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Genes 2019, 10(4), 253; https://doi.org/10.3390/genes10040253

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

1
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20007, USA
2
The Jackson Laboratory, Farmington, CT 06032, USA
3
Pacific Biosciences, Menlo Park, CA 94025, USA
4
Icahn School of Medicine at Mount Sinai, Institute for Genomics and Multi-scale Biology, New York, NY 10029, USA
5
Biomedical Center, National Reference Laboratory, Kigali, Rwanda
*
Author to whom correspondence should be addressed.
Received: 19 February 2019 / Revised: 19 March 2019 / Accepted: 22 March 2019 / Published: 27 March 2019
(This article belongs to the Special Issue Advances in Single Molecule, Real-Time (SMRT) Sequencing)
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Abstract

Hematopoietic 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 messenger RNA (mRNA) isoforms transcribed from the ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was evaluated 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, and 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. View Full-Text
Keywords: full length RNAseq; mRNA isoforms; protein isoforms; bone marrow cell subpopulations full length RNAseq; mRNA isoforms; protein isoforms; bone marrow cell subpopulations
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Deslattes Mays, A.; Schmidt, M.; Graham, G.; Tseng, E.; Baybayan, P.; Sebra, R.; Sanda, M.; Mazarati, J.-B.; Riegel, A.; Wellstein, A. Single-Molecule Real-Time (SMRT) Full-Length RNA-Sequencing Reveals Novel and Distinct mRNA Isoforms in Human Bone Marrow Cell Subpopulations. Genes 2019, 10, 253.

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