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

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

Identification of AUF1 (heterogeneous nuclear ribonucleoprotein D) as a component of the alpha-globin mRNA stability complex.

mRNA turnover is an important regulatory component of gene expression and is significantly influenced by ribonucleoprotein (RNP) complexes which form on the mRNA. Studies of human alpha-globin mRNA stability have identified a specific RNP complex (alpha-complex) which forms on the 3' untranslated region (3'UTR) of the mRNA and appears to regulate the erythrocyte-specific accumulation of alpha-globin mRNA. One of the protein activities in this multiprotein complex is a poly(C)-binding activity which consists of two proteins, alphaCP1 and alphaCP2. Neither of these proteins, individually or as a pair, can bind the alpha-globin 3'UTR unless they are complexed with the remaining non-poly(C) binding proteins of the alpha-complex. With the yeast two-hybrid screen, a second alpha-complex protein was identified. This protein is a member of the previously identified A+U-rich (ARE) binding/degradation factor (AUF1) family of proteins, which are also known as the heterogeneous nuclear RNP (hnRNP) D proteins. We refer to these proteins as AUF1/hnRNP-D. Thus, a protein implicated in ARE-mediated mRNA decay is also an integral component of the mRNA stabilizing alpha-complex. The interaction of AUF1/hnRNP-D is more efficient with alphaCP1 relative to alphaCP2 both in vitro and in vivo, suggesting that the alpha-complex might be dynamic rather than a fixed complex. AUF1/hnRNP-D could, therefore, be a general mRNA turnover factor involved in both stabilization and decay of mRNA.     

Complementary change in cis determinants and trans factors in the evolution of an mRNP stability complex.

RNA-protein (RNP) complexes play significant roles in the fate and expression of mRNAs. The prolonged half-life of human alpha-globin mRNA, a major determinant of normal erythroid differentiation, is dependent on the assembly of a sequence-specific 3'-untranslated region (3'UTR) RNP (alpha-complex). We demonstrate that the stability of murine alpha-globin mRNA is controlled by a parallel mechanism. Unexpectedly, however, the respective 3'UTR RNP complexes that stabilize the h(alpha)- and m(alpha)-globin mRNAs differ in structure. While the cis determinants in both species are encoded in polypyrimidine tracks, the human determinant is C-rich (CCUCC motif) while the mouse alpha-3'UTR consists of an equal distribution of Cs and Us (CCUUCU motif). The protein components of the corresponding human and murine alpha-complexes differ in a complementary manner: the previously described 39 kDa poly(C) binding protein (PCBP) present in the human alpha-complex is replaced in the mouse alpha-complex by a 48 kDa cytoplasmic poly(CU) binding protein (CUBP). These results reveal that drift in the primary sequences of the alpha-globin mRNA 3'UTR polypyrimidine tracks in a comparison between mouse and human is paralleled by an alteration in the composition of the corresponding trans-acting components. Surprisingly, these structurally distinct complexes appear to perform the identical function of stabilizing the corresponding alpha-globin mRNAs.     

The poly(A)-binding protein and an mRNA stability protein jointly regulate an endoribonuclease activity

We previously identified a sequence-specific erythroid cell-enriched endoribonuclease (ErEN) activity involved in the turnover of the stable alpha-globin mRNA. We now demonstrate that ErEN activity is regulated by the poly(A) tail. The unadenylated alpha-globin 3' untranslated region (3'UTR) was an efficient substrate for ErEN cleavage, while the polyadenylated 3'UTR was inefficiently cleaved in an in vitro decay assay. The influence of the poly(A) tail was mediated through the poly(A)-binding protein (PABP) bound to the poly(A) tail, which can inhibit ErEN activity. ErEN cleavage of an adenylated alpha-globin 3'UTR was accentuated upon depletion of PABP from the cytosolic extract, while addition of recombinant PABP reestablished the inhibition of endoribonuclease cleavage. PABP inhibited ErEN activity indirectly through an interaction with the alphaCP mRNA stability protein. Sequestration of alphaCP resulted in an increase of ErEN cleavage activity, regardless of the polyadenylation state of the RNA. Using electrophoretic mobility shift assays, PABP was shown to enhance the binding efficiency of alphaCP to the alpha-globin 3'UTR, which in turn protected the ErEN target sequence. Conversely, the binding of PABP to the poly(A) tail was also augmented by alphaCP, implying that a stable higher-order structural network is involved in stabilization of the alpha-globin mRNA. Upon deadenylation, the interaction of PABP with alphaCP would be disrupted, rendering the alpha-globin 3'UTR more susceptible to endoribonuclease cleavage. The data demonstrated a specific role for PABP in protecting the body of an mRNA in addition to demonstrating PABP's well-characterized effect of stabilizing the poly(A) tail.     

Purification and RNA binding properties of the polycytidylate-binding proteins alphaCP1 and alphaCP2

Regulation of mRNA turnover is a critical control mechanism of gene expression and is influenced by ribonucleoprotein (RNP) complexes that form on cis elements. All mRNAs have an intrinsic half-life and in many cases these half-lives can be altered by a variety of stimuli that are manifested through the formation or disruption of an RNP structure. The stability of alpha-globin mRNA is determined by elements in the 3' untranslated region that are bound by an RNP complex (alpha-complex) which appears to control the erythroid-specific accumulation of alpha-globin mRNA. The alpha-complex could consist of up to six distinct proteins or protein families. One of these families is a prominent polycytidylate binding activity which consists of two highly homologous proteins, alpha-complex proteins 1 and 2 (alphaCP1 and alphaCP2). This article focuses on various methodologies for the detection and manipulation of alphaCP1 and alphaCP2 binding to RNA and details means of isolating and characterizing mRNA bound by these proteins to study mRNA turnover and its regulation.     

An mRNA Stability Complex Functions with Poly(A)-Binding Protein To Stabilize mRNA In Vitro

The stable globin mRNAs provide an ideal system for studying the mechanism governing mammalian mRNA turnover. alpha-Globin mRNA stability is dictated by sequences in the 3' untranslated region (3'UTR) which form a specific ribonucleoprotein complex (alpha-complex) whose presence correlates with mRNA stability. One of the major protein components within this complex is a family of two polycytidylate-binding proteins, alphaCP1 and alphaCP2. Using an in vitro-transcribed and polyadenylated alpha-globin 3'UTR, we have devised an in vitro mRNA decay assay which reproduces the alpha-complex-dependent mRNA stability observed in cells. Incubation of the RNA with erythroleukemia K562 cytosolic extract results in deadenylation with distinct intermediates containing a periodicity of approximately 30 nucleotides, which is consistent with the binding of poly(A)-binding protein (PABP) monomers. Disruption of the alpha-complex by sequestration of alphaCP1 and alphaCP2 enhances deadenylation and decay of the mRNA, while reconstitution of the alpha-complex stabilizes the mRNA. Similarly, PABP is also essential for the stability of mRNA in vitro, since rapid deadenylation resulted upon its depletion. An RNA-dependent interaction between alphaCP1 and alphaCP2 with PABP suggests that the alpha-complex can directly interact with PABP. Therefore, the alpha-complex is an mRNA stability complex in vitro which could function at least in part by interacting with PABP.     

Detection and characterization of a 3' untranslated region ribonucleoprotein complex associated with human alpha-globin mRNA stability.

The highly stable nature of globin mRNA is of central importance to erythroid cell differentiation. We have previously identified cytidine-rich (C-rich) segments in the human alpha-globin mRNA 3' untranslated region (alpha-3'UTR) which are critical in the maintenance of mRNA stability in transfected erythroid cells. In the present studies, we have detected trans-acting factors which interact with these cis elements to mediate this stabilizing function. A sequence-specific ribonucleoprotein (RNP) complex is assembled after incubation of the alpha-3'UTR with a variety of cytosolic extracts. This so-called alpha-complex is sequence specific and is not formed on the 3'UTR of either beta-globin or growth hormone mRNAs. Furthermore, base substitutions within the C-rich stretches which destabilize alpha-globin mRNA in vivo result in a parallel disruption of the alpha-complex in vitro. Competition studies with a series of homoribopolymers reveals a striking sensitivity of alpha-complex formation to poly(C), suggesting the presence of a poly(C)-binding activity within the alpha-complex. Three predominant proteins are isolated by alpha-3'UTR affinity chromatography. One of these binds directly to poly(C). This cytosolic poly(C)-binding protein is distinct from previously described nuclear poly(C)-binding heterogeneous nuclear RNPs and is necessary but not sufficient for alpha-complex formation. These data suggest that a messenger RNP complex formed by interaction of defined segments within the alpha-3'UTR with a limited number of cytosolic proteins, including a potentially novel poly(C)-binding protein, is of functional importance in establishing high-level stability of alpha-globin mRNA.     

Control of protein expression through mRNA stability in calcium signalling

Specific sequences (cis-acting elements) in the 3'-untranslated region (UTR) of RNA, together with stabilizing and destabilizing proteins (trans-acting factors), determine the mRNA stability, and consequently, the level of expression of several proteins. Such interactions were discovered initially for short-lived mRNAs encoding cytokines and early genes like c-jun and c-myc. However, they may also determine the fate of more stable mRNAs in a tissue and disease-dependent manner. The interactions between the cis-acting elements and the trans-acting factors may also be modulated by Ca(2+) either directly or via a control of the phosphorylation status of the trans-acting factors. We focus initially on the basic concepts in mRNA stability with the trans-acting factors AUF1 (destabilizing) and HuR (stabilizing). Sarco/endoplasmic reticulum Ca(2+) pumps, SERCA2a (cardiac and slow twitch muscles) and SERCA2b (most cells including smooth muscle cells), are pivotal in Ca(2+) mobilization during signal transduction. SERCA2a and SERCA2b proteins are encoded by relatively stable mRNAs that contain cis-acting stability determinants in their 3'-regions. We present several pathways where 3'-UTR mediated mRNA decay is key to Ca(2+) signalling: SERCA2a and beta-adrenergic receptors in heart failure, renin-angiotensin system, and parathyroid hormones. Other examples discussed include cytokines vascular endothelial growth factor, endothelin and endothelial nitric oxide synthase. Roles of Ca(2+) and Ca(2+)-binding proteins in mRNA stability are also discussed. We anticipate that these novel modes of control of protein expression will form an emerging area of research that may explore the central role of Ca(2+) in cell function during development and in disease.     

Epidermal growth factor increases the interaction between nucleolin and heterogeneous nuclear ribonucleoprotein K/poly(C) binding protein 1 complex to regulate the gastrin mRNA turnover

Gastrin, a gastrointestinal hormone responsible for gastric acid secretion, has been confirmed as a growth factor for gastrointestinal tract malignancies. High expression of gastrin mRNA was observed in pancreatic and colorectal cancer; however, the mechanism is unclear. Epidermal growth factor (EGF) was found to increase gastrin mRNA stability, indicating mRNA turnover regulation mechanism is involved in the control of gastrin mRNA expression. Using biotin-labeled RNA probe pull-down assay combined with mass spectrometry analysis, we identified the heterogeneous nuclear ribonucleoprotein K (hnRNP K) and poly(C) binding protein 1 (PCBP1) bound with the C-rich region in gastrin mRNA 3′ untranslated region. Nucleolin bound with the AGCCCU motif and interacted with hnRNP K were also demonstrated. Under EGF treatment, we observed the amount of nucleolin interacting with hnRNP K and gastrin mRNA increased. Using small interfering RNA technology to define their functional roles, we found hnRNP K, PCBP1, and nucleolin were all responsible for stabilizing gastrin mRNA. Moreover, nucleolin plays a crucial role in mediating the increased gastrin mRNA stability induced by EGF signaling. Besides, we also observed hnRNP K/PCBP1 complex bound with the C-rich region in the gastrin mRNA increased nucleolin binding with gastrin mRNA. Finally, a novel binding model was proposed.