The normal expression of Hb A in humans requires the high-level stability of α - and β-globin mRNAs in both transcriptionally active and transcriptionally silenced erythroid progenitors. In contrast to α -globin–whose stability is known to be enhanced by an mRNA-protein (mRNP) complex that assembles on a specific pyrimidine-rich track within its 3'UTR–the structure(s) and mechanism(s) that underlie the high stability of human β-globin mRNA remain poorly defined. We recently identified two RNA-binding proteins, AUF-1 and YB-1, that regulate levels of β-globin mRNA in erythroid progenitors by assembling a cytoplasm-restricted mRNP 'β-complex' on its 3'UTR. The function of the β-complex was predicted by in vitro analyses that mapped its binding to a cis-acting determinant of β-globin mRNA stability, and by in vivo siRNA studies demonstrating that simultaneous knockdown of AUF-1 and YB-1 coordinately ablated the β-complex and coordinately reduced the accumulation of β-globin mRNA in K562 cells. The biological importance of the β-complex was subsequently confirmed in human hematopoietic stem cells, where shRNA-mediated knock-down of AUF-1 or YB-1 effected lower levels of β-globin mRNA in cells induced to the erythroid lineage, again implicating their participation in post-transcriptional mechanism(s) regulating the stability of β-globin mRNA.
To unambiguously link β-complex activity to β-globin mRNA half-life, we conducted formal in vivo mRNA stability analyses in K562 cells using a β-globin mRNA-specific tetracycline-conditional transcriptional chase strategy. A derivative β-globin mRNA carrying a 5-nt substitution that totally disrupts β-complex assembly (βMut mRNA) displayed a lower half-life than wild-type β-globin mRNA (βWT mRNA), confirming the participation of the β-complex in post-transcriptional regulatory processes. Parallel poly(A) tail length analyses indicated a possible mechanism for this activity, revealing that the βMut mRNA had a shortened steady-state poly(A) tail that truncated faster than the poly(A) tail on βWT mRNA, suggesting a functional interaction between the β-complex and poly(A) tail-associated factors. This observation is fully consistent with the known importance of deadenylation to processes regulating the decay of heterologous mRNAs in several other experimental systems. Subsequent studies supported our model for β-complex/poly(A) tail interaction: electrophoretic gel mobility-shift analyses demonstrated that the β-complex readily assembles on polyadenylated β-globin 3'UTRs but not on corresponding deadenylated 3'UTRs, while RNA affinity capture experiments using K562 cytoplasmic extracts demonstrated that a polyadenylated βWT 3'UTR retains poly(A) binding protein (PABP), while a similar β-complex-deficient βMut 3'UTR fails to bind PABP. Ongoing co-immunoprecipation studies are expected to determine whether the β-complex and PABP are tethered by an interval of mRNA or, alternately, interact directly via a protein-protein interaction.
Based upon our previous structural and functional analyses indicating that AUF-1 and YB-1 act redundantly to regulate the cytoplasmic level of β-globin mRNA, we are currently investigating the hypothesis that these two factors also display redundant interactions with the poly(A) tail and its trans-acting binding factors. Our initial RNA affinity analyses confirm this expectation, demonstrating that K562 extracts depleted of either AUF-1 or YB-1 (using an shRNA-knock-down strategy) both maintained the ability to assemble a β-complex as well as facilitate PABP binding to a the polyadenylated βWT 3'UTR. We are presently testing AUF-1 and YB-1 for corresponding functional redundancy (i.e., their abilities to independently induce βWT mRNA stability) using in vivo mRNA tethering experiments in which AUF-1 or YB-1 can be structurally modified to promote their independent interaction with the β-complex binding site.
Altogether, these experiments demonstrate that the β-complex, through its component mRNA-binding factors AUF-1 and YB-1, effects the high stability of β-globin mRNA by interacting with PABP. A detailed structural and mechanistic description of this process will be invaluable to the design of novel therapeutics for patients with congenital disorders of β-globin gene expression.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.