David T. Fritz

An in Vitro Assay to Study Regulated mRNA Stability

The examination of posttranscriptional regulation of mRNA in mammalian cells is critical to discovering the role that mRNA plays in the initiation and maintenance of cellular processes. The complexity of the system defies a holistic approach and, therefore, we have devised an in vitro mRNA turnover assay that enables us to elucidate the factors involved in mRNA deadenylation and degradation. Our system, using an S100 HeLa extract and in vitro transcribed RNAs, accurately mimics the end products of mRNA turnover, which have been previously described using in vivo studies and, in addition, allows for the detailed study of factors that may play a role in regulated deadenylation and degradation. Another important aspect of our system is the ease with which it can be manipulated. We can provide any synthetic RNA molecule to the assay to test for specific sequence activity. Furthermore, the results are clear and accurately interpretable. We have demonstrated that our in vitro system accurately deadenylates and decays a capped and polyadenylated RNA molecule in a processive manner without nonspecific nuclease activity. Finally, we have demonstrated regulated instability in vitro using the AU-rich elements (AREs) from tumor necrosis factor-α (TNF-α) and granulocyte macrophage colony stimulating factor (GM-CSF) embedded within the RNA molecule. The presence of the AREs increased the deadenylation and the decay rates seen in vivo. We feel that this system can be expanded and adapted to examine a variety of mRNA regulatory events in mammalian cells.

Differences in Fat and Muscle Mass Associated With a Functional Human Polymorphism in a Post-Transcriptional BMP2 Gene Regulatory Element

A classic morphogen, bone morphogenetic protein 2 (BMP2) regulates the differentiation of pluripotent mesenchymal cells. High BMP2 levels promote osteogenesis or chondrogenesis and low levels promote adipogenesis. BMP2 inhibits myogenesis. Thus, BMP2 synthesis is tightly controlled. Several hundred nucleotides within the 3′ untranslated regions of BMP2 genes are conserved from mammals to fishes indicating that the region is under stringent selective pressure. Our analyses indicate that this region controls BMP2 synthesis by post‐transcriptional mechanisms. A common A to C single nucleotide polymorphism (SNP) in the BMP2 gene (rs15705, +A1123C) disrupts a putative post‐transcriptional regulatory motif within the human ultra‐conserved sequence. In vitro studies indicate that RNAs bearing the A or C alleles have different protein binding characteristics in extracts from mesenchymal cells. Reporter genes with the C allele of the ultra‐conserved sequence were differentially expressed in mesenchymal cells. Finally, we analyzed MRI data from the upper arm of 517 healthy individuals aged 18–41 years. Individuals with the C/C genotype were associated with lower baseline subcutaneous fat volumes (P = 0.0030) and an increased gain in skeletal muscle volume (P = 0.0060) following resistance training in a cohort of young males. The rs15705 SNP explained 2–4% of inter‐individual variability in the measured parameters. The rs15705 variant is one of the first genetic markers that may be exploited to facilitate early diagnosis, treatment, and/or prevention of diseases associated with poor fitness. Furthermore, understanding the mechanisms by which regulatory polymorphisms influence BMP2 synthesis will reveal novel pharmaceutical targets for these disabling conditions. J. Cell. Biochem. 107: 1073–1082, 2009.

Messenger RNA decay in mammalian cells: the exonuclease perspective.

The majority of messenger RNA (mRNA) decay in mammalian cells appears to be the work of a series of RNA exoribonucleases. A set of multiple poly(A)-specific deadenylases has been identified, some, if not most, of which are likely to play a role in the key first step of mRNA turnover—the regulated shortening of the poly (A) tail. After deadenylation, the transcript likely gets degraded by either a 5′-to-3′ or a 3′-to-5′ exonucleolytic pathway. Interestigly, multiple exonucleases have been identified for both of these pathways that appear to form multicomponent complexes with diverse roles in cellular biology. Therefore these enzymes appear not only to be important components of the mRNA turnover machinery, but also may function in a networked fashion in the post-transcriptional control of gene expression.

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

BMP2 (bone morphogenetic protein 2) is a multifunctional member of the transforming growth factor-β 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 site- and 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.

An autonomous BMP2 regulatory element in mesenchymal cells

BMP2 is a morphogen that controls mesenchymal cell differentiation and behavior. For example, BMP2 concentration controls the differentiation of mesenchymal precursors into myocytes, adipocytes, chondrocytes, and osteoblasts. Sequences within the 3′untranslated region (UTR) of the Bmp2 mRNA mediate a post‐transcriptional block of protein synthesis. Interaction of cell and developmental stage‐specific trans‐regulatory factors with the 3′UTR is a nimble and versatile mechanism for modulating this potent morphogen in different cell types. We show here, that an ultra‐conserved sequence in the 3′UTR functions independently of promoter, coding region, and 3′UTR context in primary and immortalized tissue culture cells and in transgenic mice. Our findings indicate that the ultra‐conserved sequence is an autonomously functioning post‐transcriptional element that may be used to modulate the level of BMP2 and other proteins while retaining tissue specific regulatory elements. J. Cell. Biochem. 112: 666–674, 2011.

A conserved post‐transcriptional BMP2 switch in lung cells

An ultra‐conserved sequence in the bone morphogenetic protein 2 (BMP2) 3′ untranslated region (UTR) markedly represses BMP2 expression in non‐transformed lung cells. In contrast, the ultra‐conserved sequence stimulates BMP2 expression in transformed lung cells. The ultra‐conserved sequence functions as a post‐transcriptional cis‐regulatory switch. A common single‐nucleotide polymorphism (SNP, rs15705, +A1123C), which has been shown to influence human morphology, disrupts a conserved element within the ultra‐conserved sequence and altered reporter gene activity in non‐transformed lung cells. This polymorphism changed the affinity of the BMP2 RNA for several proteins including nucleolin, which has an increased affinity for the C allele. Elevated BMP2 synthesis is associated with increased malignancy in mouse models of lung cancer and poor lung cancer patient prognosis. Understanding the cis‐ and trans‐regulatory factors that control BMP2 synthesis is relevant to the initiation or progression of pathologies associated with abnormal BMP2 levels. J. Cell. Biochem. 110: 509–521, 2010.

An In Vivo Map of Bone Morphogenetic Protein 2 Post-transcriptional Repression in the Heart

The Bmp2 3′untranslated region (UTR) sequence bears a sequence conserved between mammals and fishes that can post‐transcriptionally activate or repress protein synthesis. We developed a map of embryonic cells in the mouse where this potent Bmp2 regulatory sequence functions by using a lacZ reporter transgene with a 3′UTR bearing two loxP sites flanking the ultra‐conserved sequence. Cre‐recombinase‐mediated deletion of the ultra‐conserved sequence caused strong ectopic expression in proepicardium, epicardium and epicardium‐derived cells (EPDC) and in tissues with known epicardial contributions (coronary vessels and valves). Transient transfections of reporters in the epicardial/mesothelial cell (EMC) line confirmed this repression. Ectopic expression of the recombined transgene also occurred in the aorta, outlet septum, posterior cardiac plexus, cardiac and extracardiac nerves and neural ganglia. Bmp2 is dynamically regulated in the developing heart. 3′UTR‐mediated mechanisms that restrain BMP2 synthesis may be relevant to congenital heart and vasculature malformations and to adult diseases involving aberrant BMP2 synthesis. genesis 49:841–850, 2011.

Repressive BMP2 gene regulatory elements near the BMP2 promoter.

The level of bone morphogenetic protein 2 (BMP2) profoundly influences essential cell behaviors such as proliferation, differentiation, apoptosis, and migration. The spatial and temporal pattern of BMP2 synthesis, particular in diverse embryonic cells, is highly varied and dynamic. We have identified GC-rich sequences within the BMP2 promoter region that strongly repress gene expression. These elements block the activity of a highly conserved, osteoblast enhancer in response to FGF2 treatment. Both positive and negative gene regulatory elements control BMP2 synthesis. Detecting and mapping the repressive motifs is essential because they impede the identification of developmentally regulated enhancers necessary for normal BMP2 patterns and concentration.

Competing Repressive Factors Control Bone Morphogenetic Protein 2 (BMP2) in Mesenchymal Cells

The amount, timing, and location of bone morphogenetic protein 2 (BMP2) synthesis influences the differentiation of pluripotent mesenchymal cells in embryos and adults. The BMP2 3′untranslated region (3′UTR) contains a highly conserved AU‐rich element (ARE) embedded in a sequence that commonly represses gene expression in mesenchymal cells. Computational analyses indicate that this site also may bind several microRNAs (miRNAs). Although miRNAs frequently target AU‐rich regions, this ARE is unusual because the miRNAs directly span the ARE. We began to characterize the factors that may regulate Bmp2 expression via this complex site. The activating protein HuR (Hu antigen R, ELAVL1, HGNC:3312) directly binds this ARE and can activate gene expression. An miRNA was demonstrated to reverse HuR‐mediated activation. Mutational and RNA‐interference evidence also supports an AUF1 (AU‐factor‐1, HNRNPD, HGNC:5036) contribution to the observed repressive activity of the 3′UTR in mesenchymal cells. A limited number of studies describe how miRNAs interact with ARE‐binding proteins that bind adjacent sites. This study is among the first to describe protein/miRNA interactions at the same site. J. Cell. Biochem. 117: 439–447, 2016.

Using emerging genome data to identify conserved bone morphogenetic protein (Bmp) 2 gene expression mechanisms

Comparing genomes from diverse species has revealed surprisingly high conservation between proteins of vastly different organisms, e.g. 75% of pufferfish proteins have human counterparts. Thus subtle variation in the expression of master developmental control genes, like Bone Morphogenetic Protein (Bmp)2, is central to species differentiation. Understanding the evolution of the complex transcriptional and post-transcriptional mechanisms required to precisely regulate such genes requires novel, interdisciplinary approaches. We describe here our efforts towards defining Bmp2 gene expression determinants by combining functional assays with computational analyses of emerging genome data. Our results suggest that both primary sequence and more subtle parameters such as nucleotide composition control Bmp2 expression at the post-transcriptional level.