Matrix GLA protein (MGP) is ubiquitously expressed with high accumulation in bone and cartilage, where it was found to associate with bone morphogenetic proteins (BMP) during protein purification. To test whether MGP affects BMP-induced differentiation, three sets of experiments were performed. First, pluripotent C3H10T1/2 cells transfected with human MPG (hMGP) or antisense to hMGP (AS-hMGP) were treated with BMP-2. In cells overexpressing hMGP, osteogenic and chondrogenic differentiation was inhibited indicating decreased BMP-2 activity. Conversely, in cells overexpressing AS-hMGP, BMP-2 activity was enhanced. Second, cells were prepared from homozygous and heterozygous MPG-deficient mice aortas. When treated with BMP-2, these cells underwent chondrogenic and osteogenic differentiation, respectively, whereas controls did not. Third, FLAG-tagged hMGP with the same biological effect as native hMGP inhibited BMP-induced differentiation, when exogenously added to culture media. Together, these results suggest that MGP modulates BMP activity. To test whether hMGP fragments would retain the effect of full-length hMGP, three subdomains were overexpressed in C3H10T1/2 cells. In cells expressing the mid-region, alone (amino acids (aa) 35-54) or in combination with the N terminus (aa 1-54) but not the C terminus (aa 35-84), osteogenic differentiation was enhanced and occurred even without added BMP-2. Thus, two subdomains had the opposite effect of full-length hMGP, possibly due to different expression levels or domain characteristics.Matrix GLA protein (MGP) 1 is a small ubiquitous matrix protein containing carboxyglutamic acid (GLA) (calculated mass of mature protein 10.4 kDa), initially isolated from bone and characterized by Price et al. (1). Results from other investigators suggest that MGP affects differentiation in developing cartilage and bone. Luo et al. (2) observed that MGP is expressed in early and late stages of chondrogenic differentiation but not in the intermediate stage. Yagami et al. (3) found that MGP had an effect on mineralization in chondrocytes that was dependent on cell stage; it affected mineralization in hypertrophic chondrocytes but not in proliferative chondrocytes. They also found that overexpression of MGP in developing limb buds delayed chondrocyte maturation and blocked endochondral ossification. In MGP null (MGPϪ/Ϫ) mice (4), the epiphyseal growth plates of bones showed inappropriate calcification in the layer of proliferating chondrocytes that failed to differentiate into hypertrophic chondrocytes. In addition, the mice unexpectedly developed severe vascular calcification, resulting from a replacement of the aortic medial layer by chondrocytelike cells, producing a typical cartilage matrix that progressively calcified.When Urist and colleagues (5) first discovered bone morphogenetic protein (BMP), they observed a tight association with MGP in vitro during protein purification requiring strong denaturants to break. Although complex formation was not shown in vivo or in situ, it was sug...

The carboxyl-terminal repeat domain (CTD) of RNA polymerase II is thought to help coordinate events during RNA metabolism. The mammalian CTD consists of 52 imperfectly repeated heptads followed by 10 additional residues at the C terminus. The CTD is required for cleavage and polyadenylation in vitro. We studied poly(A)-dependent termination in vivo using CTD truncation mutants. Poly(A)-dependent termination occurs in two steps, pause and release. We found that the CTD is required for release, the first 25 heptads being sufficient. Neither the final 10 amino acids nor the variant heptads of the second half of the CTD were required. No part of the CTD was required for poly(A)-dependent pausing-the poly(A) signal could communicate directly with the body of the polymerase. By removing the CTD, pausing could be observed without being obscured by release. Poly(A)-dependent pausing appeared to operate by slowing down the polymerase, such as by down-regulation of a positive elongation factor. Although the first 25 heptads supported undiminished poly(A)-dependent termination, they did not efficiently support events near the promoter involved in abortive elongation. However, the second half of the CTD, including the final 10 amino acids, was sufficient for these functions.

A useful method for studying the function of the mammalian RNA polymerase II takes advantage of the extreme sensitivity of its largest subunit, Rpb1, to a-amanitin. Mutations of interest are introduced into an a-amanitin-resistant version of Rpb1, which is then expressed ectopically in cells. The phenotypes of these cells are then examined after inhibiting the endogenous wild-type polymerase with a-amanitin. Here, we show that cells that are enabled to grow in a-amanitin by expression of an a-amanitinresistant Rpb1 exhibit changes in cell physiology that can lead to misleading experimental outcomes. The changes we have characterized include the accelerated degradation of some proteins, such as DSIF160, and the reduced rate of synthesis of others. In one series of experiments, we examined an a-amanitin-resistant construct, with a mutant C-terminal domain (CTD), that was unable to direct poly(A)-dependent transcription termination in cells growing in a-amanitin. The potential interpretation that the termination defect in this construct is due to the mutation in the CTD was rejected when the construct was found to be termination-competent in cells grown in the absence of a-amanitin. Instead, it appears that certain termination factors become limiting when the cells are grown in a-amanitin, presumably due to the a-amanitin-induced degradation we have characterized and/or to the inadequate transcription of certain genes by the a-amanitin-resistant Rpb1-containing polymerase.