cometadata/datacite-titles-descriptions-related-identifiers huggingface dataset:
DOI: 10.6084/m9.figshare.24567028
title: Determinants of DNMT2/TRDMT1 preference for substrates tRNA and DNA during the evolution

description: RNA methyltransferase DNMT2/TRDMT1 is the most conserved member of the DNMT family from bacteria to plants and mammals. In previous studies, we found some determinants for tRNA recognition of DNMT2/TRDMT1, but the preference mechanism of this enzyme for substrates tRNA and DNA remains to be explored. In the present study, CFT-containing target recognition domain (TRD) and target recognition extension domain (TRED) in DNMT2/TRDMT1 play a crucial role in the substrate DNA and RNA selection during the evolution. Moreover, the classical substrate tRNA for DNMT2/TRDMT1 had a characteristic sequence CUXXCAC in the anticodon loop. Position 35 was occupied by U, making cytosine-38 (C38) twist into the loop, whereas C, G or A was located at position 35, keeping the C38-flipping state. Hence, the substrate preference could be modulated by the easily flipped state of target cytosine in tRNA, as well as TRD and TRED. Additionally, DNMT2/TRDMT1 cancer mutant activity was collectively mediated by five enzymatic characteristics, which might impact gene expressions. Importantly, G155C, G155V and G155S mutations reduced enzymatic activities and showed significant associations with diseases using seven prediction methods. Altogether, these findings will assist in illustrating the substrate preference mechanism of DNMT2/TRDMT1 and provide a promising therapeutic strategy for cancer.
DOI: 10.6084/m9.figshare.24567028.v1
title: Determinants of DNMT2/TRDMT1 preference for substrates tRNA and DNA during the evolution

description: RNA methyltransferase DNMT2/TRDMT1 is the most conserved member of the DNMT family from bacteria to plants and mammals. In previous studies, we found some determinants for tRNA recognition of DNMT2/TRDMT1, but the preference mechanism of this enzyme for substrates tRNA and DNA remains to be explored. In the present study, CFT-containing target recognition domain (TRD) and target recognition extension domain (TRED) in DNMT2/TRDMT1 play a crucial role in the substrate DNA and RNA selection during the evolution. Moreover, the classical substrate tRNA for DNMT2/TRDMT1 had a characteristic sequence CUXXCAC in the anticodon loop. Position 35 was occupied by U, making cytosine-38 (C38) twist into the loop, whereas C, G or A was located at position 35, keeping the C38-flipping state. Hence, the substrate preference could be modulated by the easily flipped state of target cytosine in tRNA, as well as TRD and TRED. Additionally, DNMT2/TRDMT1 cancer mutant activity was collectively mediated by five enzymatic characteristics, which might impact gene expressions. Importantly, G155C, G155V and G155S mutations reduced enzymatic activities and showed significant associations with diseases using seven prediction methods. Altogether, these findings will assist in illustrating the substrate preference mechanism of DNMT2/TRDMT1 and provide a promising therapeutic strategy for cancer.
DOI: 10.6084/m9.figshare.22689657
title: Prior metabolite extraction fully preserves RNAseq quality and enables integrative multi-‘omics analysis of the liver metabolic response to viral infection

description: Here, we provide an in-depth analysis of the usefulness of single-sample metabolite/RNA extraction for multi-‘omics readout. Using pulverized frozen livers of mice injected with lymphocytic choriomeningitis virus (LCMV) or vehicle (Veh), we isolated RNA prior (RNA) or following metabolite extraction (MetRNA). RNA sequencing (RNAseq) data were evaluated for differential expression analysis and dispersion, and differential metabolite abundance was determined. Both RNA and MetRNA clustered together by principal component analysis, indicating that inter-individual differences were the largest source of variance. Over 85% of LCMV <i>versus</i> Veh differentially expressed genes were shared between extraction methods, with the remaining 15% evenly and randomly divided between groups. Differentially expressed genes unique to the extraction method were attributed to randomness around the 0.05 FDR cut-off and stochastic changes in variance and mean expression. In addition, analysis using the mean absolute difference showed no difference in the dispersion of transcripts between extraction methods. Altogether, our data show that prior metabolite extraction preserves RNAseq data quality, which enables us to confidently perform integrated pathway enrichment analysis on metabolomics and RNAseq data from a single sample. This analysis revealed pyrimidine metabolism as the most LCMV-impacted pathway. Combined analysis of genes and metabolites in the pathway exposed a pattern in the degradation of pyrimidine nucleotides leading to uracil generation. In support of this, uracil was among the most differentially abundant metabolites in serum upon LCMV infection. Our data suggest that hepatic uracil export is a novel phenotypic feature of acute infection and highlight the usefulness of our integrated single-sample multi-‘omics approach.
DOI: 10.6084/m9.figshare.22689657.v1
title: Prior metabolite extraction fully preserves RNAseq quality and enables integrative multi-‘omics analysis of the liver metabolic response to viral infection

description: Here, we provide an in-depth analysis of the usefulness of single-sample metabolite/RNA extraction for multi-‘omics readout. Using pulverized frozen livers of mice injected with lymphocytic choriomeningitis virus (LCMV) or vehicle (Veh), we isolated RNA prior (RNA) or following metabolite extraction (MetRNA). RNA sequencing (RNAseq) data were evaluated for differential expression analysis and dispersion, and differential metabolite abundance was determined. Both RNA and MetRNA clustered together by principal component analysis, indicating that inter-individual differences were the largest source of variance. Over 85% of LCMV <i>versus</i> Veh differentially expressed genes were shared between extraction methods, with the remaining 15% evenly and randomly divided between groups. Differentially expressed genes unique to the extraction method were attributed to randomness around the 0.05 FDR cut-off and stochastic changes in variance and mean expression. In addition, analysis using the mean absolute difference showed no difference in the dispersion of transcripts between extraction methods. Altogether, our data show that prior metabolite extraction preserves RNAseq data quality, which enables us to confidently perform integrated pathway enrichment analysis on metabolomics and RNAseq data from a single sample. This analysis revealed pyrimidine metabolism as the most LCMV-impacted pathway. Combined analysis of genes and metabolites in the pathway exposed a pattern in the degradation of pyrimidine nucleotides leading to uracil generation. In support of this, uracil was among the most differentially abundant metabolites in serum upon LCMV infection. Our data suggest that hepatic uracil export is a novel phenotypic feature of acute infection and highlight the usefulness of our integrated single-sample multi-‘omics approach.
DOI: 10.6084/m9.figshare.24189998
title: Characterization of CoCas9 nuclease from <i>Capnocytophaga ochracea</i>

description: Cas9 nucleases are widely used for genome editing and engineering. Cas9 enzymes encoded by CRISPR-Cas defence systems of various prokaryotic organisms possess different properties such as target site preferences, size, and DNA cleavage efficiency. Here, we biochemically characterized CoCas9 from <i>Capnocytophaga ochracea</i>, a bacterium that inhabits the oral cavity of humans and contributes to plaque formation on teeth. CoCas9 recognizes a novel 5’-NRRWC-3’ PAM and efficiently cleaves DNA <i>in vitro</i>. Functional characterization of CoCas9 opens ways for genetic engineering of <i>C. ochracea</i> using its endogenous CRISPR-Cas system. The novel PAM requirement makes CoCas9 potentially useful in genome editing applications.
DOI: 10.6084/m9.figshare.24189998.v1
title: Characterization of CoCas9 nuclease from <i>Capnocytophaga ochracea</i>

description: Cas9 nucleases are widely used for genome editing and engineering. Cas9 enzymes encoded by CRISPR-Cas defence systems of various prokaryotic organisms possess different properties such as target site preferences, size, and DNA cleavage efficiency. Here, we biochemically characterized CoCas9 from <i>Capnocytophaga ochracea</i>, a bacterium that inhabits the oral cavity of humans and contributes to plaque formation on teeth. CoCas9 recognizes a novel 5’-NRRWC-3’ PAM and efficiently cleaves DNA <i>in vitro</i>. Functional characterization of CoCas9 opens ways for genetic engineering of <i>C. ochracea</i> using its endogenous CRISPR-Cas system. The novel PAM requirement makes CoCas9 potentially useful in genome editing applications.
DOI: 10.6084/m9.figshare.23622476
title: Characteristics of Human and Microbiome RNA Profiles in Saliva

description: Saliva is a convenient non-invasive source of liquid biopsy to monitor human health and diagnose diseases. In particular, extracellular vesicles (EVs) in saliva can potentially reveal clinically relevant information for systemic health. Recent studies have shown that RNA in saliva EVs could be exploited as biomarkers for disease diagnosis. However, there is no standardized protocol for profiling RNA in saliva EV nor clear guideline on selecting saliva fractions for biomarker analysis. To address these issues, we established a robust protocol for small RNA profiling from fractionated saliva. With this method, we performed comprehensive small RNA sequencing of four saliva fractions, including cell-free saliva (CFS), EV-depleted saliva (EV-D), exosome (EXO), and microvesicle (MV) from ten healthy volunteers. By comparing the expression profiles of total RNA from these fractions, we found that MV was most enriched in microbiome RNA (76.2% of total reads on average), whereas EV-D was notably enriched in human RNA (70.3% of total reads on average). As for human RNA composition, CFS and EV-D were both enriched in snoRNA and tRNA compared with the two EV fractions (EXO and MV, <i>P</i> &lt; 0.05). Interestingly, EXO and MV had highly correlated expression profiles for various noncoding RNAs such as miRNA, tRNA, and yRNA. Our study revealed unique characteristics of circulating RNAs in various saliva fractions, which provides a guideline on preparing saliva samples to study specific RNA biomarkers of interest.
DOI: 10.6084/m9.figshare.23622476.v1
title: Characteristics of Human and Microbiome RNA Profiles in Saliva

description: Saliva is a convenient non-invasive source of liquid biopsy to monitor human health and diagnose diseases. In particular, extracellular vesicles (EVs) in saliva can potentially reveal clinically relevant information for systemic health. Recent studies have shown that RNA in saliva EVs could be exploited as biomarkers for disease diagnosis. However, there is no standardized protocol for profiling RNA in saliva EV nor clear guideline on selecting saliva fractions for biomarker analysis. To address these issues, we established a robust protocol for small RNA profiling from fractionated saliva. With this method, we performed comprehensive small RNA sequencing of four saliva fractions, including cell-free saliva (CFS), EV-depleted saliva (EV-D), exosome (EXO), and microvesicle (MV) from ten healthy volunteers. By comparing the expression profiles of total RNA from these fractions, we found that MV was most enriched in microbiome RNA (76.2% of total reads on average), whereas EV-D was notably enriched in human RNA (70.3% of total reads on average). As for human RNA composition, CFS and EV-D were both enriched in snoRNA and tRNA compared with the two EV fractions (EXO and MV, <i>P</i> &lt; 0.05). Interestingly, EXO and MV had highly correlated expression profiles for various noncoding RNAs such as miRNA, tRNA, and yRNA. Our study revealed unique characteristics of circulating RNAs in various saliva fractions, which provides a guideline on preparing saliva samples to study specific RNA biomarkers of interest.
DOI: 10.6084/m9.figshare.22591252
title: L1CAM immunocapture generates a unique extracellular vesicle population with a reproducible miRNA fingerprint

description: Micro RNAs (miRNAs) are short, non-coding RNAs with significant potential as diagnostic and prognostic biomarkers. However, a lack of reproducibility across studies has hindered their introduction into clinical settings. Inconsistencies between studies include a lack of consensus on the miRNAs associated with a specific disease and the direction of regulation. These differences may reflect the heterogenous nature of pathologies with multiple phenotypes, such as amyotrophic lateral sclerosis (ALS). It is also possible that discrepancies are due to different sampling, processing, and analysis protocols across labs. Using miRNA extracted from L1CAM immunoaffinity purified extracellular vesicles (neural-enriched extracellular vesicles or NEE), we thrice replicated an 8-miRNA fingerprint diagnostic of ALS, which includes the miRNA species and direction of regulation. We aimed to determine if the extra purification steps required to generate NEE created a unique extracellular vesicle (EV) fraction that might contribute to the robustness and replicability of our assay. We compared three fractions from control human plasma: 1) total heterogenous EVs (T), 2) L1CAM/neural enriched EVs (NEE), and 3) the remaining total-minus-NEE fraction (T-N). Each fraction was characterized for size, total protein content, and protein markers, then total RNA was extracted, and qPCR was run on 20 miRNAs. We report that the miRNA expression within NEE was different enough compared to T and T-N to justify the extra steps required to generate this fraction. We conclude that L1CAM immunocapture generates a unique fraction of EVs that consistently and robustly replicates a miRNA fingerprint which differentiates ALS patients from controls.
DOI: 10.6084/m9.figshare.22591252.v1
title: L1CAM immunocapture generates a unique extracellular vesicle population with a reproducible miRNA fingerprint

description: Micro RNAs (miRNAs) are short, non-coding RNAs with significant potential as diagnostic and prognostic biomarkers. However, a lack of reproducibility across studies has hindered their introduction into clinical settings. Inconsistencies between studies include a lack of consensus on the miRNAs associated with a specific disease and the direction of regulation. These differences may reflect the heterogenous nature of pathologies with multiple phenotypes, such as amyotrophic lateral sclerosis (ALS). It is also possible that discrepancies are due to different sampling, processing, and analysis protocols across labs. Using miRNA extracted from L1CAM immunoaffinity purified extracellular vesicles (neural-enriched extracellular vesicles or NEE), we thrice replicated an 8-miRNA fingerprint diagnostic of ALS, which includes the miRNA species and direction of regulation. We aimed to determine if the extra purification steps required to generate NEE created a unique extracellular vesicle (EV) fraction that might contribute to the robustness and replicability of our assay. We compared three fractions from control human plasma: 1) total heterogenous EVs (T), 2) L1CAM/neural enriched EVs (NEE), and 3) the remaining total-minus-NEE fraction (T-N). Each fraction was characterized for size, total protein content, and protein markers, then total RNA was extracted, and qPCR was run on 20 miRNAs. We report that the miRNA expression within NEE was different enough compared to T and T-N to justify the extra steps required to generate this fraction. We conclude that L1CAM immunocapture generates a unique fraction of EVs that consistently and robustly replicates a miRNA fingerprint which differentiates ALS patients from controls.
DOI: 10.6084/m9.figshare.23294486
title: Hybrid-seq deciphers the complex transcriptional profile of the human <i>BRCA1</i> DNA repair associated gene

description: Breast Cancer Gene 1 (BRCA1) is a tumour suppressor protein that modulates multiple biological processes including genomic stability and DNA damage repair. Although the main BRCA1 protein is well characterized, further proteomics studies have already identified additional BRCA1 isoforms with lower molecular weights. However, the accurate nucleotide sequence determination of their corresponding mRNAs is still a barrier, mainly due to the increased mRNA length of <i>BRCA1</i> (~5.5 kb) and the limitations of the already implemented sequencing approaches. In the present study, we designed and employed a multiplexed hybrid sequencing approach (Hybrid-seq), based on nanopore and semi-conductor sequencing, aiming to detect <i>BRCA1</i> alternative transcripts in a panel of human cancer and non-cancerous cell lines. The implementation of the described Hybrid-seq approach led to the generation of highly accurate long sequencing reads that enabled the identification of a wide spectrum of <i>BRCA1</i> splice variants (<i>BRCA1</i> sv.7 – sv.52), thus deciphering the transcriptional landscape of the human <i>BRCA1</i> gene. In addition, demultiplexing of the sequencing data unveiled the expression profile and abundance of the described <i>BRCA1</i> mRNAs in breast, ovarian, prostate, colorectal, lung and brain cancer as well as in non-cancerous human cell lines. Finally, <i>in silico</i> analysis supports that multiple detected mRNAs harbour open reading frames, being highly expected to encode putative protein isoforms with conserved domains, thus providing new insights into the complex roles of BRCA1 in genomic stability and DNA damage repair.
DOI: 10.6084/m9.figshare.23294486.v1
title: Hybrid-seq deciphers the complex transcriptional profile of the human <i>BRCA1</i> DNA repair associated gene

description: Breast Cancer Gene 1 (BRCA1) is a tumour suppressor protein that modulates multiple biological processes including genomic stability and DNA damage repair. Although the main BRCA1 protein is well characterized, further proteomics studies have already identified additional BRCA1 isoforms with lower molecular weights. However, the accurate nucleotide sequence determination of their corresponding mRNAs is still a barrier, mainly due to the increased mRNA length of <i>BRCA1</i> (~5.5 kb) and the limitations of the already implemented sequencing approaches. In the present study, we designed and employed a multiplexed hybrid sequencing approach (Hybrid-seq), based on nanopore and semi-conductor sequencing, aiming to detect <i>BRCA1</i> alternative transcripts in a panel of human cancer and non-cancerous cell lines. The implementation of the described Hybrid-seq approach led to the generation of highly accurate long sequencing reads that enabled the identification of a wide spectrum of <i>BRCA1</i> splice variants (<i>BRCA1</i> sv.7 – sv.52), thus deciphering the transcriptional landscape of the human <i>BRCA1</i> gene. In addition, demultiplexing of the sequencing data unveiled the expression profile and abundance of the described <i>BRCA1</i> mRNAs in breast, ovarian, prostate, colorectal, lung and brain cancer as well as in non-cancerous human cell lines. Finally, <i>in silico</i> analysis supports that multiple detected mRNAs harbour open reading frames, being highly expected to encode putative protein isoforms with conserved domains, thus providing new insights into the complex roles of BRCA1 in genomic stability and DNA damage repair.
DOI: 10.6084/m9.figshare.23629334
title: Using <i>sno-</i><i>lncRNAs</i> as potential markers for Prader-Willi syndrome diagnosis

description: The genetic disorder Prader-Willi syndrome (PWS) is mainly caused by the loss of multiple paternally expressed genes in chromosome 15q11-q13 (the PWS region). Early diagnosis of PWS is essential for timely treatment, leading to effectively easing some clinical symptoms. Molecular approaches for PWS diagnosis at the DNA level are available, but the diagnosis of PWS at the RNA level has been limited. Here, we show that a cluster of paternally transcribed snoRNA-ended long noncoding RNAs (<i>sno-lncRNAs</i>, <i>sno-lncRNA1–5</i>) derived from the <i>SNORD116</i> locus in the PWS region can serve as diagnostic markers. In particular, quantification analysis has revealed that 6,000 copies of <i>sno-lncRNA3</i> are present in 1 μL whole blood samples from non-PWS individuals. <i>sno-lncRNA</i>3 is absent in all examined whole blood samples of 8 PWS individuals compared to 42 non-PWS individuals and dried blood samples of 35 PWS individuals compared to 24 non-PWS individuals. Further developing a new CRISPR-MhdCas13c system for RNA detection with a sensitivity of 10 molecules per μL has ensured <i>sno-lncRNA3</i> detection in non-PWS, but not PWS individuals. Together, we suggest that the absence of <i>sno-lncRNA3</i> represents a potential marker for PWS diagnosis that can be detected by both RT-qPCR and CRISPR-MhdCas13c systems with only microlitre amount of blood samples. Such an RNA-based sensitive and convenient approach may facilitate the early detection of PWS.
DOI: 10.6084/m9.figshare.23629334.v1
title: Using <i>sno-</i><i>lncRNAs</i> as potential markers for Prader-Willi syndrome diagnosis

description: The genetic disorder Prader-Willi syndrome (PWS) is mainly caused by the loss of multiple paternally expressed genes in chromosome 15q11-q13 (the PWS region). Early diagnosis of PWS is essential for timely treatment, leading to effectively easing some clinical symptoms. Molecular approaches for PWS diagnosis at the DNA level are available, but the diagnosis of PWS at the RNA level has been limited. Here, we show that a cluster of paternally transcribed snoRNA-ended long noncoding RNAs (<i>sno-lncRNAs</i>, <i>sno-lncRNA1–5</i>) derived from the <i>SNORD116</i> locus in the PWS region can serve as diagnostic markers. In particular, quantification analysis has revealed that 6,000 copies of <i>sno-lncRNA3</i> are present in 1 μL whole blood samples from non-PWS individuals. <i>sno-lncRNA</i>3 is absent in all examined whole blood samples of 8 PWS individuals compared to 42 non-PWS individuals and dried blood samples of 35 PWS individuals compared to 24 non-PWS individuals. Further developing a new CRISPR-MhdCas13c system for RNA detection with a sensitivity of 10 molecules per μL has ensured <i>sno-lncRNA3</i> detection in non-PWS, but not PWS individuals. Together, we suggest that the absence of <i>sno-lncRNA3</i> represents a potential marker for PWS diagnosis that can be detected by both RT-qPCR and CRISPR-MhdCas13c systems with only microlitre amount of blood samples. Such an RNA-based sensitive and convenient approach may facilitate the early detection of PWS.