American Society of Hematology

Figure 2.

$PUM1 binds to γ-globin mRNA and decreases γ-globin (HBG1) mRNA stability and translational efficiency, and the blood from an individual harboring a novel heterozygous mutation in PUM1 RNA-binding domain displays elevated percentages of HbF and F cells. (A) qRT-PCR analysis of RNA levels of the indicated transcripts in the immunoprecipitated eluates after RIP of PUM1 in HUDEP2 cells (n = 4). Bar graphs show mean ± standard error of the mean. (B) qRT-PCR analysis of 5-EU incorporated mRNAs of indicated genes at 0-hour pulse and 24-hour chase time points after incorporation in HUDEP2 cells infected with either PUM1 or NTC lentiviral shRNAs showed an increase in the stability of HBG1 but not HBG2 or HBB globins at the 24-hour time point upon PUM1 knockdown (n = 2). Bar graphs show mean ± SD. (C) Polysome fraction analysis in HUDEP2 cells infected with either PUM1 or NTC lentiviral shRNAs showing relative distribution of HBG1 mRNA in each of the 14 fractions. Fraction 8 corresponds to the monosome fraction (80S), whereas fractions 10 to 14 correspond to the polysome fractions. A decrease in the distribution of HBG1 mRNA in the monosomal fraction with a corresponding increase in the polysomal fraction was observed upon PUM1 knockdown, suggesting an increase in translational efficiency in these samples. (D) Ratio of the polysomes (mRNA pooled from fractions 10 to 12) to the monosomes (mRNA in fraction 8) in the polysome fraction analysis in panel C is shown (n = 2). Bar graphs show mean ± SD. (E) Left: DNA sequencing chromatogram of the blood from a patient with PUM1-associated developmental disability, ataxia, and seizure (PADDAS) who has a novel heterozygous mutation (c.3267_3270delTCAC) and normal (parent) blood. Right: The structure of the RNA-binding domain of PUM1 bound to RNA. The region altered by the mutation p.(His1090Profs∗16) is shown in yellow and magenta. (F) HPLC analysis of Hb. Red arrows indicate HbF levels. (G) F cells were stained by using a modified Kleihauer-Betke procedure in the blood of a patient with PADDAS and normal (parent) blood. Red arrows indicate the F cells. Data were analyzed by using a two-sided Student t test. Scale bar represents 30 μm. ∗P < .05; ∗∗P < .005.$

PUM1 binds to γ-globin mRNA and decreases γ-globin (HBG1) mRNA stability and translational efficiency, and the blood from an individual harboring a novel heterozygous mutation in PUM1 RNA-binding domain displays elevated percentages of HbF and F cells. (A) qRT-PCR analysis of RNA levels of the indicated transcripts in the immunoprecipitated eluates after RIP of PUM1 in HUDEP2 cells (n = 4). Bar graphs show mean ± standard error of the mean. (B) qRT-PCR analysis of 5-EU incorporated mRNAs of indicated genes at 0-hour pulse and 24-hour chase time points after incorporation in HUDEP2 cells infected with either PUM1 or NTC lentiviral shRNAs showed an increase in the stability of HBG1 but not HBG2 or HBB globins at the 24-hour time point upon PUM1 knockdown (n = 2). Bar graphs show mean ± SD. (C) Polysome fraction analysis in HUDEP2 cells infected with either PUM1 or NTC lentiviral shRNAs showing relative distribution of HBG1 mRNA in each of the 14 fractions. Fraction 8 corresponds to the monosome fraction (80S), whereas fractions 10 to 14 correspond to the polysome fractions. A decrease in the distribution of HBG1 mRNA in the monosomal fraction with a corresponding increase in the polysomal fraction was observed upon PUM1 knockdown, suggesting an increase in translational efficiency in these samples. (D) Ratio of the polysomes (mRNA pooled from fractions 10 to 12) to the monosomes (mRNA in fraction 8) in the polysome fraction analysis in panel C is shown (n = 2). Bar graphs show mean ± SD. (E) Left: DNA sequencing chromatogram of the blood from a patient with PUM1-associated developmental disability, ataxia, and seizure (PADDAS) who has a novel heterozygous mutation (c.3267_3270delTCAC) and normal (parent) blood. Right: The structure of the RNA-binding domain of PUM1 bound to RNA. The region altered by the mutation p.(His1090Profs∗16) is shown in yellow and magenta. (F) HPLC analysis of Hb. Red arrows indicate HbF levels. (G) F cells were stained by using a modified Kleihauer-Betke procedure in the blood of a patient with PADDAS and normal (parent) blood. Red arrows indicate the F cells. Data were analyzed by using a two-sided Student t test. Scale bar represents 30 μm. ∗P < .05; ∗∗P < .005.

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