Jian Kong

Assembly of the α-Globin mRNA Stability Complex Reflects Binary Interaction between the Pyrimidine-Rich 3′ Untranslated Region Determinant and Poly(C) Binding Protein αCP

ABSTRACT Globin mRNAs accumulate to 95% of total cellular mRNA during terminal erythroid differentiation, reflecting their extraordinary stability. The stability of human α-globin mRNA is paralleled by formation of a sequence-specific RNA-protein (RNP) complex at a pyrimidine-rich site within its 3′ untranslated region (3′UTR), the α-complex. The proteins of the α-complex are widely expressed. The α-complex or a closely related complex also assembles at pyrimidine-rich 3′UTR segments of other stable mRNAs. These data suggest that the α-complex may constitute a general determinant of mRNA stability. One or more αCPs, members of a family of hnRNP K-homology domain poly(C) binding proteins, are essential constituents of the α-complex. The ability of αCPs to homodimerize and their reported association with additional RNA binding proteins such as AU-rich binding factor 1 (AUF1) and hnRNP K have suggested that the α-complex is a multisubunit structure. In the present study, we have addressed the composition of the α-complex. An RNA titration recruitment assay revealed that αCPs were quantitatively incorporated into the α-complex in the absence of associated AUF1 and hnRNP K. A high-affinity direct interaction between each of the three major αCP isoforms and the α-globin 3′UTR was detected, suggesting that each of these proteins might be sufficient for α-complex assembly. This sufficiency was further supported by the sequence-specific binding of recombinant αCPs to a spectrum of RNA targets. Finally, density sedimentation analysis demonstrated that the α-complex could accommodate only a single αCP. These data established that a single αCP molecule binds directly to the α-globin 3′UTR, resulting in a simple binary structure for the α-complex.

In Vivo Association of the Stability Control Protein αCP with Actively Translating mRNAs

ABSTRACT Posttranscriptional controls play a major role in eucaryotic gene expression. These controls are mediated by sequence-specific interactions of cis-acting determinants in target mRNAs with one or more protein factors. The positioning of a subset of these mRNA-protein (RNP) complexes within the 3′ untranslated region (3′ UTR) may allow them to remain associated with the mRNA during active translation. Robust expression of human α-globin mRNA during erythroid differentiation has been linked to formation of a binary complex between a KH-domain protein, αCP, and a 3′ UTR C-rich motif. Detection of this “α-complex” has been limited to in vitro studies, and the functional state of the α-globin mRNA targeted by αCP has not been defined. In the present study we demonstrate that a significant fraction of αCP is associated with polysomal mRNA. Targeted analysis of the polysomal RNP complexes revealed that αCP is specifically bound to actively translating α-globin mRNA. The bound αCP is restricted to the poly(C)-rich 3′ UTR motif and is dislodged when ribosomes are allowed to enter this region. These data validate the general importance of the 3′ UTR as a sheltered site for RNP complexes and support a specific model in which the stabilizing function of αCP is mediated on actively translating target mRNAs.

The KH-Domain Protein αCP Has a Direct Role in mRNA Stabilization Independent of Its Cognate Binding Site

ABSTRACT Previous studies suggest that high-level stability of a subset of mammalian mRNAs is linked to a C-rich motif in the 3′ untranslated region (3′UTR). High-level expression of human α-globin mRNA (hα-globin mRNA) in erythroid cells has been specifically attributed to formation of an RNA-protein complex comprised of a 3′UTR C-rich motif and an associated 39-kDa poly(C) binding protein, αCP. Documentation of this RNA-protein α-complex has been limited to in vitro binding studies, and its impact has been monitored by alterations in steady-state mRNA. Here we demonstrate that αCP is stably bound to hα-globin mRNA in vivo, that α-complex assembly on the hα-globin mRNA is restricted to the 3′UTR C-rich motif, and that α-complex assembly extends the physical half-life of hα-globin mRNA selectively in erythroid cells. Significantly, these studies also reveal that an artificially tethered αCP has the same mRNA-stabilizing activity as the native α-complex. These data demonstrate a unique contribution of the α-complex to hα-globin mRNA stability and support a model in which the sole function of the C-rich motif is to selectively tether αCP to a subset of mRNAs. Once bound, αCP appears to be fully sufficient to trigger downstream events in the stabilization pathway.

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 α-globin mRNA is bound at a C-rich motif in the 3′ untranslated region (3′UTR) by the KH domain protein α-globin poly(C)-binding protein (αCP). This “α-complex” is essential to cytoplasmic stability of α-globin mRNA in erythroid cells. Here we report that the 3′UTR α-complex also serves an independent nuclear role as a splice enhancer. Consistent with this role, we find that αCP binds α-globin transcripts prior to splicing. Surprisingly, this binding occurs at C-rich sites within intron I as well as at the 3′UTR C-rich determinant. The intronic and 3′UTR αCP complexes appear to have distinct effects on splicing. While intron I complexes repress intron I excision, the 3′UTR 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 3′UTR αCP complex may serve to “prepackage” α-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.

A cell type-restricted mRNA surveillance pathway triggered by ribosome extension into the 3' untranslated region.

The accuracy of eukaryotic gene expression is monitored at multiple levels. Surveillance pathways have been identified that degrade messenger RNAs containing nonsense mutations, harboring stalled ribosomes or lacking termination codons. Here we report a previously uncharacterized surveillance pathway triggered by ribosome extension into the 3′ untranslated region. This ribosome extension–mediated decay, REMD, accounts for marked repression of protein synthesis from a human α-globin gene containing a prevalent antitermination mutation. REMD can be mechanistically distinguished from other surveillance pathways by its functional linkage to accelerated deadenylation, by its independence from the NMD factor Upf1 and by cell-type restriction. This unusual pathway of mRNA surveillance is likely to act as a modifier of additional genetic defects and may reflect post-transcriptional controls particular to erythroid and other differentiated cell lineages.

An RNA–protein complex links enhanced nuclear 3′ processing with cytoplasmic mRNA stabilization

Post‐transcriptional controls are critical to gene regulation. These controls are frequently based on sequence‐specific binding of trans‐acting proteins to cis‐acting motifs on target RNAs. Prior studies have revealed that the KH‐domain protein, αCP, binds to a 3′ UTR C‐rich motif of hα‐globin mRNA and contributes to its cytoplasmic stability. Here, we report that this 3′ UTR αCP complex regulates the production of mature α‐globin mRNA by enhancing 3′ processing of the hα‐globin transcript. We go on to demonstrate that this nuclear activity reflects enhancement of both the cleavage and the polyadenylation reactions and that αCP interacts in vivo with core components of the 3′ processing complex. Consistent with its nuclear processing activity, our studies reveal that αCP assembles co‐transcriptionally at the hα‐globin chromatin locus and that this loading is selectively enriched at the 3′ terminus of the gene. The demonstrated linkage of nuclear processing with cytoplasmic stabilization via a common RNA–protein complex establishes a basis for integration of sequential controls critical to robust and sustained expression of a target mRNA.

Shared Stabilization Functions of Pyrimidine-Rich Determinants in the Erythroid 15-lipoxygenase and α-globin mRNAs

ABSTRACT The poly(C)-binding proteins, αCPs, comprise a set of highly conserved KH-domain factors that participate in mRNA stabilization and translational controls in developmental and viral systems. Two prominent models of αCP function link these controls to late stages of erythroid differentiation: translational silencing of 15-lipoxygenase (Lox) mRNA and stabilization of α-globin mRNA. These two controls are mediated via association of αCPs with structurally related C-rich 3′-untranslated region elements: the differentiation control elements (DICE) in Lox mRNA and the pyrimidine-rich motifs in α-globin mRNA. In the present report a set of mRNA translation and stability assays are used to determine how these two αCP-containing complexes, related in structure and position, mediate distinct posttranscriptional controls. While the previously reported translational silencing by the DICE is not evident in our studies, we find that the two determinants mediate similar levels of mRNA stabilization in erythroid cells. In both cases this stabilization is sensitive to interference by a nuclear-restricted αCP decoy but not by the same decoy restricted to the cytoplasm. These data support a general role for αCPs in stabilizing a subset of erythroid mRNAs. The findings also suggest that initial binding of αCP to target mRNAs occurs in the nucleus. Assembly of stabilizing mRNP complexes in the nucleus prior to export may maximize their impact on cytoplasmic events.

Cytoplasmic Poly(A) Binding Protein C4 Serves a Critical Role in Erythroid Differentiation

ABSTRACT The expression of an mRNA is strongly impacted by its 3′ poly(A) tail and associated poly(A)-binding proteins (PABPs). Vertebrates encode six PABP isoforms that vary in abundance, distribution, developmental control, and subcellular localization. Here we demonstrate that the minor PABP isoform PABPC4 is expressed in erythroid cells and impacts the steady-state expression of a subset of erythroid mRNAs. Motif analyses reveal a high-value AU-rich motif in the 3′ untranslated regions (UTRs) of PABPC4-impacted mRNAs. This motif enhances the association of PABPC4 with mRNAs containing critically shortened poly(A) tails. This association may serve to protect a subset of mRNAs from accelerated decay. Finally, we demonstrate that selective depletion of PABPC4 in an erythroblast cell line inhibits terminal erythroid maturation with corresponding alterations in the erythroid gene expression. These observations lead us to conclude that PABPC4 plays an essential role in posttranscriptional control of a major developmental pathway.

Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). The level of sensitivity of a PTC-containing mRNA to NMD is multifactorial. We have previously shown that human β-globin mRNAs carrying PTCs in close proximity to the translation initiation AUG codon escape NMD. This was called the ‘AUG-proximity effect’. The present analysis of nonsense codons in the human α-globin mRNA illustrates that the determinants of the AUG-proximity effect are in fact quite complex, reflecting the ability of the ribosome to re-initiate translation 3′ to the PTC and the specific sequence and secondary structure of the translated ORF. These data support a model in which the time taken to translate the short ORF, impacted by distance, sequence, and structure, not only modulates translation re-initiation, but also impacts on the exact boundary of AUG-proximity protection from NMD.

An RNA-protein complex links enhanced nuclear 3′ processing with cytoplasmic mRNA stabilization: A novel upstream enhancer of 3′ processing

Post‐transcriptional controls are critical to gene regulation. These controls are frequently based on sequence‐specific binding of trans‐acting proteins to cis‐acting motifs on target RNAs. Prior studies have revealed that the KH‐domain protein, αCP, binds to a 3′ UTR C‐rich motif of hα‐globin mRNA and contributes to its cytoplasmic stability. Here, we report that this 3′ UTR αCP complex regulates the production of mature α‐globin mRNA by enhancing 3′ processing of the hα‐globin transcript. We go on to demonstrate that this nuclear activity reflects enhancement of both the cleavage and the polyadenylation reactions and that αCP interacts in vivo with core components of the 3′ processing complex. Consistent with its nuclear processing activity, our studies reveal that αCP assembles co‐transcriptionally at the hα‐globin chromatin locus and that this loading is selectively enriched at the 3′ terminus of the gene. The demonstrated linkage of nuclear processing with cytoplasmic stabilization via a common RNA–protein complex establishes a basis for integration of sequential controls critical to robust and sustained expression of a target mRNA.