EGO, a novel, noncoding RNA gene, regulates eosinophil granule protein transcript expression

Abstract

Gene expression profiling of early eosinophil development shows increased transcript levels of proinflammatory cytokines, chemokines, transcription factors, and a novel gene, EGO (eosinophil granule ontogeny). EGO is nested within an intron of the inositol triphosphate receptor type 1 (ITPR1) gene and is conserved at the nucleotide level; however, the largest open reading frame (ORF) is 86 amino acids. Sucrose density gradients show that EGO is not associated with ribosomes and therefore is a noncoding RNA (ncRNA). EGO transcript levels rapidly increase following interleukin-5 (IL-5) stimulation of CD34(+) hematopoietic progenitors. EGO RNA also is highly expressed in human bone marrow and in mature eosinophils. RNA silencing of EGO results in decreased major basic protein (MBP) and eosinophil derived neurotoxin (EDN) mRNA expression in developing CD34(+) hematopoietic progenitors in vitro and in a CD34(+) cell line model. Therefore, EGO is a novel ncRNA gene expressed during eosinophil development and is necessary for normal MBP and EDN transcript expression.

Figure 1

Conservation and gene structure of EGO. (A) Vista tracks of mouse and chicken on the UCSC browser showing conservation of the ITPR-1 region. The arrow indicates the EGO region. ITPR-1 exons are shown as vertical lines. The y axis ranges from 50%-100% percent identity. (B) Vista tracks of mouse and chicken on the UCSC browser at the same magnification as panel C. (C) The structure of EGO transcripts is shown. The representative cDNA clones (BCO35947 and AW970881) from BLAT (http://genome.ucsc.edu) are shown in black. The putative 5′ end of EGO-A is shown in gray. Note that EGO transcripts are shown 3′ to 5′ because they are on the minus strand of genomic DNA.

Figure 2

Northern blots of EGO transcripts. Northern blots of TF-1 cell poly A+ RNA hybridized with radiolabeled probe specific for panel A, EGO-A (1 kb), and panel B. EGO-B (1.7 kb). Sequenced splice junction of EGO-B is 4767665 TTCTATCAG…. GCACGATGGT 4766655 and 5′ end of EGO as determined by PCR is 4768306 TTCAAACAG (Figure S2).

Figure 3

Real-time Q-RT-PCR of CD34⁺ RNA from sucrose density gradient fractions. (A) OD260. (B) α-tubulin. (C) EGO-A. (D) EGO-B. Expression level as fold change is shown on the y axis. Standard errors of PCR triplicates are shown.

Figure 4

Real-time Q-RT-PCR of transcript expression following cytokine stimulation of CD34⁺ cells. (A) EGO-A, UCB CD34⁺ cells. (B) EGO-B, UCB CD34⁺ cells. (C) MBP transcripts following IL-5 or epoietin-α stimulation of UCB CD34⁺ cells. (D) EGO-A and B transcripts following IL-5 stimulation of bone marrow CD34⁺ cells. EGO-A, solid line. EGO-B, dashed line. Standard errors of PCR triplicates are shown.

Figure 5

Expression of EGO in peripheral blood eosinophils. (A) Real-time Q-RT-PCR of unstimulated peripheral blood eosinophils. (B). Real-time Q-RT-PCR of IL-5–stimulated peripheral blood eosinophils for EGO-A, solid line and EGO-B, dashed line. Standard errors of PCR triplicates are shown.

Figure 6

Real-time Q-RT-PCR of EGO transcripts derived from human tissues. (A) Fold expression levels of EGO-A in various tissues relative to brain. (B) Fold expression levels of EGO-B relative to brain. CD34⁺ cells and bone marrow mononuclear cell RNA was normalized to the average level of α-tubulin in the tissue panel. UC CD34⁺, umbilical cord CD34⁺; BM CD34+, bone marrow CD34⁺; BMMC, bone marrow mononuclear cells. Standard errors of PCR triplicates are shown.

Figure 7

EGO RNA silencing decreases EDN and MBP transcript levels. (A) TF-1 cells transfected with negative control plasmid, pSil Neg (black), or shRNA targeting both EGO transcripts, pSil 20-2 (gray). Results shown are from experiment 5 in Table 2. (B) UCB CD34⁺ cells transfected with negative control plasmid, pSil Neg (black), or shRNA targeting both EGO transcripts, pSil 20-2 (gray). Standard errors of PCR triplicates are shown.