved by segmental duplication, causes a human disease.

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April 21, 2020  |  

Sequence properties of certain GC rich avian genes, their origins and absence from genome assemblies: case studies.

More and more eukaryotic genomes are sequenced and assembled, most of them presented as a complete model in which missing chromosomal regions are filled by Ns and where a few chromosomes may be lacking. Avian genomes often contain sequences with high GC content, which has been hypothesized to be at the origin of many missing sequences in these genomes. We investigated features of these missing sequences to discover why some may not have been integrated into genomic libraries and/or sequenced.The sequences of five red jungle fowl cDNA models with high GC content were used as queries to search publicly available datasets of Illumina and Pacbio sequencing reads. These were used to reconstruct the leptin, TNFa, MRPL52, PCP2 and PET100 genes, all of which are absent from the red jungle fowl genome model. These gene sequences displayed elevated GC contents, had intron sizes that were sometimes larger than non-avian orthologues, and had non-coding regions that contained numerous tandem and inverted repeat sequences with motifs able to assemble into stable G-quadruplexes and intrastrand dyadic structures. Our results suggest that Illumina technology was unable to sequence the non-coding regions of these genes. On the other hand, PacBio technology was able to sequence these regions, but with dramatically lower efficiency than would typically be expected.High GC content was not the principal reason why numerous GC-rich regions of avian genomes are missing from genome assembly models. Instead, it is the presence of tandem repeats containing motifs capable of assembling into very stable secondary structures that is likely responsible.

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