Characterization of the nuclear and cytosolic transcriptomes in human brain tissue reveals new insights into the subcellular distribution of RNA transcripts

Zaghlool, Ammar; Niazi, Adnan; Björklund, Åsa K.; Westholm, Jakub Orzechowski; Ameur, Adam; Feuk, Lars

doi:10.1101/2020.04.08.031419

Cited by 6 publications

(9 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For cell composition (Figure 2B), we recovered the same main cell groups across compartments, as also reflected by the accuracy of a multiclass random forest classifier trained on scRNA-seq data in predicting cell types on snRNA-Seq data (Methods, Figure 2C-E) and vice versa (Figure S8F-H), and in overall similarity of cell type intrinsic (pseudobulk) profiles of protein coding genes (average Spearman ρ across cell types = 0.58 (skin), 0.69 (prostate), 0.53 (lung), Table S4). Notable divergences include the greater expression in cells vs. nuclei profiles of a stress/dissociation signature (Denisenko et al, 2020;van den Brink et al, 2017) (Wilcoxon rank-sum test, Benjamini-Hochberg FDR < 10 -16 , Figure 2F, Figure S10A,B), as we previously reported (Slyper et al, 2020), and of ribosomal and nuclear-encoded mitochondrial protein genes (Figure 2G, Methods, linear model), consistent with their longer half-lives and higher cytoplasmic levels (Rabani et al, 2014;Zaghlool et al, 2021).…”

Section: Snrna-seq Protocols Perform Well Across Tissues and Correspond To Scrna-seqsupporting

confidence: 77%

Single-nucleus cross-tissue molecular reference maps to decipher disease gene function

Eraslan

Drokhlyansky

Anand

et al. 2021

Preprint

View full text Add to dashboard Cite

Understanding the function of genes and their regulation in tissue homeostasis and disease requires knowing the cellular context in which genes are expressed in tissues across the body. Single cell genomics allows the generation of detailed cellular atlases in human tissues, but most efforts are focused on single tissue types. Here, we establish a framework for profiling multiple tissues across the human body at single-cell resolution using single nucleus RNA-Seq (snRNA-seq), and apply it to 8 diverse, archived, frozen tissue types (three donors per tissue). We apply four snRNA-seq methods to each of 25 samples from 16 donors, generating a cross-tissue atlas of 209,126 nuclei profiles, and benchmark them vs. scRNA-seq of comparable fresh tissues. We use a conditional variational autoencoder (cVAE) to integrate an atlas across tissues, donors, and laboratory methods. We highlight shared and tissue-specific features of tissue-resident immune cells, identifying tissue-restricted and non-restricted resident myeloid populations. These include a cross-tissue conserved dichotomy between LYVE1- and HLA class II-expressing macrophages, and the broad presence of LAM-like macrophages across healthy tissues that is also observed in disease. For rare, monogenic muscle diseases, we identify cell types that likely underlie the neuromuscular, metabolic, and immune components of these diseases, and biological processes involved in their pathology. For common complex diseases and traits analyzed by GWAS, we identify the cell types and gene modules that potentially underlie disease mechanisms. The experimental and analytical frameworks we describe will enable the generation of large-scale studies of how cellular and molecular processes vary across individuals and populations.

show abstract

Section: Snrna-seq Protocols Perform Well Across Tissues and Correspond To Scrna-seqsupporting

confidence: 77%

Single-nucleus cross-tissue molecular reference maps to decipher disease gene function

Eraslan

Drokhlyansky

Anand

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…To first assay the efficiency with which Halo-seq can enrich localized RNAs in C2bbe1 monolayers, we targeted the cytoplasmic compartment for analysis with Halo-seq. Because differences between the cytoplasmic and nuclear transcriptomes are well characterized (Engel et al, 2021; Zaghlool et al, 2021), we reasoned that this would provide a good benchmark of the technique in C2bbe1 monolayers. We fused a HaloTag domain to a cytoplasmically restricted NF-kappa B subunit, P65.…”

Section: Resultsmentioning

confidence: 99%

RNA localization mechanisms transcend cell morphology

Goering

Arora

Taliaferro

2022

Preprint

View full text Add to dashboard Cite

RNA molecules are localized to specific subcellular regions through interactions between RNA regulatory elements and RNA binding proteins (RBPs). Generally, our knowledge of the mechanistic details behind the localization of a given RNA is restricted to a particular cell type. Here, we show that RNA/RBP interactions that regulate RNA localization in one cell type predictably regulate localization in other cell types with vastly different morphologies. To determine transcriptome-wide RNA spatial distributions across the apicobasal axis of human intestinal epithelial cells, we used our recently developed RNA proximity labeling technique, Halo-seq. We found that mRNAs encoding ribosomal proteins (RP mRNAs) were strongly localized to the basal pole of these cells. Using reporter transcripts and single molecule RNA FISH, we found that pyrimidine-rich TOP motifs in the 5′ UTRs of RP mRNAs were sufficient to drive basal RNA localization. Interestingly, the same TOP motifs were also sufficient to drive RNA localization to the neurites of mouse neuronal cells. In both cell types, the regulatory activity of the TOP motif was dependent on it being at the extreme 5′ end of the transcript, was abolished upon perturbation of the TOP-binding protein LARP1, and was reduced upon inhibition of kinesins. To extend these findings, we compared subcellular RNAseq data from neuronal and epithelial cells. We found that the basal compartment of epithelial cells and the projections of neuronal cells were enriched for highly similar sets of RNAs, indicating that broadly similar mechanisms may be transporting RNAs in both cell types. These findings identify the first RNA element known to regulate RNA localization across the apicobasal axis of epithelial cells, establish LARP1 as an RNA localization regulator, and demonstrate that RNA localization mechanisms cut across cell morphologies.

show abstract

“…6b,d). These results show high overlap with studies in other tissues that identi ed differential enrichment of mRNA molecules in the nuclear and cytoplasmic compartments [35][36][37]. For instance, it was demonstrated in Zaghlool, Niazi [37] that many nuclearencoded-mitochondrial protein (NEMPs) mRNAs are signi cantly enriched in the cytosolic compartment across various human tissue types.…”

Section: Resultsmentioning

confidence: 58%

“…These results show high overlap with studies in other tissues that identi ed differential enrichment of mRNA molecules in the nuclear and cytoplasmic compartments [35][36][37]. For instance, it was demonstrated in Zaghlool, Niazi [37] that many nuclearencoded-mitochondrial protein (NEMPs) mRNAs are signi cantly enriched in the cytosolic compartment across various human tissue types. To determine if NEMPs mRNAs were also preferentially localized in the ATAC-SN fraction of human T cells, we retrieved a full list of genes (n = 1158) encoding proteins associated with mitochondrial localization from Mitocharta2.0 [38] and conducted gene enrichment analysis for differentially expressed genes either up-or down-regulated in the ATAC-SN fraction and human NEMPs.…”

Section: Resultsmentioning

confidence: 58%

Parallel recovery of chromatin accessibility and gene expression dynamics from frozen human Regulatory T cells

Wong

Harbison

Hope

et al. 2022

Preprint

View full text Add to dashboard Cite

The epigenome and transcriptome constitute a critical element of a tightly regulated, cell-type specific gene expression program, and subtle perturbations in the regulation of this program can result in pathology. Epigenetic features such as DNA accessibility dictate transcriptional regulation in a cell type- and cell state- specific manner, and mapping this in health vs. disease in clinically relevant material is opening the door to new mechanistic insights and new targets for therapy. Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) allows profiling of chromatin accessibility with low cell input, making it amenable to the clinical setting, such as peripheral blood from clinical trials, and this makes it applicable to rare cell populations, such as regulatory T (Treg) cells. However, there is little known about the compatibility of the assay on materials recovered from cryopreserved rare cell populations. In the context of tolerance or autoimmunity, regulatory T cells play a critical role in maintaining immune homeostasis, and loss of numbers or function is linked to many diseases, making them a clinically relevant population to analyse using genomic platforms. Here we demonstrate the robustness and reproducibility of an ATAC-seq protocol comparing fresh or cryopreserved primary Treg cells, and comparing their profile in the steady state and in response to stimulation. We extend this method to explore the feasibility of conducting simultaneous quantitation of chromatin accessibility and transcriptome from a single aliquot of 50,000 Treg cells from cryopreserved PBMCs. Profiling of chromatin accessibility and gene expression in parallel within the same pool of cells controls for cellular heterogeneity and will be particularly beneficial for experiments constrained by limited input material, such as biobanked PBMC from clinical trials. This approach will be complementary to single-cell experiments as libraries used to profile chromatin accessibility and transcriptome are derived from the same population of cells, controlling for stochastic gene fluctuation in different cells in a population at any given time. Overall, we observed a high correlation of accessibility patterns and transcription factor (TF) dynamics between fresh Treg cells and cells recovered from cryopreservation samples. The distribution of fragment size, enrichment of transcription start sites (TSS) and genomic features of thawed Treg cells recapitulate that of the fresh cells. Furthermore, highly consistent global chromatin and transcriptional changes in response to stimulation were observed in both fresh and frozen samples. Lastly, highly similar transcriptomic profiles were obtained from whole cells and from the supernatants recovered from ATAC-seq reactions. This report highlights the feasibility of applying these techniques to profile the epigenomic landscape of cells recovered from cryopreservation biorepositories. Implementation of this approach is suitable in biorepositories and will contribute to advances in the field of translational research and personalized medicine.

show abstract

Characterization of the nuclear and cytosolic transcriptomes in human brain tissue reveals new insights into the subcellular distribution of RNA transcripts

Cited by 6 publications

References 55 publications

Single-nucleus cross-tissue molecular reference maps to decipher disease gene function

Single-nucleus cross-tissue molecular reference maps to decipher disease gene function

RNA localization mechanisms transcend cell morphology

Parallel recovery of chromatin accessibility and gene expression dynamics from frozen human Regulatory T cells

Contact Info

Product

Resources

About