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Frontiers in Microbiology 2024Lymphatic filariasis is caused by parasitic nematodes and is a leading cause of disability worldwide. Many filarial worms contain the bacterium as an obligate...
Lymphatic filariasis is caused by parasitic nematodes and is a leading cause of disability worldwide. Many filarial worms contain the bacterium as an obligate endosymbiont. RNA sequencing is a common technique used to study their molecular relationships and to identify potential drug targets against the nematode and bacteria. Ribosomal RNA (rRNA) is the most abundant RNA species, accounting for 80-90% of the RNA in a sample. To reduce sequencing costs, it is necessary to remove ribosomal reads through poly-A enrichment or ribosomal depletion. Bacterial RNA does not contain a poly-A tail, making it difficult to sequence both the nematode and from the same library preparation using standard poly-A selection. Ribosomal depletion can utilize species-specific oligonucleotide probes to remove rRNA through pull-down or degradation methods. While species-specific probes are commercially available for many commonly studied model organisms, there are currently limited depletion options for filarial parasites. Here, we performed total RNA sequencing from containing the symbiont (Bm) and designed ssDNA depletion probes against their rRNA sequences. We compared the total RNA library to poly-A enriched, Terminator 5'-Phosphate-Dependent Exonuclease treated, NEBNext Human/Bacteria rRNA depleted and our custom nematode probe depleted libraries. The custom nematode depletion library had the lowest percentage of ribosomal reads across all methods, with a 300-fold decrease in rRNA when compared to the total RNA library. The nematode depletion libraries also contained the highest percentage of mRNA reads, resulting in a 16-1,000-fold increase in bacterial reads compared to the other enrichment and depletion methods. Finally, we found that the depletion probes can remove rRNA from the filarial worm and the majority of rRNA from the more distantly related free living nematode . These custom filarial probes will allow for future dual RNA-seq experiments between nematodes and their bacterial symbionts from a single sequencing library.
PubMed: 38832111
DOI: 10.3389/fmicb.2024.1418032 -
BMC Genomics May 2024Direct RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platforms can produce reads covering up to full-length gene transcripts, while containing...
BACKGROUND
Direct RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platforms can produce reads covering up to full-length gene transcripts, while containing decipherable information about RNA base modifications and poly-A tail lengths. Although many published studies have been expanding the potential of dRNA-seq, its sequencing accuracy and error patterns remain understudied.
RESULTS
We present the first comprehensive evaluation of sequencing accuracy and characterisation of systematic errors in dRNA-seq data from diverse organisms and synthetic in vitro transcribed RNAs. We found that for sequencing kits SQK-RNA001 and SQK-RNA002, the median read accuracy ranged from 87% to 92% across species, and deletions significantly outnumbered mismatches and insertions. Due to their high abundance in the transcriptome, heteropolymers and short homopolymers were the major contributors to the overall sequencing errors. We also observed systematic biases across all species at the levels of single nucleotides and motifs. In general, cytosine/uracil-rich regions were more likely to be erroneous than guanines and adenines. By examining raw signal data, we identified the underlying signal-level features potentially associated with the error patterns and their dependency on sequence contexts. While read quality scores can be used to approximate error rates at base and read levels, failure to detect DNA adapters may be a source of errors and data loss. By comparing distinct basecallers, we reason that some sequencing errors are attributable to signal insufficiency rather than algorithmic (basecalling) artefacts. Lastly, we generated dRNA-seq data using the latest SQK-RNA004 sequencing kit released at the end of 2023 and found that although the overall read accuracy increased, the systematic errors remain largely identical compared to the previous kits.
CONCLUSIONS
As the first systematic investigation of dRNA-seq errors, this study offers a comprehensive overview of reproducible error patterns across diverse datasets, identifies potential signal-level insufficiency, and lays the foundation for error correction methods.
Topics: Sequence Analysis, RNA; Nanopore Sequencing; Nanopores; Humans; Animals; RNA; High-Throughput Nucleotide Sequencing
PubMed: 38807060
DOI: 10.1186/s12864-024-10440-w -
RNA Biology Jan 2024Although circular RNAs (circRNAs) play important roles in regulating gene expression, the understanding of circRNAs in livestock animals is scarce due to the significant...
Although circular RNAs (circRNAs) play important roles in regulating gene expression, the understanding of circRNAs in livestock animals is scarce due to the significant challenge to characterize them from a biological sample. In this study, we assessed the outcomes of bovine circRNA identification using six enrichment approaches with the combination of ribosomal RNAs removal (); linear RNAs degradation (); linear RNAs and RNAs with structured 3' ends degradation (); ribosomal RNAs coupled with linear RNAs elimination (); ribosomal RNA, linear RNAs and RNAs with poly (A) tailing elimination (); and ribosomal RNA, linear RNAs and RNAs with structured 3' ends elimination (), respectively. RNA-sequencing analysis revealed that different approaches led to varied ratio of uniquely mapped reads, false-positive rate of identifying circRNAs, and the number of circRNAs per million clean reads ( <0.05). Out of 2,285 and 2,939 highly confident circRNAs identified in liver and rumen tissues, respectively, 308 and 260 were commonly identified from five methods, with Ribo-RTP method identified the highest number of circRNAs. Besides, 507 of 4,051 identified bovine highly confident circRNAs had shared splicing sites with human circRNAs. The findings from this work provide optimized methods to identify bovine circRNAs from cattle tissues for downstream research of their biological roles in cattle.
Topics: Cattle; RNA, Circular; Animals; RNA, Ribosomal; Sequence Analysis, RNA; Liver; Rumen; Computational Biology; Gene Expression Profiling; Humans
PubMed: 38797889
DOI: 10.1080/15476286.2024.2356334 -
NPJ Parkinson's Disease May 2024A biallelic (AAGGG) expansion in the poly(A) tail of an AluSx3 transposable element within the gene RFC1 is a frequent cause of cerebellar ataxia, neuropathy, vestibular...
A biallelic (AAGGG) expansion in the poly(A) tail of an AluSx3 transposable element within the gene RFC1 is a frequent cause of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS), and more recently, has been reported as a rare cause of Parkinson's disease (PD) in the Finnish population. Here, we investigate the prevalence of RFC1 (AAGGG) expansions in PD patients of non-Finnish European ancestry in 1609 individuals from the Parkinson's Progression Markers Initiative study. We identified four PD patients carrying the biallelic RFC1 (AAGGG) expansion and did not identify any carriers in controls.
PubMed: 38789445
DOI: 10.1038/s41531-024-00723-0 -
Microbial Cell (Graz, Austria) 2024In , polyadenylated forms of mature (and not precursor) small non-coding RNAs (sncRNAs) those fail to undergo proper 3'-end maturation are subject to an active...
In , polyadenylated forms of mature (and not precursor) small non-coding RNAs (sncRNAs) those fail to undergo proper 3'-end maturation are subject to an active degradation by Rrp6p and Rrp47p, which does not require the involvement of core exosome and TRAMP components. In agreement with this finding, Rrp6p/Rrp47p is demonstrated to exist as an exosome-independent complex, which preferentially associates with mature polyadenylated forms of these sncRNAs. Consistent with this observation, a C-terminally truncated version of Rrp6p (Rrp6p-ΔC2) lacking physical association with the core nuclear exosome supports their decay just like its full-length version. Polyadenylation is catalyzed by both the canonical and non-canonical poly(A) polymerases, Pap1p and Trf4p. Analysis of the polyadenylation profiles in WT and -Δ strains revealed that the majority of the polyadenylation sites correspond to either one to three nucleotides upstream or downstream of their mature ends and their poly(A) tails ranges from 10-15 adenylate residues. Most interestingly, the accumulated polyadenylated snRNAs are functional in the -Δ strain and are assembled into spliceosomes. Thus, Rrp6p-Rrp47p defines a core nuclear exosome-independent novel RNA turnover system in baker's yeast targeting imperfectly processed polyadenylated sncRNAs that accumulate in the absence of Rrp6p.
PubMed: 38783922
DOI: 10.15698/mic2024.05.823 -
Scientific Reports May 2024Peptide deformylase can catalyse the removal of formyl groups from the N-terminal formyl methionine of the primary polypeptide chain. The peptide deformylase genes of a...
Peptide deformylase can catalyse the removal of formyl groups from the N-terminal formyl methionine of the primary polypeptide chain. The peptide deformylase genes of a few herbaceous plants have been studied to some extent, but the peptide deformylase genes of woody plants have not been studied. In this study, we isolated EuPDF1B from Eucommia ulmoides Oliv. The full-length sequence of EuPDF1B is 1176 bp long with a poly-A tail and contains an open reading frame of 831 bp that encodes a protein of 276 amino acids. EuPDF1B was localized to the chloroplast. qRT‒PCR analysis revealed that this gene was expressed in almost all tissues tested but mainly in mature leaves. Moreover, the expression of EuPDF1B was enhanced by ABA, MeJA and GA and inhibited by shading treatment. The expression pattern of EuPDF1B was further confirmed in EuPDF1Bp: GUS transgenic tobacco plants. Among all the transgenic tobacco plants, EuPDF1Bp-3 showed the highest GUS histochemical staining and activity in different tissues. This difference may be related to the presence of enhancer elements in the region from - 891 bp to - 236 bp of the EuPDF1B promoter. In addition, the expression of the chloroplast gene psbA and the net photosynthetic rate, fresh weight and height of tobacco plants overexpressing EuPDF1B were greater than those of the wild-type tobacco plants, suggesting that EuPDF1B may promote the growth of transgenic tobacco plants. This is the first time that PDF and its promoter have been cloned from woody plants, laying a foundation for further analysis of the function of PDF and the regulation of its expression.
Topics: Eucommiaceae; Plants, Genetically Modified; Gene Expression Regulation, Plant; Cloning, Molecular; Amidohydrolases; Nicotiana; Chloroplasts; Plant Proteins; Plant Leaves; Phylogeny; Amino Acid Sequence; Cyclopentanes; Oxylipins
PubMed: 38773239
DOI: 10.1038/s41598-024-62512-2 -
PLoS Genetics May 2024Ataxin-2 (ATXN2) is a gene implicated in spinocerebellar ataxia type II (SCA2), amyotrophic lateral sclerosis (ALS) and Parkinsonism. The encoded protein is a...
Ataxin-2 (ATXN2) is a gene implicated in spinocerebellar ataxia type II (SCA2), amyotrophic lateral sclerosis (ALS) and Parkinsonism. The encoded protein is a therapeutic target for ALS and related conditions. ATXN2 (or Atx2 in insects) can function in translational activation, translational repression, mRNA stability and in the assembly of mRNP-granules, a process mediated by intrinsically disordered regions (IDRs). Previous work has shown that the LSm (Like-Sm) domain of Atx2, which can help stimulate mRNA translation, antagonizes mRNP-granule assembly. Here we advance these findings through a series of experiments on Drosophila and human Ataxin-2 proteins. Results of Targets of RNA Binding Proteins Identified by Editing (TRIBE), co-localization and immunoprecipitation experiments indicate that a polyA-binding protein (PABP) interacting, PAM2 motif of Ataxin-2 may be a major determinant of the mRNA and protein content of Ataxin-2 mRNP granules. Experiments with transgenic Drosophila indicate that while the Atx2-LSm domain may protect against neurodegeneration, structured PAM2- and unstructured IDR- interactions both support Atx2-induced cytotoxicity. Taken together, the data lead to a proposal for how Ataxin-2 interactions are remodelled during translational control and how structured and non-structured interactions contribute differently to the specificity and efficiency of RNP granule condensation as well as to neurodegeneration.
Topics: Ataxin-2; Animals; Humans; Ribonucleoproteins; Drosophila Proteins; Drosophila melanogaster; RNA, Messenger; Poly(A)-Binding Proteins; Animals, Genetically Modified; Cytoplasmic Granules; Amyotrophic Lateral Sclerosis; Protein Biosynthesis; RNA-Binding Proteins; Intrinsically Disordered Proteins; Nerve Tissue Proteins; DNA-Binding Proteins
PubMed: 38768217
DOI: 10.1371/journal.pgen.1011251 -
International Journal of Biological... Jun 2024Leishmania is one of the most common diseases between human and animals, caused by Leishmania infantum parasite. Here, we have developed an ultra-selective turn-on...
Leishmania is one of the most common diseases between human and animals, caused by Leishmania infantum parasite. Here, we have developed an ultra-selective turn-on fluorescent probe based on an aptamer and Chitosan-CD nanocomposite. The CD used in this study were synthesized using Quercus cap extract and a microwave-assisted approach. The Chitosan-CD nanocomposite was optimized using several microscopic and spectroscopic techniques to possess a bright fluorescence emission before adding aptamer and totally quenched fluorescence after addition of aptamer. The designed probe was proficient in the detection and quantification Leishmania infantum parasite by selective targeting of poly(A) binding protein (PABP) on the surface of the parasite. The designed fluorescent biosensor with high sensitivity, excellent selectivity, and a limit of detection (LOD) of 94 cells/mL of the Leishmania infantum parasite as well as a linear response in the ranges of 188-750 cells/mL and 3000-6000 cells/mL (R ≥ 0.98 for both linear ranges). Additionally, the selectivity of the designed probe was evaluated in the presence of different pathogenic species such as Trypanosoma brucei parasite and Staphylococcus aureus bacteria, as well as LiIF2α and LiP2a and BSA proteins as interference substances. The results of this study shows that using Chitosan-CD nanocomposite is a great strategy for developing selective turn-on probes with extraordinary accuracy and sensitivity in identifying Leishmania infantum parasite, especially in the early stages of the disease, and it is promising for the future clinical applications.
Topics: Leishmania infantum; Chitosan; Nanocomposites; Aptamers, Nucleotide; Biosensing Techniques; Carbon; Limit of Detection; Fluorescent Dyes; Humans
PubMed: 38763252
DOI: 10.1016/j.ijbiomac.2024.132483 -
BioRxiv : the Preprint Server For... May 2024elements are non-autonomous Short INterspersed Elements (SINEs) derived from the gene that are present at over one million copies in human genomic DNA. mobilizes by a...
elements are non-autonomous Short INterspersed Elements (SINEs) derived from the gene that are present at over one million copies in human genomic DNA. mobilizes by a mechanism known as retrotransposition, which requires the Long INterspersed Element-1 (LINE-1 or L1) -encoded protein (ORF2p). Here, we demonstrate that HeLa strains differ in their capacity to support retrotransposition. Human elements retrotranspose efficiently in HeLa-HA and HeLa-CCL2 ( -permissive) strains, but not in HeLa-JVM or HeLa-H1 ( -nonpermissive) strains. A similar pattern of retrotransposition was observed for other -derived SINEs and -derived SINEs. In contrast, mammalian LINE-1s, a zebrafish LINE, a human - ( ) element, and an -containing messenger RNA can retrotranspose in all four HeLa strains. Using an reverse transcriptase-based assay, we show that RNAs associate with ORF2p and are converted into cDNAs in both -permissive and -nonpermissive HeLa strains, suggesting that - and -derived SINE RNAs use strategies to 'hijack' L1 ORF2p that are distinct from those used by elements and -containing mRNAs. These data further suggest ORF2p associates with the RNA poly(A) tract in both -permissive and -nonpermissive HeLa strains, but that retrotransposition is blocked after this critical step in -nonpermissive HeLa strains.
PubMed: 38746229
DOI: 10.1101/2024.05.03.592410 -
BMC Plant Biology May 2024Riccia fluitans, an amphibious liverwort, exhibits a fascinating adaptation mechanism to transition between terrestrial and aquatic environments. Utilizing nanopore...
BACKGROUND
Riccia fluitans, an amphibious liverwort, exhibits a fascinating adaptation mechanism to transition between terrestrial and aquatic environments. Utilizing nanopore direct RNA sequencing, we try to capture the complex epitranscriptomic changes undergone in response to land-water transition.
RESULTS
A significant finding is the identification of 45 differentially expressed genes (DEGs), with a split of 33 downregulated in terrestrial forms and 12 upregulated in aquatic forms, indicating a robust transcriptional response to environmental changes. Analysis of N6-methyladenosine (m6A) modifications revealed 173 m6A sites in aquatic and only 27 sites in the terrestrial forms, indicating a significant increase in methylation in the former, which could facilitate rapid adaptation to changing environments. The aquatic form showed a global elongation bias in poly(A) tails, which is associated with increased mRNA stability and efficient translation, enhancing the plant's resilience to water stress. Significant differences in polyadenylation signals were observed between the two forms, with nine transcripts showing notable changes in tail length, suggesting an adaptive mechanism to modulate mRNA stability and translational efficiency in response to environmental conditions. This differential methylation and polyadenylation underline a sophisticated layer of post-transcriptional regulation, enabling Riccia fluitans to fine-tune gene expression in response to its living conditions.
CONCLUSIONS
These insights into transcriptome dynamics offer a deeper understanding of plant adaptation strategies at the molecular level, contributing to the broader knowledge of plant biology and evolution. These findings underscore the sophisticated post-transcriptional regulatory strategies Riccia fluitans employs to navigate the challenges of aquatic versus terrestrial living, highlighting the plant's dynamic adaptation to environmental stresses and its utility as a model for studying adaptation mechanisms in amphibious plants.
Topics: Transcriptome; Sequence Analysis, RNA; Nanopore Sequencing; Marchantia; Gene Expression Regulation, Plant; RNA, Plant; Adaptation, Physiological; Epigenesis, Genetic
PubMed: 38745128
DOI: 10.1186/s12870-024-05114-4