scholarly journals NanoSINC-seq dissects the isoform diversity in subcellular compartments of single cells

2021 ◽  
Vol 7 (15) ◽  
pp. eabe0317
Author(s):  
Yusuke Oguchi ◽  
Yuka Ozaki ◽  
Mahmoud N. Abdelmoez ◽  
Hirofumi Shintaku
Keyword(s):  
Single Cells ◽  
Full Length ◽  
Protein Isoforms ◽  
Mrna Isoforms ◽  
Transcriptional Noise ◽  
Subcellular Compartments ◽  
Cytoplasmic Rna ◽  
Novel Approach ◽  
Isoform Diversity ◽  
Nanopore Sequencer

Alternative mRNA isoforms play a key role in generating diverse protein isoforms. To dissect isoform usage in the subcellular compartments of single cells, we introduced an novel approach, nanopore sequencing coupled with single-cell integrated nuclear and cytoplasmic RNA sequencing, that couples microfluidic fractionation, which separates cytoplasmic RNA from nuclear RNA, with full-length complementary DNA (cDNA) sequencing using a nanopore sequencer. Leveraging full-length cDNA reads, we found that the nuclear transcripts are notably more diverse than cytoplasmic transcripts. Our findings also indicated that transcriptional noise emanating from the nucleus is regulated across the nuclear membrane and then either attenuated or amplified in the cytoplasm depending on the function involved. Overall, our results provide the landscape that shows how the transcriptional noise arising from the nucleus propagates to the cytoplasm.

scholarly journals Single molecule real time (SMRT) full length RNA-sequencing reveals novel and distinct mRNA isoforms in human bone marrow cell subpopulations

2019 ◽  
Author(s):  
Anne Deslattes Mays ◽  
Marcel O. Schmidt ◽  
Garrett T. Graham ◽  
Elizabeth Tseng ◽  
Primo Baybayan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Real Time ◽  
Rna Sequencing ◽  
Single Molecule ◽  
Marrow Cell ◽  
Full Length ◽  
Protein Isoforms ◽  
Mrna Isoforms ◽  
Transcript Isoforms ◽  
Cell Subpopulations

AbstractHematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single molecule, real time (SMRT) full length RNA sequencing. This analysis revealed a ∼5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of mRNA isoforms transcribed from the ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was validated by mass spectrometry and validated previously unknown protein isoforms predicted e.g. for EEF1A1. These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g. CFD, GATA2, HLA-A, B & C) also distinguished cell subpopulations but were only detectable by full length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis.

scholarly journals Full-length transcriptome reconstruction reveals a large diversity of RNA and protein isoforms in rat hippocampus

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Xi Wang ◽  
Xintian You ◽  
Julian D. Langer ◽  
Jingyi Hou ◽  
Fiona Rupprecht ◽  
...  
Keyword(s):  
Gene Annotation ◽  
Full Length ◽  
Protein Isoforms ◽  
Rat Hippocampus ◽  
Reading Frame ◽  
Functional Studies ◽  
Third Generation Sequencing ◽  
Isoform Diversity ◽  
Polysome Profiling ◽  
Ribosome Footprinting

Abstract Gene annotation is a critical resource in genomics research. Many computational approaches have been developed to assemble transcriptomes based on high-throughput short-read sequencing, however, only with limited accuracy. Here, we combine next-generation and third-generation sequencing to reconstruct a full-length transcriptome in the rat hippocampus, which is further validated using independent 5´ and 3´-end profiling approaches. In total, we detect 28,268 full-length transcripts (FLTs), covering 6,380 RefSeq genes and 849 unannotated loci. Based on these FLTs, we discover co-occurring alternative RNA processing events. Integrating with polysome profiling and ribosome footprinting data, we predict isoform-specific translational status and reconstruct an open reading frame (ORF)-eome. Notably, a high proportion of the predicted ORFs are validated by mass spectrometry-based proteomics. Moreover, we identify isoforms with subcellular localization pattern in neurons. Collectively, our data advance our knowledge of RNA and protein isoform diversity in the rat brain and provide a rich resource for functional studies.

scholarly journals scCAT-seq:single-cell identification and quantification of mRNA isoforms by cost-effective short-read sequencing of cap and tail

2019 ◽  
Author(s):  
Youjin Hu ◽  
Jiawei Zhong ◽  
Yuhua Xiao ◽  
Zheng Xing ◽  
Katherine Sheu ◽  
...  
Keyword(s):  
Single Cell ◽  
Learning Algorithm ◽  
Single Cells ◽  
Full Length ◽  
Translation Efficiency ◽  
Mrna Isoforms ◽  
Short Read ◽  
Short Read Sequencing ◽  
Long Read ◽  
Identification And Quantification

AbstractThe differences in transcription start sites (TSS) and transcription end sites (TES) among gene isoforms can affect the stability, localization, and translation efficiency of mRNA. Isoforms also allow a single gene different functions across various tissues and cells However, methods for efficient genome-wide identification and quantification of RNA isoforms in single cells are still lacking. Here, we introduce single cell Cap And Tail sequencing (scCAT-seq). In conjunction with a novel machine learning algorithm developed for TSS/TES characterization, scCAT-seq can demarcate transcript boundaries of RNA transcripts, providing an unprecedented way to identify and quantify single-cell full-length RNA isoforms based on short-read sequencing. Compared with existing long-read sequencing methods, scCAT-seq has higher efficiency with lower cost. Using scCAT-seq, we identified hundreds of previously uncharacterized full-length transcripts and thousands of alternative transcripts for known genes, quantitatively revealed cell-type specific isoforms with alternative TSSs/TESs in dorsal root ganglion (DRG) neurons, mature oocytes and ageing oocytes, and generated the first atlas of the non-human primate cornea. The approach described here can be widely adapted to other short-read or long-read methods to improve accuracy and efficiency in assessing RNA isoform dynamics among single cells.

scholarly journals Single-Molecule Real-Time (SMRT) Full-Length RNA-Sequencing Reveals Novel and Distinct mRNA Isoforms in Human Bone Marrow Cell Subpopulations

Genes ◽  
2019 ◽  
Vol 10 (4) ◽  
pp. 253 ◽  
Author(s):  
Anne Deslattes Mays ◽  
Marcel Schmidt ◽  
Garrett Graham ◽  
Elizabeth Tseng ◽  
Primo Baybayan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Real Time ◽  
Rna Sequencing ◽  
Cell Population ◽  
Single Molecule ◽  
Full Length ◽  
Protein Isoforms ◽  
Mrna Isoforms ◽  
Transcript Isoforms ◽  
Cell Subpopulations

Hematopoietic cells are continuously replenished from progenitor cells that reside in thebone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomesof freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineagepositive)cells by single-molecule real-time (SMRT) full-length RNA-sequencing. This analysisrevealed a ~5-fold higher number of transcript isoforms than previously detected and showed adistinct composition of individual transcript isoforms characteristic for bone marrowsubpopulations. A detailed analysis of messenger RNA (mRNA) isoforms transcribed from theANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predictedfrom the transcriptome analysis was evaluated by mass spectrometry and validated previouslyunknown protein isoforms predicted e.g., for EEF1A1. These protein isoforms distinguished thelineage negative cell population from the lineage positive cell population. Finally, transcriptisoforms expressed from paralogous gene loci (e.g., CFD, GATA2, HLA-A, B, and C) alsodistinguished cell subpopulations but were only detectable by full-length RNA sequencing. Thus,qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cellsubpopulations indicating complex transcriptional regulation and protein isoform generationduring hematopoiesis.

scholarly journals Two methods for full-length RNA sequencing for low quantities of cells and single cells

2012 ◽  
Vol 110 (2) ◽  
pp. 594-599 ◽  
Author(s):  
X. Pan ◽  
R. E. Durrett ◽  
H. Zhu ◽  
Y. Tanaka ◽  
Y. Li ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Single Cells ◽  
Full Length

Identification of Gαs messenger ribonucleic acid splice variants in human granulosa cells

1997 ◽  
Vol 18 (1) ◽  
pp. 27-35 ◽  
Author(s):  
G N Europe-Finner ◽  
E Cartwright ◽  
J Bellinger ◽  
H J Mardon ◽  
D H Barlow ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Ovarian Function ◽  
Splice Variants ◽  
Consensus Sequence ◽  
Phosphorylation Site ◽  
Follicular Development ◽  
Protein Isoforms ◽  
Mrna Isoforms ◽  
Messenger Ribonucleic Acid

ABSTRACT Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by Gαs-linked receptors. In this paper we have investigated the expression of Gαs mRNA splice variants in relation to expression of Gαs protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all Gαs mRNA isoforms as well as quantifying total amounts of Gαs mRNA. Granulosa cells express the message for Gαs-Large and Gαs-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of Gαs-Large and Gαs-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in Gαs variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between Gαs variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.

scholarly journals Regulation, diversity and function of MECP2 exon and 3′UTR isoforms

2020 ◽  
Vol 29 (R1) ◽  
pp. R89-R99
Author(s):  
Deivid Carvalho Rodrigues ◽  
Marat Mufteev ◽  
James Ellis
Keyword(s):  
Dna Binding ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Alternative Polyadenylation ◽  
Cell Types ◽  
Protein Isoforms ◽  
Translation Efficiency ◽  
Mrna Isoforms ◽  
Exon 2 ◽  
Messenger Ribonucleic Acid

Abstract The methyl-CpG-binding protein 2 (MECP2) is a critical global regulator of gene expression. Mutations in MECP2 cause neurodevelopmental disorders including Rett syndrome (RTT). MECP2 exon 2 is spliced into two alternative messenger ribonucleic acid (mRNA) isoforms encoding MECP2-E1 or MECP2-E2 protein isoforms that differ in their N-termini. MECP2-E2, isolated first, was used to define the general roles of MECP2 in methyl-deoxyribonucleic acid (DNA) binding, targeting of transcriptional regulatory complexes, and its disease-causing impact in RTT. It was later found that MECP2-E1 is the most abundant isoform in the brain and its exon 1 is also mutated in RTT. MECP2 transcripts undergo alternative polyadenylation generating mRNAs with four possible 3′untranslated region (UTR) lengths ranging from 130 to 8600 nt. Together, the exon and 3′UTR isoforms display remarkable abundance disparity across cell types and tissues during development. These findings indicate discrete means of regulation and suggest that protein isoforms perform non-overlapping roles. Multiple regulatory programs have been explored to explain these disparities. DNA methylation patterns of the MECP2 promoter and first intron impact MECP2-E1 and E2 isoform levels. Networks of microRNAs and RNA-binding proteins also post-transcriptionally regulate the stability and translation efficiency of MECP2 3′UTR isoforms. Finally, distinctions in biophysical properties in the N-termini between MECP2-E1 and E2 lead to variable protein stabilities and DNA binding dynamics. This review describes the steps taken from the discovery of MECP2, the description of its key functions, and its association with RTT, to the emergence of evidence revealing how MECP2 isoforms are differentially regulated at the transcriptional, post-transcriptional and post-translational levels.

Denervation-induced changes in myosin heavy chain expression in the rat diaphragm muscle

2003 ◽  
Vol 95 (2) ◽  
pp. 611-619 ◽  
Author(s):  
Paige C. Geiger ◽  
Jeffrey P. Bailey ◽  
Wen-Zhi Zhan ◽  
Carlos B. Mantilla ◽  
Gary C. Sieck
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Protein Isoforms ◽  
Diaphragm Muscle ◽  
Northern Analysis ◽  
Mrna Levels ◽  
Rat Diaphragm ◽  
Mrna Isoforms

Unilateral denervation (Dnv) of the rat diaphragm muscle (Diam) markedly alters expression of myosin heavy chain (MHC) isoforms. After 2 wk of Diam Dnv, MHC content per half-sarcomere decreases in fibers expressing MHC2X and MHC2B. We hypothesized that changes in MHC protein expression parallel changes in MHC mRNA expression. Relative MHC isoform mRNA levels were determined by Northern analysis after 1, 3, 7, and 14 days of Dnv of the rat Diam. MHC protein expression was determined by SDS-PAGE. Changes in MHC isoform protein and mRNA expression were not concurrent. Expression of MHCSlow and MHC2X mRNA isoforms decreased dramatically by 3 days of Dnv, whereas that of MHC2A and MHC2B did not change. Expression of all MHC protein isoforms decreased by 3 days of Dnv. We observed a differential effect of rat Diam Dnv on MHC isoform protein and mRNA expression. The time course of the changes in MHC isoform mRNA and protein expression suggests a predominant effect of altered protein turnover rates on MHC protein expression instead of altered transcription after Dnv.

scholarly journals Quantifying BRCA1 and BRCA2 mRNA Isoform Expression Levels in Single Cells

2019 ◽  
Vol 20 (3) ◽  
pp. 693 ◽  
Author(s):  
Vanessa Lattimore ◽  
John Pearson ◽  
Arthur Morley-Bunker ◽  
kConFab Investigators ◽  
Amanda Spurdle ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Single Cells ◽  
In Situ Hybridisation ◽  
Brca1 And Brca2 ◽  
Mrna Isoforms ◽  
Absolute Expression ◽  
Mrna Isoform ◽  
Splicing Patterns ◽  
Diagnostic Setting

BRCA1 and BRCA2 spliceogenic variants are often associated with an elevated risk of breast and ovarian cancers. Analyses of BRCA1 and BRCA2 splicing patterns have traditionally used technologies that sample a population of cells but do not account for the variation that may be present between individual cells. This novel proof of concept study utilises RNA in situ hybridisation to measure the absolute expression of BRCA1 and BRCA2 mRNA splicing events in single lymphoblastoid cells containing known spliceogenic variants (BRCA1c.671-2 A>G or BRCA2c.7988 A>T). We observed a large proportion of cells (>42%) in each sample that did not express mRNA for the targeted gene. Increased levels (average mRNA molecules per cell) of BRCA2 ∆17_18 were observed in the cells containing the known spliceogenic variant BRCA2c.7988 A>T, but cells containing BRCA1c.671-2 A>G were not found to express significantly increased levels of BRCA1 ∆11, as had been shown previously. Instead, we show for each variant carrier sample that a higher proportion of cells expressed the targeted splicing event compared to control cells. These results indicate that BRCA1/2 mRNA is expressed stochastically, suggesting that previously reported results using RT-PCR may have been influenced by the number of cells with BRCA1/2 mRNA expression and may not represent an elevation of constitutive mRNA expression. Detection of mRNA expression in single cells allows for a more comprehensive understanding of how spliceogenic variants influence the expression of mRNA isoforms. However, further research is required to assess the utility of this technology to measure the expression of predicted spliceogenic BRCA1 and BRCA2 variants in a diagnostic setting.

J0540101 Purity of cytoplasmic RNA extracted from single cells via electrical lysis and isotachophoresis

2015 ◽  
Vol 2015 (0) ◽  
pp. _J0540101--_J0540101-
Author(s):  
Shota HATA ◽  
Ryuji YOKOKAWA ◽  
Hidetoshi KOTERA ◽  
Hirofumi SHINTAKU
Keyword(s):  
Single Cells ◽  
Cytoplasmic Rna ◽  
Electrical Lysis
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