MicroRNA-96 Directly Inhibits γ-Globin Expression in Human Erythropoiesis

Fetal hemoglobin, HbF (α2γ2), is the main hemoglobin synthesized up to birth, but it subsequently declines and adult hemoglobin, HbA (α2β2), becomes predominant. Several studies have indicated that expression of the HbF subunit γ-globin might be regulated post-transcriptionally. This could be confered by ∼22-nucleotide long microRNAs that associate with argonaute proteins to specifically target γ-globin mRNAs and inhibit protein expression. Indeed, applying immunopurifications, we found that γ-globin mRNA was associated with argonaute 2 isolated from reticulocytes that contain low levels of HbF (<1%), whereas association was significantly lower in reticulocytes with high levels of HbF (90%). Comparing microRNA expression in reticulocytes from cord blood and adult blood, we identified several miRNAs that were preferentially expressed in adults, among them miRNA-96. The overexpression of microRNA-96 in human ex vivo erythropoiesis decreased γ-globin expression by 50%, whereas the knock-down of endogenous microRNA-96 increased γ-globin expression by 20%. Moreover, luciferase reporter assays showed that microRNA-96 negatively regulates expression of γ-globin in HEK293 cells, which depends on a seedless but highly complementary target site located within the coding sequence of γ-globin. Based on these results we conclude that microRNA-96 directly suppresses γ-globin expression and thus contributes to HbF regulation

The Relative Levels Of Alpha(2)-, Alpha(1)-, And Zeta-Mrna In Hb H Patients With Different Deletional And Nondeletional Alpha-Thalassemia Determinants

We have analyzed the alpha(2)/alpha(1)-, alpha/beta-, zeta/(alpha + zeta)-mRNA ratios in the: retic-ulocytes of 40 patients with Hb H disease. 21 patients had deletional Hb H disease (- - / - alpha), namely combinations of one of four types of alpha-thal-1 (MED-I, MED-II, -(alpha)20.5, SEA) and one of two types of alpha-thal-2(- 3.7 or - 4.2 kb): 13 had Hb H disease because of combinations of one of these alpha-thal-1 deletions with either a 5 nt deletion at the 5' splicing site of IVS-I, or a terminating codon mutation (Hb CS), or a poly(A) mutation, and six were homozygous for either a poly(A) mutation or the 5 nt deletion. Significant differences were observed between the deletional types (- - /- alpha; alpha(2)/alpha(1) ratio of zero; alpha/beta ratio of similar to 1) and non-deletional types (- - /alpha(T) alpha; alpha(2)/alpha(1) ratio of 0.05-0.3 for those with T = the 5 nt deletion or the terminating codon mutant, and similar to 1.0 for those with T = a poly(A) mutation; alpha/beta ratio in all types of similar to 0.7). Comparable data were found for the nondeletional alpha-thal-2 homozygotes. The noted differences were highly significant and the determination of the two ratios may be diagnostically of considerable value. The Low alpha(2)/alpha(1)-mRNA ratio in the two patients- with - - /alpha(-5nt)alpha and the one patient with alpha(-5nt)alpha/alpha(-5nt)alpha indicates the presence of minute amounts of alpha(2)-mRNA; apparently splicing at the donor site is greatly impaired by this a deletion but not eliminated. The high alpha(2)/alpha(1)-mRNA ratio in the four patients with - -/alpha(PA-2)alpha and the five patients with alpha(PA-1)alpha/alpha(PA-1)alpha (PA-1 and PA-2 are poly(A) mutations) is due to the presence of an elongated alpha(2)-mRNA which uses an alternate location as a polyadenylation site. The relative levels of zeta-mRNA varied considerably the highest levels were found in patients with the -(alpha)20.5/-alpha (SEA)/ - alpha deletional types but not in those with the - (alpha)20.5/-alpha or - -(SEA)/-alpha deletional types but not in those with the -(alpha)20.5/alpha(PA-2)alpha, -(alpha)20.5/alpha(-5nt)alpha, or - -(SEA)/alpha(CS)alpha nondeletional types. No definitive explanation can be given for these differences; perhaps certain sequences that are part of some of the alpha-thal-1 deletions are important for the suppresstion of the zeta-globin gene.WoSScopu