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.
Highlights
Alternative splicing of pre-messenger RNA generates multiple transcript isoforms from a single gene that can code for proteins with distinct functions
We first focus on the analysis of two representative genes that are abundantly expressed in hematopoietic cells (EEF1A1 and ANXA1)
Distinct novel transcript isoforms of EEF1A1 detected in bone marrow cell subpopulations by full-length
Summary
Alternative splicing of pre-messenger RNA (mRNA) generates multiple transcript isoforms from a single gene that can code for proteins with distinct functions. For >90% of human genes, an average of five transcript isoforms are predicted, suggesting a challenging complexity of the protein-coding transcriptome [8]. Unbiased methods such as RNA sequencing are crucial for unraveling the transcriptome complexity in different tissues or cells under diverse physiologic or pathologic conditions [9,10,11]. Bone marrow continuously replenishes differentiated blood cells from a small pool of stem cells Studies of this process have provided many of the concepts of tissue regeneration from resident stem cells [12,13,14]. We compared the transcriptomes of progenitor and differentiated bone marrow cell populations using single-molecule real-time (SMRT) full-length RNA-seq of unfragmented complementary DNAs (cDNAs) abbreviated here as “full length RNA-seq”
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