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.     

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.     

Finding the right RNA: identification of cellular mRNA substrates for RNA-binding proteins.

Defects in RNA-binding proteins have been implicated in human genetic disorders. However, efforts in understanding the functions of these proteins have been hampered by the inability to obtain their mRNA substrates. To identify cognate cellular mRNAs associated with an RNA-binding protein, we devised a strategy termed isolation of specific nucleic acids associated with proteins (SNAAP). The SNAAP technique allows isolation and subsequent identification of these mRNAs. To assess the validity of this approach, we utilized cellular mRNA and protein from K562 cells and alphaCP1, a protein implicated in a-globin mRNA stability, as a model system. Immobilization of an RNA-binding protein with the glutathione-S-transferase (GST) domain enables isolation of mRNA within an mRNP context and the identity of the bound mRNAs is determined by the differential display assay. The specificity of protein-RNA interactions was considerably enhanced when the interactions were carried out in the presence of cellular extract rather than purified components. Two of the mRNAs specifically bound by alphaCP1 were mRNAs encoding the transmembrane receptor protein, TAPA-1, and the mitochondrial cytochrome c oxidase subunit II enzyme, coxII. A specific poly(C)-sensitive complex formed on the TAPA-1 and coxII 3' UTRs consistent with the binding of aCP1. Furthermore, direct binding of purified alphaCP proteins to these 3' UTRs was demonstrated and the binding sites determined. These results support the feasibility of the SNAAP technique and suggest a broad applicability for the approach in identifying mRNA targets for clinically relevant RNA-binding proteins that will provide insights into their possible functions.     

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.     

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.     

Structure and RNA binding of the third KH domain of poly(C)-binding protein 1

Poly(C)-binding proteins (CPs) are important regulators of mRNA stability and translational regulation. They recognize C-rich RNA through their triple KH (hn RNP K homology) domain structures and are thought to carry out their function though direct protection of mRNA sites as well as through interactions with other RNA-binding proteins. We report the crystallographically derived structure of the third domain of alphaCP1 to 2.1 A resolution. alphaCP1-KH3 assumes a classical type I KH domain fold with a triple-stranded beta-sheet held against a three-helix cluster in a betaalphaalphabetabetaalpha configuration. Its binding affinity to an RNA sequence from the 3'-untranslated region (3'-UTR) of androgen receptor mRNA was determined using surface plasmon resonance, giving a K(d) of 4.37 microM, which is indicative of intermediate binding. A model of alphaCP1-KH3 with poly(C)-RNA was generated by homology to a recently reported RNA-bound KH domain structure and suggests the molecular basis for oligonucleotide binding and poly(C)-RNA specificity.     

The gene for histone RNA hairpin binding protein is located on human chromosome 4 and encodes a novel type of RNA binding protein.

The hairpin structure at the 3' end of animal histone mRNAs controls histone RNA 3' processing, nucleocytoplasmic transport, translation and stability of histone mRNA. Functionally overlapping, if not identical, proteins binding to the histone RNA hairpin have been identified in nuclear and polysomal extracts. Our own results indicated that these hairpin binding proteins (HBPs) bind their target RNA as monomers and that the resulting ribonucleoprotein complexes are extremely stable. These features prompted us to select for HBP-encoding human cDNAs by RNA-mediated three-hybrid selection in Saccharomyces cerevesiae. Whole cell extract from one selected clone contained a Gal4 fusion protein that interacted with histone hairpin RNA in a sequence- and structure-specific manner similar to a fraction enriched for bovine HBP, indicating that the cDNA encoded HBP. DNA sequence analysis revealed that the coding sequence did not contain any known RNA binding motifs. The HBP gene is composed of eight exons covering 19.5 kb on the short arm of chromosome 4. Translation of the HBP open reading frame in vitro produced a 43 kDa protein with RNA binding specificity identical to murine or bovine HBP. In addition, recombinant HBP expressed in S. cerevisiae was functional in histone pre-mRNA processing, confirming that we have indeed identified the human HBP gene.     

Regulatory Role of the Conserved Stem-Loop Structure at the 5′ End of Collagen α1(I) mRNA

Three fibrillar collagen mRNAs, alpha1(I), alpha2(I), and alpha1(III), are coordinately upregulated in the activated hepatic stellate cell (hsc) in liver fibrosis. These three mRNAs contain sequences surrounding the start codon that can be folded into a stem-loop structure. We investigated the role of this stem-loop structure in expression of collagen alpha1(I) reporter mRNAs in hsc's and fibroblasts. The stem-loop dramatically decreases accumulation of mRNAs in quiescent hsc's and to a lesser extent in activated hsc's and fibroblasts. The stem-loop decreases mRNA stability in fibroblasts. In activated hsc's and fibroblasts, a protein complex binds to the stem-loop, and this binding requires the presence of a 7mG cap on the RNA. Placing the 3' untranslated region (UTR) of collagen alpha1(I) mRNA in a reporter mRNA containing this stem-loop further increases the steady-state level in activated hsc's. This 3' UTR binds alphaCP, a protein implicated in increasing stability of collagen alpha1(I) mRNA in activated hsc's (B. Stefanovic, C. Hellerbrand, M. Holcik, M. Briendl, S. A. Liebhaber, and D. A. Brenner, Mol. Cell. Biol. 17:5201-5209, 1997). A set of protein complexes assembles on the 7mG capped stem-loop RNA, and a 120-kDa protein is specifically cross-linked to this structure. Thus, collagen alpha1(I) mRNA is regulated by a complex interaction between the 5' stem-loop and the 3' UTR, which may optimize collagen production in activated hsc's.