Enhanced alternative splicing of the FLVCR1 gene in Diamond Blackfan anemia disrupts FLVCR1 expression and function that are critical for erythropoiesis
Michelle A. Rey, Simon P. Duffy, Jennifer K. Brown, James A. Kennedy, John E. Dick, Yigal Dror, Chetankumar S. Tailor
Haematologica November 2008 93: 1617-1626; doi:10.3324/haematol.13359

Author Affiliations

  1. Michelle A. Rey1,2,
  2. Simon P. Duffy1,2,
  3. Jennifer K. Brown1,2,
  4. James A. Kennedy2,3,
  5. John E. Dick2,3,
  6. Yigal Dror1 and
  7. Chetankumar S. Tailor1,2⇓
  1. 1 Program in Cell Biology, The Hospital for Sick Children, Toronto, ON
  2. 2 Department of Molecular Genetics, University of Toronto, Toronto, ON
  3. 3 Division of Cell and Molecular Biology, University Health Network, Suite 8-355 Toronto Medical Discovery Tower, Toronto, ON, Canada
  1. Correspondence: Chetankumar S. Tailor, The Hospital for Sick Children, Program in Cell Biology, Room 7129 Elm Wing, 555 University Avenue, Toronto, ON, M5G 1X8, Canada. E-mail: chetankumar.tailor{at}sickkids.ca
View Abstract

ARTICLE FIGURES & DATA

Figures

  • Figure 1.
    Figure 1.

    Susceptibility of human TE671 cells expressing FeLV-C or FeLV-B Env proteins to FeLV-C and FeLV-B infection. FeLV-C (C Env) or FeLV-BEnv (BEnv) genes were introduced into TE671 cells by retroviral infection, and infected cells were selected using G418. Transduced cells were then tested for susceptibility to infection by β-galactosidase encoding FeLV-C (white) and FeLV-B (black). Titers are the average of three experiments and are represented as colony-forming units (cfu) per milliliter of virus supernatant.

  • Figure 2.
    Figure 2.

    Erythroid and myeloid cell development of vector-, FeLV-B Env- or FeLV-C Env- transduced lineage-depleted cord blood cells. FeLV-C and FeLV-B Env genes were introduced into lineage depleted cord blood cells by retroviral infection and cells were subsequently cultured in either erythroid or myeloid growth medium. Surface expression of CD36, GlyA, CD14, CD15 and CD45 was analyzed by flow cytometry after 13 days of culture in the respective growth media. (A) CD36 and GlyA surface expression on vector- and FeLV Env-transduced cells grown in erythroid growth medium. The percentages of cells expressing GlyA are shown in the upper two quadrants. (B) CD14 and CD45 expression on FeLV Env-and vector- transduced cells grown in erythroid growth medium. CD45- cells are represented in the two left quadrants. (C) CD14 (monocyte/macrophage) and CD15 (myelocyte/granulocyte) expression on FeLV Env- and vector- transduced cells grown in myeloid growth medium. The percentages of cells expressing CD14 and CD15 markers are shown.

  • Figure 3.
    Figure 3.

    Alternatively spliced FLVCR1 isoforms isolated from Diamond Blackfan anemia and normal immature erythroid cells. Alternatively spliced FLVCR1 isoforms were isolated from five Diamond Blackfan anemia (D1–D5) and ten normal (N1–N10) samples by PCR using primers encoding FLVCR1 transmembrane (TM) 1 and 12 (black arrows). Amplified sequences were cloned into a PCR cloning vector and subsequently sequenced. FLVCR1 exons (E) 1–10 are shown as boxes. FLVCR1 sequences are labeled E2− (exon 2 deleted), E2−E6− (exon 2 and 6 deleted), E3− (exon 3 deleted), E3−E6− (exon 3 and 6 deleted) and E6- (exon 6 deleted). The premature termination codon is denoted by an asterisk. A diagram of the topology of potential proteins encoded by the alternatively spliced FLVCR1 sequences is also shown. Normally spliced FLVCR1 encodes a cell surface protein predicted to contain 12 transmembrane spanning segments with six presumptive extracellular loops (loop numbers are indicated above the loops). The Diamond Blackfan anemia and normal samples from which the FLVCR1 sequences were isolated are also denoted.

  • Figure 4.
    Figure 4.

    Expression and functional analysis of the E3− and E3−E6−FLVCR1 encoded proteins. HA-tagged normal FLVCR1, and E3− and E3−E6− FLVCR1 sequences were introduced into murine Mus. dunni tail fibroblast (MDTF) cells by retroviral infection. Cells were subsequently tested for susceptibility to β-galactosidase expressing FeLV-C, and E3− and E3−E6− FLVCR1 protein expression analyzed by western blotting and by confocal microscopy (A) Susceptibility of murine MDTF cells expressing human FLVCR1, E3− or E3−E6− encoded proteins to β-galactosidase expressing FeLV-C. Control represents parental MDTF cells. Infection titers are the averages of three experiments and are represented as colony-forming units (cfu) per milliliter of virus supernatant. The arrow indicates zero infection titer (B) Western blot analysis of HA-tagged E3−, E3−E6−, and hFLVCR1 proteins in cell lysate fractions from FLVCR1-transduced MDTF cells. (C) Confocal immunofluorescence microscope image of MDTF cells (control), and MDTF cells expressing HA-tagged human FLVCR1, E3− or E3−E6− FLVCR1. The white arrows depict surface localization. The nucleus of the cell is stained with DAPI.

  • Figure 5.
    Figure 5.

    Quantification of normal and alternatively spliced FLVCR1 transcript expression in Diamond Blackfan anemia and normal immature erythroid cells. Total RNA from CD71high cells isolated from Diamond Blackfan anemia and normal bone marrow was used to generate cDNA, and subsequent real time PCR was carried out using specific exon-specific FLVCR1 primers (A) The specific E1/αE1 and E2/αE3 primers used in the real-time PCR assays are shown in the inset. Total FLVCR1 transcript expression, relative to actin B gene expression, is shown for five Diamond Blackfan anemia (striped) and ten normal (black) cell samples. Total FLVCR1 transcript expression was determined by real-time PCR using the E1/αE1 primers (B) Percent of E2 and E3-containing FLVCR1 transcripts. E2 and E3-containing FLVCR1 transcripts were quantified by real-time PCR using E2/αE3 primers and the percentage of transcripts containing E2/E3 calculated relative to total FLVCR1 transcript expression.

  • Figure 6.
    Figure 6.

    FLVCR1 alternative splicing and protein expression profile in RPS19 down-regulated human K562 cells. RPS19 expression was down-regulated by expression of R1 and R2 RPS19 shRNA. shRNA against the luciferase gene was used as a control. shRNA were introduced into K562 cells by retroviral infection and transduced cells selected using puromycin. RPS19 and FLVCR1 gene expression was subsequently determined by real-time PCR using gene-specific primers. Total RPS19 and FLVCR1 protein expression was determined by western blot analysis using specific anti-RPS19 and anti-FLVCR1 monoclonal antibodies. (A) RPS19 gene expression in R1 and R2 RPS19 shRNA-expressing human K562 cells relative to expression in Luc shRNA-expressing K562 cells. (B) Western blot showing RPS19 protein expression in R1, R2 and Luc shRNA-expressing cells. RPS19 protein expression is shown relative to β-actin expression. (C) Total FLVCR1 transcript expression in R1 and R2 cells relative to expression in Luc cells. Total FLVCR1 transcript expression was determined by real-time PCR using the E1/αE1 primers (Figure 5A) (D) Percentage of FLVCR1 transcripts containing E2 and E3 in R1, R2 and Luc cells relative to total FLVCR1 transcript expression. (E) Western blot showing total cellular expression of FLVCR1 protein in R1, R2 and Luc cells. FLVCR1 expression is shown relative to β-actin expression.

Back to top
Email
Print
Citation Tools

Jump To

What about you?
Tell us your interests and get all the new contents of Haematologica in advance