Lineage- and Developmental Stage-Specific Patterns Of Auf-1 Isoform Expression Contribute To The Regulation Of Erythroid-Specific mRNAs

Post-transcriptional events that regulate the stabilities of one or more specific mRNAs are increasingly recognized for their importance to cell development and differentiation: genome-wide analyses attribute ∼50% of changes in gene expression to alterations in the stabilities of their encoded mRNAs. Processes that differentially regulate the half-lives of mRNAs are particularly important in definitive erythropoiesis, as they control the relative levels of actively translating transcripts in the interval when the nucleus is transcriptionally silenced and is ultimately extruded. We recently identified AUF-1 (AU-binding factor 1; hnRNP D) as a trans-acting factor that stabilizes human β-globin mRNA, and noted previous reports that this RNA-binding protein also regulates the half-lives of other mRNAs encoding factors that are critical to normal erythropoiesis, including p16^INK4a (CDKN2A), p21 (CDKN1A), cyclin D1 (CCND1), and thymidylate synthase. Based upon this evidence, we posited that AUF-1 plays a role in post-transcriptional erythropoiesis that is far broader than previously imagined. Our analyses of umbilical cord blood CD34+ cells validated this expectation, demonstrating the presence of two AUF-1 isoforms (p45^AUF1 and p40^AUF1) that differ by the inclusion/exclusion of exon 7-encoded sequence. Cells that are induced to erythroid differentiation initially express p45^AUF1 and p40^AUF1 at equal levels, but shift after several days to express only the p40^AUF1 isoform. In contrast, cells that are induced to granulocyte-monocyte differentiation initially express only p45^AUF1, and continue to express this single isoform through the remainder of development. These observations demonstrate lineage-specific AUF-1 isoform expression that is likely to be important to both erythroid and non-erythroid developmental programs. We subsequently investigated the functional consequences of the erythropoietic AUF-1 isoform switch by analyzing the abilities of p45^AUF1 and p40^AUF1 to stabilize informative erythroid-specific and -restricted mRNAs. Using an AUF-1 isoform-specific siRNA knockdown strategy, we demonstrated that a reduction in p40^AUF-1 (but not p45^AUF-1) effected a two-fold decrease in β-globin mRNA in K562 cells that we engineered to express this gene at high levels. p45^AUF1 and p40^AUF1 knockdown had a different effect on mRNAs encoding cyclin D1 and p21, both of which were unaffected by single-isoform depletion but--in contrast to β-globin mRNA--were increased by coordinate knockdown of both p45^AUF1 and p40^AUF1. While we expected that the erythropoietic AUF-1 isoform switch was likely due to alternative splicing of exon 7 from the AUF-1 pre-mRNA, we observed instead that the switch results from the transfer of p40^AUF1 from the nucleus to the cytoplasm during the developmental interval that immediately precedes nuclear extrusion. This observation implicates developmentally regulated trafficking of p40^AUF1 during erythropoiesis that is consistent with its demonstrated post-transcriptional mRNA-stabilizing effects. Finally, we assessed expression of HuR--a competitive antagonist of AUF-1 that has been implicated in stabilizing the mRNAs encoding FasL and cyclin E1 (CCNE1)--in erythroid-induced CD34+ cells, and observed that levels of HuR mRNA increase significantly during the developmental interval that coincides with the erythropoietic p45:p40^AUF1 isoform switch. Collectively, the lineage- and developmental-stage specific expression of p45^AUF1, p40^AUF1, and HuR--combined with their mRNA-specific binding properties--illustrate both the existence and the complexity of post-transcriptional regulatory programs that contribute to the normal development of both erythroid and nonerythroid cells. We are currently conducting RNA-seq analyses in erythroid-differentiated CD34+ cells to identify transcripts that differentially bind p45^AUF1, p40^AUF1, and HuR; as well as factor-specific shRNA knock-down analyses to characterize corresponding functional effects. In sum, our present work describes a novel mechanism through which erythroid progenitors maintain dynamic regulatory control during an interval when transcriptional processes are silenced.