Annotation of nuclear lncRNAs based on chromatin interactions.

The human genome is pervasively transcribed and produces a wide variety of long non-coding RNAs (lncRNAs), constituting the majority of transcripts across human cell types. Some specific nuclear lncRNAs have been shown to be important regulatory components acting locally. As RNA-chromatin interaction and Hi-C chromatin conformation data showed that chromatin interactions of nuclear lncRNAs are determined by the local chromatin 3D conformation, we used Hi-C data to identify potential target genes of lncRNAs. RNA-protein interaction data suggested that nuclear lncRNAs act as scaffolds to recruit regulatory proteins to target promoters and enhancers. Nuclear lncRNAs may therefore play a role in directing regulatory factors to locations spatially close to the lncRNA gene. We provide the analysis results through an interactive visualization web portal at https://fantom.gsc.riken.jp/zenbu/reports/#F6_3D_lncRNA.

Nuclear sequences of mitochondrial origin in domestic yak.

Mitochondrial DNA sequences are frequently transferred into the nuclear genome, generating nuclear mitochondrial DNA sequences (NUMTs). Here, we analysed, for the first time, NUMTs in the domestic yak genome. We obtained 499 alignment matches covering 340.2 kbp of the yak nuclear genome. After a merging step, we identified 167 NUMT regions with a total length of ~ 503 kbp, representing 0.02% of the nuclear genome. We discovered copies of all mitochondrial regions and found that most NUMT regions are intergenic or intronic and mostly untranscribed. 98 different NUMT regions from domestic yak showed high homology with cow and/or wild yak genomes, suggesting selection or hybridization between domestic/wild yak and cow. To rule out the possibility that the identified NUMTs could be artifacts of the domestic yak genome assembly, we validated experimentally five NUMT regions by PCR amplification. As NUMT regions show high similarity to the mitochondrial genome can potentially pose a risk to domestic yak DNA mitochondrial studies, special care is therefore needed to select primers for PCR amplification of mitochondrial DNA sequences.

Deep sequencing of Phox2a nuclei reveals five classes of anterolateral system neurons.

The anterolateral system (ALS) is a major ascending pathway from the spinal cord that projects to multiple brain areas and underlies the perception of pain, itch, and skin temperature. Despite its importance, our understanding of this system has been hampered by the considerable functional and molecular diversity of its constituent cells. Here, we use fluorescence-activated cell sorting to isolate ALS neurons belonging to the Phox2a-lineage for single-nucleus RNA sequencing. We reveal five distinct clusters of ALS neurons (ALS1-5) and document their laminar distribution in the spinal cord using in situ hybridization. We identify three clusters of neurons located predominantly in laminae I-III of the dorsal horn (ALS1-3) and two clusters with cell bodies located in deeper laminae (ALS4 and ALS5). Our findings reveal the transcriptional logic that underlies ALS neuronal diversity in the adult mouse and uncover the molecular identity of two previously identified classes of projection neurons. We also show that these molecular signatures can be used to target groups of ALS neurons using retrograde viral tracing. Overall, our findings provide a valuable resource for studying somatosensory biology and targeting subclasses of ALS neurons.

A nuclear genome assembly of an extinct flightless bird, the little bush moa.

We present a draft genome of the little bush moa (Anomalopteryx didiformis)-one of approximately nine species of extinct flightless birds from Aotearoa, New Zealand-using ancient DNA recovered from a fossil bone from the South Island. We recover a complete mitochondrial genome at 249.9× depth of coverage and almost 900 megabases of a male moa nuclear genome at ~4 to 5× coverage, with sequence contiguity sufficient to identify more than 85% of avian universal single-copy orthologs. We describe a diverse landscape of transposable elements and satellite repeats, estimate a long-term effective population size of ~240,000, identify a diverse suite of olfactory receptor genes and an opsin repertoire with sensitivity in the ultraviolet range, show that the wingless moa phenotype is likely not attributable to gene loss or pseudogenization, and identify potential function-altering coding sequence variants in moa that could be synthesized for future functional assays. This genomic resource should support further studies of avian evolution and morphological divergence.

Isolation of Nuclei from Human Intermuscular Adipose Tissue and Downstream Single-Nuclei RNA Sequencing.

Intermuscular adipose tissue (IMAT) is a relatively understudied adipose depot located between muscle fibers. IMAT content increases with age and BMI and is associated with metabolic and muscle degenerative diseases; however, an understanding of the biological properties of IMAT and its interplay with the surrounding muscle fibers is severely lacking. In recent years, single-cell and nuclei RNA sequencing have provided us with cell type-specific atlases of several human tissues. However, the cellular composition of human IMAT remains largely unexplored due to the inherent challenges of its accessibility from biopsy collection in humans. In addition to the limited amount of tissue collected, the processing of human IMAT is complicated due to its proximity to skeletal muscle tissue and fascia. The lipid-laden nature of the adipocytes makes it incompatible with single-cell isolation. Hence, single nuclei RNA sequencing is optimal for obtaining high-dimensional transcriptomics at single-cell resolution and provides the potential to uncover the biology of this depot, including the exact cellular composition of IMAT. Here, we present a detailed protocol for nuclei isolation and library preparation of frozen human IMAT for single nuclei RNA sequencing. This protocol allows for the profiling of thousands of nuclei using a droplet-based approach, thus providing the capacity to detect rare and low-abundant cell types.

Active transcription and epigenetic reactions synergistically regulate meso-scale genomic organization.

In interphase nuclei, chromatin forms dense domains of characteristic sizes, but the influence of transcription and histone modifications on domain size is not understood. We present a theoretical model exploring this relationship, considering chromatin-chromatin interactions, histone modifications, and chromatin extrusion. We predict that the size of heterochromatic domains is governed by a balance among the diffusive flux of methylated histones sustaining them and the acetylation reactions in the domains and the process of loop extrusion via supercoiling by RNAPII at their periphery, which contributes to size reduction. Super-resolution and nano-imaging of five distinct cell lines confirm the predictions indicating that the absence of transcription leads to larger heterochromatin domains. Furthermore, the model accurately reproduces the findings regarding how transcription-mediated supercoiling loss can mitigate the impacts of excessive cohesin loading. Our findings shed light on the role of transcription in genome organization, offering insights into chromatin dynamics and potential therapeutic targets.

Plastid DNA is a major source of nuclear genome complexity and of RNA genes in the orphan crop moringa.

BACKGROUND: Unlike Transposable Elements (TEs) and gene/genome duplication, the role of the so-called nuclear plastid DNA sequences (NUPTs) in shaping the evolution of genome architecture and function remains poorly studied. We investigate here the functional and evolutionary fate of NUPTs in the orphan crop Moringa oleifera (moringa), featured by the highest fraction of plastid DNA found so far in any plant genome, focusing on (i) any potential biases in their distribution in relation to specific nuclear genomic features, (ii) their contribution to the emergence of new genes and gene regions, and (iii) their impact on the expression of target nuclear genes. RESULTS: In agreement with their potential mutagenic effect, NUPTs are underrepresented among structural genes, although their overall transcription levels and broadness were only lower when involved exonic regions; the occurrence of plastid DNA generally did not result in a broader expression, except among those affected in introns by older NUPTs. In contrast, we found a strong enrichment of NUPTs among specific superfamilies of retrotransposons and several classes of RNA genes, including those participating in the protein biosynthetic machinery (i.e., rRNA and tRNA genes) and a specific class of regulatory RNAs. A significant fraction of NUPT RNA genes was found to be functionally expressed, thus potentially contributing to the nuclear pool. CONCLUSIONS: Our results complete our view of the molecular factors driving the evolution of nuclear genome architecture and function, and support plastid DNA in moringa as a major source of (i) genome complexity and (ii) the nuclear pool of RNA genes.

Agent-based modeling of nuclear chromosome ensembles identifies determinants of homolog pairing during meiosis.

During meiosis, pairing of homologous chromosomes (homologs) ensures the formation of haploid gametes from diploid precursor cells, a prerequisite for sexual reproduction. Pairing during meiotic prophase I facilitates crossover recombination and homolog segregation during the ensuing reductional cell division. Mechanisms that ensure stable homolog alignment in the presence of an excess of non-homologous chromosomes have remained elusive, but rapid chromosome movements appear to play a role in the process. Apart from homolog attraction, provided by early intermediates of homologous recombination, dissociation of non-homologous associations also appears to contribute to homolog pairing, as suggested by the detection of stable non-homologous chromosome associations in pairing-defective mutants. Here, we have developed an agent-based model for homolog pairing derived from the dynamics of a naturally occurring chromosome ensemble. The model simulates unidirectional chromosome movements, as well as collision dynamics determined by attractive and repulsive forces arising from close-range physical interactions. Chromosome number and size as well as movement velocity and repulsive forces are identified as key factors in the kinetics and efficiency of homologous pairing in addition to homolog attraction. Dissociation of interactions between non-homologous chromosomes may contribute to pairing by crowding homologs into a limited nuclear area thus creating preconditions for close-range homolog attraction. Incorporating natural chromosome lengths, the model accurately recapitulates efficiency and kinetics of homolog pairing observed for wild-type and mutant meiosis in budding yeast, and can be adapted to nuclear dimensions and chromosome sets of other organisms.

Testing plastomes and nuclear ribosomal DNA sequences as the next-generation DNA barcodes for species identification and phylogenetic analysis in Acer.

BACKGROUND: Acer is a taxonomically intractable and speciose genus that contains over 150 species. It is challenging to distinguish Acer species only by morphological method due to their abundant variations. Plastome and nuclear ribosomal DNA (nrDNA) sequences are recommended as powerful next-generation DNA barcodes for species discrimination. However, their efficacies were still poorly studied. The current study will evaluate the application of plastome and nrDNA in species identification and perform phylogenetic analyses for Acer. RESULT: Based on a collection of 83 individuals representing 55 species (c. 55% of Chinese species) from 13 sections, our barcoding analyses demonstrated that plastomes exhibited the highest (90.47%) species discriminatory power among all plastid DNA markers, such as the standard plastid barcodes matK + rbcL + trnH-psbA (61.90%) and ycf1 (76.19%). And the nrDNA (80.95%) revealed higher species resolution than ITS (71.43%). Acer plastomes show abundant interspecific variations, however, species identification failure may be due to the incomplete lineage sorting (ILS) and chloroplast capture resulting from hybridization. We found that the usage of nrDNA contributed to identifying those species that were unidentified by plastomes, implying its capability to some extent to mitigate the impact of hybridization and ILS on species discrimination. However, combining plastome and nrDNA is not recommended given the cytonuclear conflict caused by potential hybridization. Our phylogenetic analysis covering 19 sections (95% sections of Acer) and 128 species (over 80% species of this genus) revealed pervasive inter- and intra-section cytonuclear discordances, hinting that hybridization has played an important role in the evolution of Acer. CONCLUSION: Plastomes and nrDNA can significantly improve the species resolution in Acer. Our phylogenetic analysis uncovered the scope and depth of cytonuclear conflict in Acer, providing important insights into its evolution.

Central dogma rates in human mitochondria.

In human cells, the nuclear and mitochondrial genomes engage in a complex interplay to produce dual-encoded oxidative phosphorylation (OXPHOS) complexes. The coordination of these dynamic gene expression processes is essential for producing matched amounts of OXPHOS protein subunits. This review focuses on our current understanding of the mitochondrial central dogma rates, highlighting the striking differences in gene expression rates between mitochondrial and nuclear genes. We synthesize a coherent model of mitochondrial gene expression kinetics, highlighting the emerging principles and emphasizing where more precise measurements would be beneficial. Such an understanding is pivotal for grasping the unique aspects of mitochondrial function and its role in cellular energetics, and it has profound implications for aging, metabolic disorders, and neurodegenerative diseases.

LncRNAs, nuclear architecture and the immune response.

Long noncoding RNAs (LncRNAs) are key regulators of gene expression and can mediate their effects in both the nucleus and cytoplasm. Some of the best-characterized lncRNAs are localized within the nucleus, where they modulate the nuclear architecture and influence gene expression. In this review, we discuss the role of lncRNAs in nuclear architecture in the context of their gene regulatory functions in innate immunity. Here, we discuss various approaches to functionally characterize nuclear-localized lncRNAs and the challenges faced in the field.

EmptyDropsMultiome discriminates real cells from background in single-cell multiomics assays.

Multiomic droplet-based technologies allow different molecular modalities, such as chromatin accessibility and gene expression (scATAC-seq and scRNA-seq), to be probed in the same nucleus. We develop EmptyDropsMultiome, an approach that distinguishes true nuclei-containing droplets from background. Using simulations, we show that EmptyDropsMultiome has higher statistical power and accuracy than existing approaches, including CellRanger-arc and EmptyDrops. On real datasets, we observe that CellRanger-arc misses more than half of the nuclei identified by EmptyDropsMultiome and, moreover, is biased against certain cell types, some of which have a retrieval rate lower than 20%.

Plastid phylogenomics and cytonuclear discordance in Rubioideae, Rubiaceae.

In this study of evolutionary relationships in the subfamily Rubioideae (Rubiaceae), we take advantage of the off-target proportion of reads generated via previous target capture sequencing projects based on nuclear genomic data to build a plastome phylogeny and investigate cytonuclear discordance. The assembly of off-target reads resulted in a comprehensive plastome dataset and robust inference of phylogenetic relationships, where most intratribal and intertribal relationships are resolved with strong support. While the phylogenetic results were mostly in agreement with previous studies based on plastome data, novel relationships in the plastid perspective were also detected. For example, our analyses of plastome data provide strong support for the SCOUT clade and its sister relationship to the remaining members of the subfamily, which differs from previous results based on plastid data but agrees with recent results based on nuclear genomic data. However, several instances of highly supported cytonuclear discordance were identified across the Rubioideae phylogeny. Coalescent simulation analysis indicates that while ILS could, by itself, explain the majority of the discordant relationships, plastome introgression may be the better explanation in some cases. Our study further indicates that plastomes across the Rubioideae are, with few exceptions, highly conserved and mainly conform to the structure, gene content, and gene order present in the majority of the flowering plants.

Single-cell mtDNA dynamics in tumors is driven by coregulation of nuclear and mitochondrial genomes.

The extent of cell-to-cell variation in tumor mitochondrial DNA (mtDNA) copy number and genotype, and the phenotypic and evolutionary consequences of such variation, are poorly characterized. Here we use amplification-free single-cell whole-genome sequencing (Direct Library Prep (DLP+)) to simultaneously assay mtDNA copy number and nuclear DNA (nuDNA) in 72,275 single cells derived from immortalized cell lines, patient-derived xenografts and primary human tumors. Cells typically contained thousands of mtDNA copies, but variation in mtDNA copy number was extensive and strongly associated with cell size. Pervasive whole-genome doubling events in nuDNA associated with stoichiometrically balanced adaptations in mtDNA copy number, implying that mtDNA-to-nuDNA ratio, rather than mtDNA copy number itself, mediated downstream phenotypes. Finally, multimodal analysis of DLP+ and single-cell RNA sequencing identified both somatic loss-of-function and germline noncoding variants in mtDNA linked to heteroplasmy-dependent changes in mtDNA copy number and mitochondrial transcription, revealing phenotypic adaptations to disrupted nuclear/mitochondrial balance.

scCDC: a computational method for gene-specific contamination detection and correction in single-cell and single-nucleus RNA-seq data.

In droplet-based single-cell and single-nucleus RNA-seq assays, systematic contamination of ambient RNA molecules biases the quantification of gene expression levels. Existing methods correct the contamination for all genes globally. However, there lacks specific evaluation of correction efficacy for varying contamination levels. Here, we show that DecontX and CellBender under-correct highly contaminating genes, while SoupX and scAR over-correct lowly/non-contaminating genes. Here, we develop scCDC as the first method to detect the contamination-causing genes and only correct expression levels of these genes, some of which are cell-type markers. Compared with existing decontamination methods, scCDC excels in decontaminating highly contaminating genes while avoiding over-correction of other genes.

Single nucleus and spatial transcriptomic profiling of healthy human hamstring tendon.

The molecular and cellular basis of health in human tendons remains poorly understood. Among human tendons, hamstring tendon has markedly low pathology and can provide a prototypic healthy tendon reference. The aim of this study was to determine the transcriptomes and location of all cell types in healthy hamstring tendon. Using single nucleus RNA sequencing, we profiled the transcriptomes of 10 533 nuclei from four healthy donors and identified 12 distinct cell types. We confirmed the presence of two fibroblast cell types, endothelial cells, mural cells, and immune cells, and identified cell types previously unreported in tendons, including different skeletal muscle cell types, satellite cells, adipocytes, and undefined nervous system cells. The location of these cell types within tendon was defined using spatial transcriptomics and imaging, and potential transcriptional networks and cell-cell interactions were analyzed. We demonstrate that fibroblasts have the highest number of potential cell-cell interactions in our dataset, are present throughout the tendon, and play an important role in the production and organization of extracellular matrix, thus confirming their role as key regulators of hamstring tendon homeostasis. Overall, our findings underscore the complexity of the cellular networks that underpin healthy human tendon function and the central role of fibroblasts as key regulators of hamstring tendon tissue homeostasis.

Analyses of Nuclear Reads Obtained Using Genome Skimming.

In this protocol paper, we review a set of methods developed in recent years for analyzing nuclear reads obtained from genome skimming. As the cost of sequencing drops, genome skimming (low-coverage shotgun sequencing of a sample) becomes increasingly a cost-effective method of measuring biodiversity at high resolution. While most practitioners only use assembled over-represented organelle reads from a genome skim, the vast majority of the reads are nuclear. Using assembly-free and alignment-free methods described in this protocol, we can compare samples to each other and reference genomes to compute distances, characterize underlying genomes, and infer evolutionary relationships.

NKX2-2 based nuclei sorting on frozen human archival pancreas enables the enrichment of islet endocrine populations for single-nucleus RNA sequencing.

BACKGROUND: Current approaches to profile the single-cell transcriptomics of human pancreatic endocrine cells almost exclusively rely on freshly isolated islets. However, human islets are limited in availability. Furthermore, the extensive processing steps during islet isolation and subsequent single cell dissolution might alter gene expressions. In this work, we report the development of a single-nucleus RNA sequencing (snRNA-seq) approach with targeted islet cell enrichment for endocrine-population focused transcriptomic profiling using frozen archival pancreatic tissues without islet isolation. RESULTS: We cross-compared five nuclei isolation protocols and selected the citric acid method as the best strategy to isolate nuclei with high RNA integrity and low cytoplasmic contamination from frozen archival human pancreata. We innovated fluorescence-activated nuclei sorting based on the positive signal of NKX2-2 antibody to enrich nuclei of the endocrine population from the entire nuclei pool of the pancreas. Our sample preparation procedure generated high-quality single-nucleus gene-expression libraries while preserving the endocrine population diversity. In comparison with single-cell RNA sequencing (scRNA-seq) library generated with live cells from freshly isolated human islets, the snRNA-seq library displayed comparable endocrine cellular composition and cell type signature gene expression. However, between these two types of libraries, differential enrichments of transcripts belonging to different functional classes could be observed. CONCLUSIONS: Our work fills a technological gap and helps to unleash frozen archival pancreatic tissues for molecular profiling targeting the endocrine population. This study opens doors to retrospective mappings of endocrine cell dynamics in pancreatic tissues of complex histopathology. We expect that our protocol is applicable to enrich nuclei for transcriptomics studies from various populations in different types of frozen archival tissues.

A new nuclear phylogeny of the tea family (Theaceae) unravels rapid radiations in genus Camellia.

Molecular analyses of rapidly radiating groups often reveal incongruence between gene trees. This mainly results from incomplete lineage sorting, introgression, and gene tree estimation error, which complicate the estimation of phylogenetic relationships. In this study, we reconstruct the phylogeny of Theaceae using 348 nuclear loci from 68 individuals and two outgroup taxa. Sequence data were obtained by target enrichment using the recently released Angiosperm 353 universal probe set applied to herbarium specimens. The robustness of the topologies to variation in data quality was established under a range of different filtering schemes, using both coalescent and concatenation approaches. Our results confirmed most of the previously hypothesized relationships among tribes and genera, while clarifying additional interspecific relationships within the rapidly radiating genus Camellia. We recovered a remarkably high degree of gene tree heterogeneity indicative of rapid radiation in the group and observed cytonuclear conflicts, especially within Camellia. This was especially pronounced around short branches, which we primarily associate with gene tree estimation error. Our analysis also indicates that incomplete lineage sorting (ILS) contributed to gene-tree conflicts and accounted for approximately 14 % of the explained variation, whereas inferred introgression levels were low. Our study advances the understanding of the evolution of this important plant family and provides guidance on the application of target capture methods and the evaluation of key processes that influence phylogenetic discordances.