Sequences involved in the control of adenovirus L1 alternative RNA splicing.

During an adenovirus infection the expression of mRNA from late region L1 is temporally regulated at the level of alternative 3' splice site selection to produce two major mRNAs encoding the 52,55K and IIIa polypeptides. The proximal 3' splice site (52,55K) is used at all times of the infectious cycle whereas the distal site (IIIa) is used exclusively late after infection. We show that a single A branch nucleotide located at position -23 is used in 52,55K splicing and that two A's located at positions -21 and -22 are used in IIIa splicing. Both 3' splice sites were active in vitro in nuclear extracts prepared from uninfected HeLa cells. However, the efficiency of IIIa splicing was only approximately 10% of 52,55K splicing. This difference in splice site activity correlated with a reduced affinity of the IIIa, relative to the 52,55K, 3' splice site for polypyrimidine tract binding proteins. Reversing the order of 3' splice sites on a tandem pre-mRNA resulted in an almost exclusive IIIa splicing indicating that the order of 3' splice site presentation is important for the outcome of alternative L1 splicing. Based on our results we suggest a cis competition model where the two 3' splice sites compete for a common RNA splicing factor(s). This may represent an important mechanism by which L1 alternative splicing is regulated

Heterogeneous nuclear ribonucleoprotein (hnRNP) binding to hormone response elements: A cause of vitamin D resistance

In previous studies, we have shown that steroid hormone resistance in New World primates occurs in the absence of abnormal expression of cognate nuclear receptors. Rather, these animals have elevated levels of heterogeneous nuclear ribonucleoproteins (hnRNPs) that act as hormone response element-binding proteins and attenuate target gene transactivation. Here we present evidence for a similar mechanism in humans via a patient with resistance to the active form of vitamin D [1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))] who presented with normal vitamin D receptor (VDR) expression. Initial cotransfection studies showed that the cells of the patient suppressed basal and hormone-induced transactivation by wild-type VDR. Electrophoretic mobility-shift assays and Western/Southwestern blot analyses indicated that this suppressive effect was due to overexpression of a nuclear protein that specifically interacts with a DNA response element known to bind retinoid X receptor–VDR heterodimers. Ab blocking in electrophoretic mobility-shift assays indicated that this dominant-negative acting protein was in the hnRNPA family of nucleic acid-binding proteins. Further studies have shown that several members of this family, most notably hnRNPA1, were able to suppress basal and 1,25(OH)(2)D(3)-induced luciferase activity. We therefore propose that this case of vitamin D resistance in a human subject is similar to that previously described for New World primates in which abnormal expression of a hormone response element-binding protein can cause target cell resistance to 1,25(OH)(2)D(3). That this protein is a member of the hnRNP family capable of interacting with double-stranded DNA highlights a potentially important new component of the complex machinery required for steroid hormone signal transduction

Inhibition of ERK1/2 and Activation of Liver X Receptor Synergistically Induce Macrophage ABCA1 Expression and Cholesterol Efflux*

ATP-binding cassette transporter A1 (ABCA1), a molecule mediating free cholesterol efflux from peripheral tissues to apoAI and high density lipoprotein (HDL), inhibits the formation of lipid-laden macrophage/foam cells and the development of atherosclerosis. ERK1/2 are important signaling molecules regulating cellular growth and differentiation. The ERK1/2 signaling pathway is implicated in cardiac development and hypertrophy. However, the role of ERK1/2 in the development of atherosclerosis, particularly in macrophage cholesterol homeostasis, is unknown. In this study, we investigated the effects of ERK1/2 activity on macrophage ABCA1 expression and cholesterol efflux. Compared with a minor effect by inhibition of other kinases, inhibition of ERK1/2 significantly increased macrophage cholesterol efflux to apoAI and HDL. In contrast, activation of ERK1/2 reduced macrophage cholesterol efflux and ABCA1 expression. The increased cholesterol efflux by ERK1/2 inhibitors was associated with the increased ABCA1 levels and the binding of apoAI to cells. The increased ABCA1 by ERK1/2 inhibitors was due to increased ABCA1 mRNA and protein stability. The induction of ABCA1 expression and cholesterol efflux by ERK1/2 inhibitors was concentration-dependent. The mechanism study indicated that activation of liver X receptor (LXR) had little effect on ERK1/2 expression and activation. ERK1/2 inhibitors had no effect on macrophage LXRα/β expression, whereas they did not influence the activation or the inhibition of the ABCA1 promoter by LXR or sterol regulatory element-binding protein (SREBP). However, inhibition of ERK1/2 and activation of LXR synergistically induced macrophage cholesterol efflux and ABCA1 expression. Our data suggest that ERK1/2 activity can play an important role in macrophage cholesterol trafficking