We have used an in vitro system that reproduces in vivo aspects of mRNA turnover to elucidate mechanisms of deadenylation. DAN, the major enzyme responsible for poly(A) tail shortening in vitro, specifically interacts with the 5' cap structure of RNA substrates, and this interaction is greatly stimulated by a poly(A) tail. Several observations suggest that cap-DAN interactions are functionally important for the networking between regulated mRNA stability and translation. First, uncapped RNA substrates are inefficiently deadenylated. Second, a stem-loop structure in the 5' UTR dramatically reduces deadenylation by interfering with cap-DAN interactions. Third, the addition of cap binding protein eIF4E inhibits deadenylation in vitro. These data provide insights into the early steps of substrate recognition that target an mRNA for degradation.

Bone morphogenetic protein (BMP) orthologs from diverse species like flies and humans are functionally interchangeable and play key roles in fundamental processes such as dorso-ventral axis formation in metazoans. Because both transcriptional and post-transcriptional mechanisms play central roles in modulating developmental protein levels, we have analyzed the 3-untranslated region (3UTR) of the Bmp 2 gene. This 3UTR is unusually long and is alternatively polyadenylated. Mouse, human, and dog mRNAs are 83-87% identical within this region. A 265-nucleotide sequence, conserved between mammals, birds, frogs, and fish, is present in Bmp2 but not Bmp4. The ability of AmphiBMP2/4, a chordate ortholog to Bmp2 and Bmp4, to align with this sequence suggests that its function may have been lost in Bmp4. Activation of reporter genes by the conserved region acts by a post-transcriptional mechanism. Mouse, human, chick, and zebrafish Bmp2 synthetic RNAs decay rapidly in extracts from cells not expressing Bmp2. In contrast, these RNAs are relatively stable in extracts from Bmp2-expressing cells. Thus, Bmp2 RNA half-lives in vitro correlate with natural Bmp2 mRNA levels. The fact that non-murine RNAs interact appropriately with the mouse decay machinery suggests that the function of these cis-regulatory regions has been conserved for 450 million years since the fish and tetrapod lineages diverged. Overall, our results suggest that the Bmp2 3UTR contains essential regulatory elements that act post-transcriptionally.

The bone morphogenetic protein (BMP)2 gene has been genetically linked to osteoporosis and osteoarthritis. We have shown that the 3'-untranslated regions (UTR) of BMP2 genes from mammals to fishes are extraordinarily conserved. This indicates that the BMP2 3'-UTR is under stringent selective pressure. We present evidence that the conserved region is a strong posttranscriptional regulator of BMP2 expression. Polymorphisms in cis-regulatory elements have been proven to influence susceptibility to a growing number of diseases. A common single nucleotide polymorphism (SNP) disrupts a putative posttranscriptional regulatory motif, an AU-rich element, within the BMP2 3'-UTR. The affinity of specific proteins for the rs15705 SNP sequence differs from their affinity for the normal human sequence. More importantly, the in vitro decay rate of RNAs with the SNP is higher than that of RNAs with the normal sequence. Such changes in mRNA:protein interactions may influence the posttranscriptional mechanisms that control BMP2 gene expression. The consequent alterations in BMP2 protein levels may influence the development or physiology of bone or other BMP2-influenced tissues.

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 maybe 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.

To understand the regulation of cap-dependent translation initiation mediated by specific 5 untranslated region (UTR) RNA-protein interactions in mammalian cells, we have studied the selective translation of influenza virus mRNAs. Previous work has shown that the host cell mRNA binding protein guanine-rich sequence factor 1 (GRSF-1) bound specifically to conserved viral 5 UTR sequences and stimulated translation of viral 5 UTR-driven mRNAs in vitro. In the present study, we have characterized the functional domains of GRSF-1 and mapped the RNA binding activity of GRSF-1 to RRM 2 (amino acids 194 to 275) with aminoterminal deletion glutathione S-transferase (GST)-GRSF-1 proteins. When these mutants were assayed for functional activity in vitro, deletion of an Ala-rich region (⌬[2-94]) appeared to diminish translational stimulation, while deletion of the Ala-rich region in addition to RRM 1 (⌬[2-194]) resulted in a 4-fold increase in translational activation over wild-type GRSF-1 (an overall 20-fold increase in activity). We have also mapped the GRSF-1 RNA binding site on influenza virus NP and NS1 5 UTRs, which was determined to be the sequence AGGGU. With polysome fractionation and cDNA microarray analysis, we have identified cellular and viral mRNAs containing putative GRSF-1 binding sites that were transcriptionally up-regulated and selectively recruited to polyribosomes following influenza virus infection. Taken together, these studies demonstrate that RRM 2 is critical for GRSF-1 RNA binding and translational activity. Further, our data suggest GRSF-1 functions by selectively recruiting cellular and viral mRNAs containing 5 UTR GRSF-1 binding sites to polyribosomes, which is mediated through interactions with cellular proteins.The ability of cells to respond to extracellular stimuli and intracellular cues, including mitogenic signals, is directly linked to the regulation of mRNA translation initiation. The control of initiation can be regulated by the specific interaction of RNA binding proteins and initiation factors (eIFs) with cisacting elements contained in both the 5Ј and 3Ј untranslated regions (UTRs) of mature mRNAs (reviewed in reference 37). Accordingly, deregulation of protein synthesis is a key mechanism in both malignant transformation and viral replication. Influenza virus infection results in the selective translation of viral mRNAs, while host cell protein synthesis is markedly attenuated (reviewed in reference 38). The subversion of the host cell protein synthetic machinery to produce high levels of influenza virus proteins, which are required for infection and replication, is dependent on conserved sequences present in the 5Ј UTRs of the influenza virus mRNAs (13).Translation initiation relies upon the interactions of transacting factors with both ribosomal RNAs and cis-acting determinants in the mRNA. Influenza virus protein synthesis is a cap-dependent process mediated by highly conserved sequences contained in the 5Ј UTRs of the viral mRNAs (15). As