The 3′ Untranslated Region Complex Involved in Stabilization of Human α-globin mRNA Assembles in the Nucleus and Serves an Independent Role as a Splice Enhancer

Abstract

Posttranscriptional controls, mediated primarily by RNA-protein complexes, have the potential to alter multiple steps in RNA processing and function. Human ␣-globin mRNA is bound at a C-rich motif in the 3 untranslated region (3UTR) by the KH domain protein ␣-globin poly(C)-binding protein (␣CP). This "␣-complex" is essential to cytoplasmic stability of ␣-globin mRNA in erythroid cells. Here we report that the 3UTR ␣-complex also serves an independent nuclear role as a splice enhancer. Consistent with this role, we find that ␣CP binds ␣-globin transcripts prior to splicing. Surprisingly, this binding occurs at C-rich sites within intron I as well as at the 3UTR C-rich determinant. The intronic and 3UTR ␣CP complexes appear to have distinct effects on splicing. While intron I complexes repress intron I excision, the 3UTR complex enhances splicing of the full-length transcript both in vivo and in vitro. In addition to its importance to splicing, nuclear assembly of the 3UTR ␣CP complex may serve to "prepackage" ␣-globin mRNA with its stabilizing complex prior to cytoplasmic export. Linking nuclear and cytoplasmic controls by the action of a particular RNA-binding protein, as reported here, may represent a modality of general importance in eukaryotic gene regulation.Posttranscriptional controls are central to the establishment and regulation of eukaryotic gene expression (15,35). Recent data highlight critical links between nuclear and cytoplasmic processes involved in these regulatory pathways. Three prominent examples illustrate how the "nuclear history" of a transcript can affect its fate in the cytoplasm. (i) Translational activity of maternal mRNAs in Xenopus oocytes reflects whether precursor transcripts have transited the nuclear splicing pathway (32). More recent studies demonstrate the same relationship in mammalian cells (4,36,37,52). The mechanism(s) that links nuclear splicing and cytoplasmic translational activity remains poorly defined. (ii) Accelerated cytoplasmic destruction of mRNAs containing premature termination codon-containing mRNAs (nonsense-mediated mRNA decay) is dependent upon the position of the premature termination codon relative to exon-exon junctions (16, 28). This nuclear "history" (i.e., where introns were located) is recorded by deposition of a multiprotein exon-junction complex at the site of each splice (16,26,53). (iii) A specific subset of cytokine mRNAs are stabilized in the cytoplasm by binding of hnRNP D p37 to a 3Ј untranslated region (3ЈUTR) AU-rich motif. Remarkably, the formation of this 3ЈUTR RNP occurs exclusively in the nucleus prior to mRNA export (7). Thus, nuclear import of a shuttling RNA-binding protein can be a prerequisite for its deposition on target transcripts and subsequent stabilization of an mRNA(s) in the cytoplasm (7). While the overall impact of "nuclear history" on the cytoplasmic fate of specific mRNAs is evident based on these and other examples (27, 43), the underlying mechanisms involved in integrating these controls and the degree to which they c...

The poly(rC)-binding protein αCP2 is a noncanonical factor in X. laevis cytoplasmic polyadenylation

“…The PR was replaced with an equal sized ''neutral'' sequence derived from the coding region of the ha-globin mRNA . This substitution is known to abolish aCP binding in mammalian erythroid cells, resulting in defects in stability , splicing (Ji et al 2007), and 39end formation (X Ji and SA Liebhaber, in prep.). The ha Neut 39 UTR ( Fig.…”

“…While of potential interest, these studies failed to demonstrate whether XaCP2 did, in fact, bind in vivo to the pp2Ac mRNA target and/or interact with the cytoplasmic polyadenylation machinery. The possibility that aCP2 plays a direct role in targeting cytoplasmic polyadenylation is of particular interest because this same protein, also known as heterogeneous nuclear ribonucleoprotein E2 (hnRNP E2)/ poly(rC) binding protein 2 (PCBP2), has been implicated in multiple post-transcriptional control pathways in mammalian cells, including splicing (Ji et al 2007), 39 end formation (X Ji and SA Liebhaber, in prep. ), translational control , and stabilization of long-lived mRNAs via poly (A) tail maintenance .…”

Controlling -globin: a review of -globin expression and its impact on -thalassemia

“…It was recently discovered that αCPs are loaded onto α-globin transcripts in the nucleus and in addition to the 3'UTR, also bind a Crich region in intron 1. 37 In HeLa cells selectively depleted of αCP, splicing efficiency of a truncated α-globin construct (comprising exon 1, intron 1 and exon 2) was increased approximately four-fold from 15% to 58%. Thus, it appears that nuclear αCP binding to intron 1 represses splicing of the α-globin transcript.…”

“…Conversely, nuclear binding of αCP to the 3'UTR appears to enhance splicing of the α-globin pre-mRNA. 37 Experiments were conducted in HeLa and MEL cells comparing WT α-globin and a mutated transcript with a modified 3'UTR which does not bind αCP (α∆PR). In both cell types, it was found that WT transcripts were spliced with higher efficiency than mutated α∆PR transcripts.…”

“…In both cell types, it was found that WT transcripts were spliced with higher efficiency than mutated α∆PR transcripts. 37 Given the apparently contradictory roles of αCP binding in different regions of α-globin transcripts, it may be interesting to determine if the affinity of αCP for each site varies with stages of cell differentiation. It is tempting to speculate that late in differentiation, αCP bound to the 3'UTR enhances splicing and stabilizes α-globin transcripts in preparation for high levels of hemoglobin synthesis.…”