Species-specific cis-Regulatory Elements in the 3′-Untranslated Region Direct Alternative Polyadenylation of Bone Morphogenetic Protein 2 mRNA

Abstract

BMP2 (bone morphogenetic protein 2) is a multifunctional member of the transforming growth factor-␤ family of growth factors. Disruption of BMP2 signaling results in developmental defects, cancers, and other diseases. BMP2 mRNAs are alternatively polyadenylated, resulting in mRNAs with distinct 3-untranslated regions. The longer mRNA contains additional putative binding sites for post-transcriptional regulatory factors, including micro-RNAs. We combined functional assays with computational analyses of emerging genome data to define siteand species-specific polyadenylation determinants. In all mouse and human cell lines tested, shorter mRNAs resulting from using the first polyadenylation signal (PA1) were more abundant than mRNAs from the second signal (PA2). However, the PA1/PA2 usage ratios were 2-3-fold higher in human than in mouse cells. Expression of human BMP2 constructs in mouse cells and mouse constructs in human cells showed that cis-regulatory elements direct species-specific 3 processing of BMP2 transcripts. A 72-nucleotide region downstream of PA2 in the mouse sequence contains two novel cis-acting elements previously hypothesized to regulate polyadenylation in a bioinformatics analysis. Mutations that humanized the mouse-specific elements lowered the affinity for cleavage stimulation factor CstF64 and significantly weakened the PA2 signal relative to the PA1 signal. Thus, we have experimentally defined for the first time cis-regulatory elements that control a species-specific difference in the 3-end processing of BMP2 and potentially of other genes.

CstF-64 is necessary for endoderm differentiation resulting in cardiomyocyte defects

“…This suggests that CstF-64 regulates the expression of paracrine factors inducing endoderm differentiation required for cardiomyocyte differentiation. Candidates include but are not limited to Afp, Ttr, Foxa2, and other downregulated genes (Figure 3B, C), and other CstF-64-regulated genes such as bone morphogenic protein 2 ( BMP2 , [43]), which has been previously been implicated in cardiac fate specification [44,45]. We propose that CstF-64 regulates the expression of several of these genes directly through polyadenylation, resulting in regulation of paracrine pathways in endoderm cells necessary to support differentiation of ESCs into cardiomyocytes.…”

Turning Bone Morphogenetic Protein 2 (BMP2) on and off in Mesenchymal Cells

“…Interestingly, genes located in gene deserts often have messenger RNAs (mRNAs) with 3'untranslated regions (UTRs) that are conserved between mammals and chick. As discussed below, Bmp2 is no exception with the entire 3'UTR conserved to chicken and a large fraction conserved to fishes [Abrams et al, ; Liu et al, ]. Such genes may require evolutionary preservation of both the DNA elements that influence transcription initiation and elongation, and the RNA elements involved in co‐ and post‐transcriptional processes.…”

“…Conservation of the two polyadenylation sites shown in Fig. D suggest that the need for alternative 3'UTRs has been retained for at least the ∼310 million years since the mammalian and avian lineages separated [Fritz et al, ; Liu et al, ]. In both human and murine cells, the upstream polyadenylation site closest to the stop codon (pA1) is used preferentially over the downstream site (pA2).…”

“…Although the human and mouse polyadenylation signals are very similar, the precise ratio of endogenous Bmp2 transcripts ending at pA1 or pA2 differs between mouse and human cells. A higher affinity of mouse cis ‐regulatory elements for the polyadenylation factor CstF‐64 accounted for this species‐specific difference, rather than species‐specific trans ‐regulatory factors [Liu et al, ]. Thus, both broadly conserved and species‐specific mechanisms regulate alternative polyadenylation of Bmp2 mRNAs [Liu et al, ].…”

Differences in fat and muscle mass associated with a functional human polymorphism in a post‐transcriptional BMP2 gene regulatory element

“…Both transcriptional and post-transcriptional aspects of BMP2 gene regulation have been studied in diverse cell types [Feng et al, 1997, 2003; Heller et al, 1999; Mundy et al, 1999; Ghosh-Choudhury et al, 2001; Zhou et al, 2003; Abrams et al, 2004; Fritz et al, 2004; Fukui et al, 2006; Xu and Rogers, 2007; Liu et al, 2008; Jiang et al, in revision]. A 265 nucleotide sequence in the 3′ untranslated region (UTR) of the BMP2 messenger RNA (mRNA) is conserved between mammals, birds, frogs, and fish [Abrams et al, 2004; Fritz et al, 2004].…”