AUF-1 and YB-1 independently regulate ß-globin mRNA in developing erythroid cells through interactions with poly(A)-binding protein.

The normal expression of ß-globin protein in mature erythrocytes is critically dependent on post-transcriptional events in erythroid progenitors that ensure the high stability of ß-globin mRNA. Previous work has revealed that these regulatory processes require AUF-1 and YB-1, two RNA-binding proteins that assemble an mRNP ß-complex on the ß-globin 3'UTR. Here, we demonstrate that the ß-complex organizes during the erythropoietic interval when both ß-globin mRNA and protein accumulate rapidly, implicating the importance of this regulatory mRNP to normal erythroid differentiation. Subsequent functional analyses link ß-complex assembly to the half-life of ß-globin mRNA in vivo, providing a mechanistic basis for this regulatory activity. AUF-1 and YB-1 appear to serve a redundant post-transcriptional function, as both ß-complex assembly and ß-globin mRNA levels are reduced by coordinate depletion of the two factors, and can be restored by independent rescue with either factor alone. Additional studies demonstrate that the ß-complex assembles more efficiently on polyadenylated transcripts, implicating a model in which the ß-complex enhances the binding of PABPC1 to the poly(A) tail, inhibiting mRNA deadenylation and consequently effecting the high half-life of ß-globin transcripts in erythroid progenitors. These data specify a post-transcriptional mechanism through which AUF1 and YB1 contribute to the normal development of erythropoietic cells, as well as to non-hematopoietic tissues in which AUF1- and YB1-based regulatory mRNPs have been observed to assemble on heterologous mRNAs.

Structural determinants of human ζ-globin mRNA stability.

BACKGROUND: The normal accumulation of adult α and ß globins in definitive erythrocytes is critically dependent upon processes that ensure that the cognate mRNAs are maintained at high levels in transcriptionally silent, but translationally active progenitor cells. The impact of these post-transcriptional regulatory events on the expression of embryonic ζ globin is not known, as its encoding mRNA is not normally transcribed during adult erythropoiesis. Recently, though, ζ globin has been recognized as a potential therapeutic for α thalassemia and sickle-cell disease, raising practical questions about constitutive post-transcriptional processes that may enhance, or possibly prohibit, the expression of exogenous or derepresssed endogenous ζ-globin genes in definitive erythroid progenitors. METHODS: The present study assesses mRNA half-life in intact cells using a pulse-chase approach; identifies cis-acting determinants of ζ-globin mRNA stability using a saturation mutagenesis strategy; establishes critical 3'UTR secondary structures using an in vitro enzymatic mapping method; and identifies trans-acting effector factors using an affinity chromatographical procedure. RESULTS: We specify a tetranucleotide 3'UTR motif that is required for the high-level accumulation of ζ-globin transcripts in cultured cells, and formally demonstrate that it prolongs their cytoplasmic half-lives. Surprisingly, the ζ-globin mRNA stability motif does not function autonomously, predicting an activity that is subject to structural constraints that we subsequently specify. Additional studies demonstrate that the ζ-globin mRNA stability motif is targeted by AUF1, a ubiquitous RNA-binding protein that enhances the half-life of adult ß-globin mRNA, suggesting commonalities in post-transcriptional processes that regulate globin transcripts at all stages of mammalian development. CONCLUSIONS: These data demonstrate a mechanism for ζ-globin mRNA stability that exists in definitive erythropoiesis and is available for therapeutic manipulation in α thalassemia and sickle-cell disease.

The RNA binding protein RBM38 (RNPC1) regulates splicing during late erythroid differentiation.

Alternative pre-mRNA splicing is a prevalent mechanism in mammals that promotes proteomic diversity, including expression of cell-type specific protein isoforms. We characterized a role for RBM38 (RNPC1) in regulation of alternative splicing during late erythroid differentiation. We used an Affymetrix human exon junction (HJAY) splicing microarray to identify a panel of RBM38-regulated alternatively spliced transcripts. Using microarray databases, we noted high RBM38 expression levels in CD71(+) erythroid cells and thus chose to examine RBM38 expression during erythroid differentiation of human hematopoietic stem cells, detecting enhanced RBM38 expression during late erythroid differentiation. In differentiated erythroid cells, we validated a subset of RBM38-regulated splicing events and determined that RBM38 regulates activation of Protein 4.1R (EPB41) exon 16 during late erythroid differentiation. Using Epb41 minigenes, Rbm38 was found to be a robust activator of exon 16 splicing. To further address the mechanism of RBM38-regulated alternative splicing, a novel mammalian protein expression system, followed by SELEX-Seq, was used to identify a GU-rich RBM38 binding motif. Lastly, using a tethering assay, we determined that RBM38 can directly activate splicing when recruited to a downstream intron. Together, our data support the role of RBM38 in regulating alternative splicing during erythroid differentiation.

AUF-1 and YB-1 are critical determinants of ß-globin mRNA expression in erythroid cells.

The normal accumulation of ß-globin protein in terminally differentiating erythroid cells is critically dependent on the high stability of its encoding mRNA. The molecular basis for this property, though, is incompletely understood. Factors that regulate ß-globin mRNA within the nucleus of early erythroid progenitors are unlikely to account for the constitutively high half-life of ß-globin mRNA in the cytoplasm of their anucleate erythroid progeny. We conducted in vitro protein-RNA binding analyses that identified a cytoplasm-restricted ß-globin messenger ribonucleoprotein (mRNP) complex in both cultured K562 cells and erythroid-differentiated human CD34(+) cells. This novel mRNP targets a specific guanine-rich pentanucleotide in a region of the ß-globin 3'untranslated region that has recently been implicated as a determinant of ß-globin mRNA stability. Subsequent affinity-enrichment analyses identified AUF-1 and YB-1, 2 cytoplasmic proteins with well-established roles in RNA biology, as trans-acting components of the mRNP. Factor-depletion studies conducted in vivo demonstrated the importance of the mRNP to normal steady-state levels of ß-globin mRNA in erythroid precursors. These data define a previously unrecognized mechanism for the posttranscriptional regulation of ß-globin mRNA during normal erythropoiesis, providing new therapeutic targets for disorders of ß-globin gene expression.

Two major 5'-untranslated regions for type XVII collagen mRNA.

BACKGROUND: Type XVII collagen is an important structural component of keratinocyte hemidesmosomes and its functional loss in genetic or autoimmune disease results in blistering of the skin. In neoplastic tissue aberrant expression is seen dependent on the stage of the tumor. While the sequence of the type XVII collagen encoding gene -COL17A1 - is now completely elucidated, the sequence of the 5'-untranslated region (UTR) of the mRNA is still unknown. Since UTRs can modulate translation efficiency, the determination of the UTR sequence is indispensable for understanding the regulation of translation of type XVII collagen mRNA. OBJECTIVE: To resolve the sequence of the 5'UTR of type XVII collagen mRNA and to analyse the promoter region for transcription motifs. METHODS: 5' Rapid amplification of cDNA ends (RACE) followed by sequence analysis and ribonuclease protection assays (RPA) were performed. RESULTS: RACE and sequence analysis revealed the presence of six different 5'UTRs for the type XVII collagen mRNA. The start points of these six transcripts differ but no alternative exons are used. The longest 5'UTR starts 220 nucleotides before the open reading frame, whereas the shortest UTR is only 89 nucleotides in length. RPA confirmed the RACE results and furthermore demonstrated that the 5'UTRs with lengths of 102 and 220 nucleotides are the two major transcripts. Transcription motif analysis of the 5' region of the COL17A gene demonstrated several binding sites for transcription factors including the Sp1 and activating protein-1 (AP-1) families. CONCLUSION: Type XVII collagen mRNA is alternatively transcribed, which may result in complex regulation of type XVII collagen.