scholarly journals RNAs as proximity labeling media for identifying nuclear speckle positions relative to the genome

2018 ◽  
Author(s):  
Weizhong Chen ◽  
Zhangming Yan ◽  
Simin Li ◽  
Norman Huang ◽  
Xuerui Huang ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Genomic Sequence ◽  
Single Cells ◽  
Regulation Of Gene Expression ◽  
Nuclear Genome ◽  
Fold Increase ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Interaction Sites ◽  
Genomic Regions

AbstractNuclear speckles are interchromatin structures enriched in RNA splicing factors. Determining their relative positions with respect to the folded nuclear genome could provide critical information on co-and post-transcriptional regulation of gene expression. However, it remains challenging to identify which parts of the nuclear genome are in proximity to nuclear speckles, due to physical separation between nuclear speckle cores and chromatin. We hypothesized that noncoding RNAs including small nuclear RNAs, 7SK and Malat1, which accumulate at the periphery of nuclear speckles (nsaRNA,nuclearspeckleassociated RNA), may extend to sufficient proximity to the nuclear genome. Leveraging a transcriptome-genome interaction assay (MARGI), we identified nsaRNA-interacting genomic sequences, which exhibited clustering patterns (nsaPeaks) in the genome, suggesting existence of relatively stable interaction sites for nsaRNAs in nuclear genome. Posttranscriptional pre-mRNAs, which are known to be clustered to nuclear speckles, exhibited proximity to nsaPeaks but rarely to other genomic regions. Furthermore, CDK9 proteins that localize to the vicinity of nuclear speckles produced ChIP-seq peaks that overlapped with nsaPeaks. Our combined DNA FISH and immunofluorescence analysis in 182 single cells revealed a 3-fold increase in odds for nuclear speckles to localize near an nsaPeak than its neighboring genomic sequence. These data suggest a model that nsaRNAs locate in sufficient proximity to nuclear genome and leave identifiable genomic footprints, thus revealing the parts of genome proximal to nuclear speckles.

scholarly journals Super-resolution imaging of a 2.5 kb non-repetitive DNA in situ in the nuclear genome using molecular beacon probes

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yanxiang Ni ◽  
Bo Cao ◽  
Tszshan Ma ◽  
Gang Niu ◽  
Yingdong Huo ◽  
...  
Keyword(s):  
Repetitive Dna ◽  
Molecular Beacon ◽  
Genomic Sequence ◽  
Single Cells ◽  
Nuclear Genome ◽  
Super Resolution ◽  
Resolution Limit ◽  
Fish Method ◽  
Resolution Imaging

High-resolution visualization of short non-repetitive DNA in situ in the nuclear genome is essential for studying looping interactions and chromatin organization in single cells. Recent advances in fluorescence in situ hybridization (FISH) using Oligopaint probes have enabled super-resolution imaging of genomic domains with a resolution limit of 4.9 kb. To target shorter elements, we developed a simple FISH method that uses molecular beacon (MB) probes to facilitate the probe-target binding, while minimizing non-specific fluorescence. We used three-dimensional stochastic optical reconstruction microscopy (3D-STORM) with optimized imaging conditions to efficiently distinguish sparsely distributed Alexa-647 from background cellular autofluorescence. Utilizing 3D-STORM and only 29–34 individual MB probes, we observed 3D fine-scale nanostructures of 2.5 kb integrated or endogenous unique DNA in situ in human or mouse genome, respectively. We demonstrated our MB-based FISH method was capable of visualizing the so far shortest non-repetitive genomic sequence in 3D at super-resolution.

scholarly journals Genomic sequences and RNA binding proteins predict RNA splicing kinetics in various single-cell contexts

2021 ◽  
Author(s):  
Ruiyan Hou ◽  
Yuanhua Huang
Keyword(s):  
Single Cell ◽  
Rna Splicing ◽  
Binding Proteins ◽  
Rna Binding ◽  
Genomic Sequence ◽  
Rna Binding Proteins ◽  
Single Cells ◽  
Regulatory Machinery ◽  
Kinetic Rates ◽  
Splicing Efficiency

RNA splicing is a key step of gene expression in higher organisms. Accurate quantification of the two-step splicing kinetics is of high interests not only for understanding the regulatory machinery, but also for estimating the RNA velocity in single cells. However, the kinetic rates remain poorly understood due to the intrinsic low content of unspliced RNAs and its stochasticity across contexts. Here, we estimated the relative splicing efficiency across a variety of single-cell RNA-Seq data with scVelo. We further extracted three large feature sets including 92 basic genomic sequence features, 65,536 octamers and 120 RNA binding proteins features and found they are highly predictive to RNA splicing efficiency across multiple tissues on human and mouse. A set of important features have been identified with strong regulatory potentials on splicing efficiency. This predictive power brings promise to reveal the complexity of RNA processing and to enhance the estimation of single-cell RNA velocity.

scholarly journals Splicing at the phase-separated nuclear speckle interface: a model

2020 ◽  
Author(s):  
Susan E Liao ◽  
Oded Regev
Keyword(s):  
Phase Separation ◽  
Rna Splicing ◽  
Splicing Factors ◽  
Sequence Motifs ◽  
Splice Sites ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Functional Roles ◽  
Hnrnp Proteins ◽  
Model Sequence

Abstract Phase-separated membraneless bodies play important roles in nucleic acid biology. While current models for the roles of phase separation largely focus on the compartmentalization of constituent proteins, we reason that other properties of phase separation may play functional roles. Specifically, we propose that interfaces of phase-separated membraneless bodies could have functional roles in spatially organizing biochemical reactions. Here we propose such a model for the nuclear speckle, a membraneless body implicated in RNA splicing. In our model, sequence-dependent RNA positioning along the nuclear speckle interface coordinates RNA splicing. Our model asserts that exons are preferentially sequestered into nuclear speckles through binding by SR proteins, while introns are excluded through binding by nucleoplasmic hnRNP proteins. As a result, splice sites at exon-intron boundaries are preferentially positioned at nuclear speckle interfaces. This positioning exposes splice sites to interface-localized spliceosomes, enabling the subsequent splicing reaction. Our model provides a simple mechanism that seamlessly explains much of the complex logic of splicing. This logic includes experimental results such as the antagonistic duality between splicing factors, the position dependence of splicing sequence motifs, and the collective contribution of many motifs to splicing decisions. Similar functional roles for phase-separated interfaces may exist for other membraneless bodies.

scholarly journals Collagen I triggers directional migration, invasion and matrix remodeling of stroma cells in a 3D spheroid model of endometriosis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anna Stejskalová ◽  
Victoria Fincke ◽  
Melissa Nowak ◽  
Yvonne Schmidt ◽  
Katrin Borrmann ◽  
...  
Keyword(s):  
Single Cells ◽  
Fold Increase ◽  
Experimental Models ◽  
Collagen I ◽  
Matrix Remodeling ◽  
Clinical Tool ◽  
Directional Migration ◽  
Endometrial Cells ◽  
Small Molecule Drugs ◽  
Actomyosin Contraction

AbstractEndometriosis is a painful gynecological condition characterized by ectopic growth of endometrial cells. Little is known about its pathogenesis, which is partially due to a lack of suitable experimental models. Here, we use endometrial stromal (St-T1b), primary endometriotic stromal, epithelial endometriotic (12Z) and co-culture (1:1 St-T1b:12Z) spheroids to mimic the architecture of endometrium, and either collagen I or Matrigel to model ectopic locations. Stromal spheroids, but not single cells, assumed coordinated directional migration followed by matrix remodeling of collagen I on day 5 or 7, resembling ectopic lesions. While generally a higher area fold increase of spheroids occurred on collagen I compared to Matrigel, directional migration was not observed in co-culture or in 12Z cells. The fold increase in area on collagen I was significantly reduced by MMP inhibition in stromal but not 12Z cells. Inhibiting ROCK signalling responsible for actomyosin contraction increased the fold increase of area and metabolic activity compared to untreated controls on Matrigel. The number of protrusions emanating from 12Z spheroids on Matrigel was decreased by microRNA miR-200b and increased by miR-145. This study demonstrates that spheroid assay is a promising pre-clinical tool that can be used to evaluate small molecule drugs and microRNA-based therapeutics for endometriosis.

scholarly journals dbVar structural variant cluster set for data analysis and variant comparison

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 673 ◽  
Author(s):  
Lon Phan ◽  
Jeffrey Hsu ◽  
Le Quang Minh Tri ◽  
Michaela Willi ◽  
Tamer Mansour ◽  
...  
Keyword(s):  
Genomic Sequence ◽  
Chromosomal Rearrangements ◽  
Copy Number Variations ◽  
Structural Variant ◽  
Variant Type ◽  
External Data ◽  
Cluster Set ◽  
Genomic Location ◽  
Complex Chromosomal Rearrangements ◽  
Genomic Regions

dbVar houses over 3 million submitted structural variants (SSV) from 120 human studies including copy number variations (CNV), insertions, deletions, inversions, translocations, and complex chromosomal rearrangements. Users can submit multiple SSVs to dbVAR  that are presumably identical, but were ascertained by different platforms and samples,  to calculate whether the variant is rare or common in the population and allow for cross validation. However, because SSV genomic location reporting can vary – including fuzzy locations where the start and/or end points are not precisely known – analysis, comparison, annotation, and reporting of SSVs across studies can be difficult. This project was initiated by the Structural Variant Comparison Group for the purpose of generating a non-redundant set of genomic regions defined by counts of concordance for all human SSVs placed on RefSeq assembly GRCh38 (RefSeq accession GCF_000001405.26). We intend that the availability of these regions, called structural variant clusters (SVCs), will facilitate the analysis, annotation, and exchange of SV data and allow for simplified display in genomic sequence viewers for improved variant interpretation. Sets of SVCs were generated by variant type for each of the 120 studies as well as for a combined set across all studies. Starting from 3.64 million SSVs, 2.5 million and 3.4 million non-redundant SVCs with count >=1 were generated by variant type for each study and across all studies, respectively. In addition, we have developed utilities for annotating, searching, and filtering SVC data in GVF format for computing summary statistics, exporting data for genomic viewers, and annotating the SVC using external data sources.

scholarly journals Analysis of Influenza B Virus NS1 Protein Trafficking Reveals a Novel Interaction with Nuclear Speckle Domains

2008 ◽  
Vol 83 (2) ◽  
pp. 701-711 ◽  
Author(s):  
Jana Schneider ◽  
Bianca Dauber ◽  
Krister Melén ◽  
Ilkka Julkunen ◽  
Thorsten Wolff
Keyword(s):  
Gene Expression ◽  
Protein Domain ◽  
Replication Capacity ◽  
Influenza B Virus ◽  
Viral Gene ◽  
Influenza B ◽  
Ns1 Protein ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
B Virus

ABSTRACT Many proteins that function in the transcription, maturation, and export of metazoan mRNAs are concentrated in nuclear speckle domains, indicating that the compartment is important for gene expression. Here, we show that the NS1 protein of influenza B virus (B/NS1) accumulates in nuclear speckles and causes rounding and morphological changes of the domains, indicating a disturbance in their normal functions. This property was located within the N-terminal 90 amino acids of the B/NS1 protein and was shown to be independent of any other viral gene product. Within this protein domain, we identified a monopartite importin α binding nuclear localization signal. Reverse-genetic analysis of this motif indicated that nuclear import and speckle association of the B/NS1 protein are required for the full replication capacity of the virus. In the late phase of virus infection, the B/NS1 protein relocated to the cytoplasm, which occurred in a CRM1-independent manner. The interaction of the B/NS1 protein with nuclear speckles may reflect a recruitment function to promote viral-gene expression. To our knowledge, this is the first functional description of a speckle-associated protein that is encoded by a negative-strand RNA virus.

scholarly journals Studying 3D genome evolution using genomic sequence

2019 ◽  
Author(s):  
Raphaël Mourad
Keyword(s):  
Genome Evolution ◽  
Genomic Sequence ◽  
Regulation Of Gene Expression ◽  
Replication Timing ◽  
Evolutionary Constraints ◽  
3D Genome ◽  
Chromatin Looping ◽  
Novel Approach ◽  
Evolutionary Studies ◽  
Ancestral Character Reconstruction

AbstractThe 3D genome is essential to numerous key processes such as the regulation of gene expression and the replication-timing program. In vertebrates, chromatin looping is often mediated by CTCF, and marked by CTCF motif pairs in convergent orientation. Comparative Hi-C recently revealed that chromatin looping evolves across species. However, Hi-C experiments are complex and costly, which currently limits their use for evolutionary studies over a large number of species. Here, we propose a novel approach to study the 3D genome evolution in vertebrates using the genomic sequence only, e.g. without the need for Hi-C data. The approach is simple and relies on comparing the distances between convergent and divergent CTCF motifs (ratio R). We show that R is a powerful statistic to detect CTCF looping encoded in the human genome sequence, thus reflecting strong evolutionary constraints encoded in DNA and associated with the 3D genome. When comparing vertebrate genomes, our results reveal that R which underlies CTCF looping and TAD organization evolves over time and suggest that ancestral character reconstruction can be used to infer R in ancestral genomes.

scholarly journals Intron and Its Splicing Mechanism and Their Connection with Human Disease

2016 ◽  
Vol 15 (3) ◽  
pp. 307-312
Author(s):  
Mine Dosay-Akbulut
Keyword(s):  
Human Genome ◽  
Human Disease ◽  
Rna Splicing ◽  
Messenger Rna ◽  
Developmental Disorders ◽  
Regulation Of Gene Expression ◽  
Medical Science ◽  
Intronless Genes ◽  
Different Types ◽  
Extra Difficulty

In the maturation mechanism of a messenger RNA, splicing play an important role with removing the noncoding introns and ligating the coding exons. Alternative splicing (AS) gives an extra difficulty to this mechanism and to the regulation of gene expression. The possible disturbing in the alternative RNA splicing mechanism can be a reason to several diseases like cancers and neurodegenerative disorders. Intronless genes (IGs) are seen in almost 3% of the human genome. Functionality of IGs has an important role in signal transduction genes and related regulatory proteins. This diversity can be reason to IG-associated diseases, especially neuropathies, developmental disorders, and cancer. The retroelements can be seen in almost half of the human genome. The known informations indicate that insertion of retroelement into exons and introns of genes promote different types of genetic disease, including cancer. The retroelement connected mutagenesis cause to fifty different types of human disease. The molecular informations and bioinformatic analyses can be used to explain the connection with splicing mutations and genetic mechanisms of several different human disease and understanding of this mechanism play an important role in the formation of treatment programme against to these diseases.Bangladesh Journal of Medical Science Vol.15(3) 2016 p.307-312

scholarly journals Studying 3D genome evolution using genomic sequence

2019 ◽  
Author(s):  
Raphaël Mourad
Keyword(s):  
Genome Evolution ◽  
High Throughput Sequencing ◽  
Genomic Sequence ◽  
Regulation Of Gene Expression ◽  
Replication Timing ◽  
Three Dimensions ◽  
Supplementary Information ◽  
3D Genome ◽  
Chromatin Looping ◽  
Novel Approach

Abstract Motivation The three dimensions (3D) genome is essential to numerous key processes such as the regulation of gene expression and the replication-timing program. In vertebrates, chromatin looping is often mediated by CTCF, and marked by CTCF motif pairs in convergent orientation. Comparative high-throughput sequencing technique (Hi-C) recently revealed that chromatin looping evolves across species. However, Hi-C experiments are complex and costly, which currently limits their use for evolutionary studies over a large number of species. Results Here, we propose a novel approach to study the 3D genome evolution in vertebrates using the genomic sequence only, e.g. without the need for Hi-C data. The approach is simple and relies on comparing the distances between convergent and divergent CTCF motifs by computing a ratio we named the 3D ratio or ‘3DR’. We show that 3DR is a powerful statistic to detect CTCF looping encoded in the human genome sequence, thus reflecting strong evolutionary constraints encoded in DNA and associated with the 3D genome. When comparing vertebrate genomes, our results reveal that 3DR which underlies CTCF looping and topologically associating domain organization evolves over time and suggest that ancestral character reconstruction can be used to infer 3DR in ancestral genomes. Availability and implementation The R code is available at https://github.com/morphos30/PhyloCTCFLooping. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Patterns of Genomic Sequence Diversity among Their Simian Immunodeficiency Viruses Suggest that L'Hoest Monkeys (Cercopithecus lhoesti) Are a Natural Lentivirus Reservoir

2000 ◽  
Vol 74 (8) ◽  
pp. 3892-3898 ◽  
Author(s):  
Brigitte E. Beer ◽  
Elizabeth Bailes ◽  
George Dapolito ◽  
Barbara J. Campbell ◽  
Robert M. Goeken ◽  
...  
Keyword(s):  
Phylogenetic Trees ◽  
Democratic Republic ◽  
Genomic Sequence ◽  
Democratic Republic Of Congo ◽  
Vervet Monkeys ◽  
Genome Sequences ◽  
Distinct Cluster ◽  
Immunodeficiency Virus ◽  
Genomic Regions ◽  
Full Length Genome

ABSTRACT Recently, we described a novel simian immunodeficiency virus (SIVlhoest) from a wild-caught L'Hoest monkey (Cercopithecus lhoesti) from a North American zoo. To investigate whether L'Hoest monkeys are the natural host for these viruses, we have screened blood samples from 14 wild animals from the Democratic Republic of Congo. Eight (57%) were found to be seropositive for SIV. Nearly full-length genome sequences were obtained for SIV isolates from three of these monkeys and compared to the original isolate and to other SIVs. The four samples of SIVlhoest formed a distinct cluster in phylogenetic trees. Two of these isolates differed on average at only about 5% of nucleotides, suggesting that they were epidemiologically linked; otherwise, the SIVlhoest isolates differed on average by 18%. Both the level of diversity and the pattern of its variation along the genome were very similar to those seen among isolates of SIVagm from vervet monkeys, pointing to similarities in the nature of, and constraints on, SIV evolution in these two species. Discordant phylogenetic relationships among the SIVlhoest isolates for different genomic regions indicated that mosaic viruses have been generated by recombination, implying that individual monkeys have been coinfected by more than one strain of SIV. Taken together, these observations provide strong evidence that L'Hoest monkeys constitute a natural reservoir for SIV.

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