Splicing of mRNA precursors (pre-mRNAs) comprises a series of ATP-dependent steps, the first of which is the stable binding of U2 snRNP at the pre-mRNA branchpoint. The basis of ATP use for the interaction between U2 snRNP and the branchpoint is unclear, and, in particular, none of the known mammalian factors required for this step have the sequence characteristics of proteins that hydrolyze ATP. Entry of U2 snRNP into the spliceosome is initiated by interaction of the essential splicing factor U2AF 65 with the pre-mRNA polypyrimidine tract. In this report we identify a new region of U2AF 6s required for function, and use this information to clone a human 56-kD U2AF 6s associated protein (UAP56). We show that UAP56 is an essential splicing factor, which is recruited to the pre-mRNA dependent on U2AF 6s, and is required for the U2 snRNP-branchpoint interaction. The sequence of UAP56 indicates it is a member of the DEAD box family of RNA-dependent ATPases, which mediate ATP hydrolysis during several steps of yeast pre-mRNA splicing. Our results reveal a new function of U2AF6S: to position a DEAD box protein required for U2 snRNP binding at the pre-mRNA branchpoint region.[Key Words: U2 snRNP; branchpoint; U2AF65; DEAD box protein; pre-mRNA splicing]Received December 30, 1996; revised version accepted June 3, 1997.Nuclear pre-mRNA splicing is catalyzed by a dynamic ribonucleoprotein, the spliceosome, whose formation involves the stepwise assembly of four (U1, U2, U5, and U4/U6) small ribonucleoproteins (snRNPs), as well as many non-snRNP protein splicing factors (for review, see Green 1991;Bennett et al. 1992;Rymond and Rosbash 1992;Moore et al. 1993; Madhani and Guthrie 1994; Nilsen t994;Kramer 1996). Several of the steps involved in spliceosome assembly require ATP hydrolysis, the first of which is the stable binding of U2 snRNP at the pre-mRNA branchpoint. ing member of this family is eIF-4A, a eukaryotic translation initiation factor that is required for mRNA binding to ribosomes. EIF-4A exhibits single-stranded RNAdependent ATPase activity, and in combination with a second initiation factor, eIF-4B, also possesses duplex RNA helicase activity. An attractive idea is that yeast DEAD box proteins are required to unwind particular RNA hybrids that are formed during spliceosome assembly and splicing. To date, however, none of the DEAD box proteins implicated in splicing have demonstrable RNA helicase activity, although several have been shown to possess RNA-dependent ATPase activity (Schwer and Guthrie 1991;Kim and Lin 1993;Xu et al. 1996).Biochemical experiments have shown that stable binding of mammalian U2 snRNP at the pre-mRNA branchpoint requires several auxiliary factors, including U2AF, SF1, SF3a, SF3b, and U1 snRNP (for review, see Moore et al. 1993;Hodges and Beggs 1994;Reed 1996). U2 snRNPbinding requires ATP hydrolysis and it is therefore puzzling that none of the forementioned mammalian splicing factors have the sequence motifs of proteins that use ATP. In yeast, the DEAD box protein PRP5 is an essenti...
Several recent reports claim the generation of insulinproducing cells from embryonic stem cells via the differentiation of progenitors that express nestin. Here, we investigate further the properties of these insulincontaining cells. We find that although differentiated cells contain immunoreactive insulin, they do not contain proinsulin-derived C-peptide. Furthermore, we find variable insulin release from these cells upon glucose addition, but C-peptide release is never detected. In addition, many of the insulin-immunoreactive cells are undergoing apoptosis or necrosis. We further show that cells cultured in the presence of a phosphoinositide 3-kinase inhibitor, which previously was reported to facilitate the differentiation of insulin ؉ cells, are not C-peptide immunoreactive but take up fluorescein isothiocyanate-labeled insulin from the culture medium. Together, these data suggest that nestin ؉ progenitor cells give rise to a population of cells that contain insulin, not as a result of biosynthesis but from the uptake of exogenous insulin. We conclude that C-peptide biosynthesis and secretion should be demonstrated to claim insulin production from embryonic stem cell progeny. Diabetes
Synthesis and structure-activity relationships of tricyclic alpha-ethoxy-phenylpropionic acid derivatives guided by in vitro PPARalpha and PPARgamma transactivation data and computer modeling led to the identification of the novel carbazole analogue, 3q, with dual PPARalpha (EC(50) = 0.36 microM) and PPARgamma (EC(50) = 0.17 microM) activity in vitro. Ten days treatment of db/db mice with 3q improved the insulin sensitivity, as measured by OGTT, better than that seen with both pioglitazone and rosiglitazone treatment, suggesting in vivo PPARgamma activity. Likewise, 3q lowered plasma triglycerides and cholesterol in high cholesterol fed rats after 4 days treatment, indicating in vivo PPARalpha activity. Investigations of the pharmacokinetics of selected compounds suggested that extended drug exposure improved the in vivo activity of in vitro active compounds.
Upper-and lower-body fat depots exhibit opposing associations with obesity-related metabolic disease. We defined the relationship between DEXA-quantified fat depots and diabetes/cardiovascular risk factors in a healthy population-based cohort (n = 3,399). Gynoid fat mass correlated negatively with insulin resistance after total fat mass adjustment, whereas the opposite was seen for abdominal fat. Paired transcriptomic analysis of gluteal subcutaneous adipose tissue (GSAT) and abdominal subcutaneous adipose tissue (ASAT) was performed across the BMI spectrum (n = 49; 21.4-45.5 kg/m 2 ). In both depots, energy-generating metabolic genes were negatively associated and inflammatory genes were positively associated with obesity. However, associations were significantly weaker in GSAT. At the systemic level, arteriovenous release of the proinflammatory cytokine interleukin-6 (n = 34) was lower from GSAT than ASAT. Isolated preadipocytes retained a depotspecific transcriptional "memory" of embryonic developmental genes and exhibited differential promoter DNA methylation of selected genes (HOTAIR, TBX5) between GSAT and ASAT. Short hairpin RNA-mediated silencing identified TBX5 as a regulator of preadipocyte proliferation and adipogenic differentiation in ASAT. In conclusion, intrinsic differences in the expression of developmental genes in regional adipocytes provide a mechanistic basis for diversity in adipose tissue (AT) function. The less inflammatory nature of lower-body AT offers insight into the opposing metabolic disease risk associations between upper-and lower-body obesity.Lower-body fat accumulation, as opposed to central obesity, is inversely associated with metabolic risk factors, including hyperinsulinemia, dyslipidemia, and hypertension (1), and is also associated with a reduced incidence of type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD) (2,3). Conversely, loss of lower-body fat during weight reduction relates to adverse changes in blood lipid and glucose profiles as well as blood pressure (4).
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