The multifunctional protein YB-1 has previously been shown to be the only protein of the cytoplasmic extract of HEK293 cells, which is able to specifically interact with imperfect RNA hairpins containing motifs that are often found in exosomal (e) RNAs. In addition, it has been revealed that similar hairpins formed by degenerate consensus sequences corresponding to three eRNA-specific motifs are responsible for the cooperative binding of YB-1 to RNA in vitro. Here, using the photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation method applied to HEK293 cells producing FLAG-labeled YB-1, we identified mRNAs cross-linked to YB-1 in vivo and then carried out a search for the aforementioned sequences in the regions of the YB-1 cross-linking sites. It turned out that many of the mRNAs found cross-linked to YB-1 encode proteins associated with various regulatory processes, including responses to stress. More than half of all cross-linked mRNAs contained degenerate consensus sequences, which were preferably located in 3\'-untranslated regions (UTRs), where most of the YB-1 cross-linking sites appeared, although not close to these sequences. Furthermore, YB-1 was mainly cross-linked to those mRNAs with degenerate consensus sequences, which could be classified as packaged because their translation levels were low compared to cellular levels. This suggests that the cooperative binding of YB-1 to mRNAs through the above sequences probably triggers the well-known multimerization of YB-l, leading to the packaging of these mRNAs. Thus, our findings indicate a previously unknown link between the degenerate consensus sequences present in the 3\'-UTRs of many cytoplasmic mRNAs and YB-1-mediated translational silencing.

Acetaldehyde has been shown to increase collagen production in cultured rat myofibroblastlike cells and to activate the mouse alpha 2(I) collagen promoter in transfected NIH 3T3 cells. Nuclear factor I (NF-I), a CCAAT binding transcription factor, is known to bind and activate the alpha 1(I) and alpha 2(I) collagen genes. Activation of the alpha 2(I) collagen promoter was not observed when the NF-I binding site of the promoter was deleted. In this study, we determined if acetaldehyde influences the binding of NF-I to the alpha 2(I) collagen promoter. Nuclear proteins extracted from NIH 3T3 cells, or myofibroblastlike cells, 36 hours after the addition of acetaldehyde (200 mumol/L) in serum-free media showed increased binding to the consensus sequence of the NF-I binding site by DNase I protection analysis and by electrophoretic mobility shift assay (EMSA) as compared with control nuclear extracts that were not exposed to acetaldehyde. Furthermore, nuclear proteins extracted from myofibroblastlike cells that had been previously exposed to acetaldehyde had a marked increase in NF-I protein, as shown by Western blot with NF-I antibodies. Antisera to NF-I resulted in a slow migrating DNA-protein-antibody complex (supershift) on EMSA. However, the NF-I antibody did not supershift all the DNA-protein complexes, and the supershift band was not increased with nuclear proteins from acetaldehyde-treated cells despite the increased binding of these nuclear protein preparations to the NF-I oligo. Therefore, nuclear proteins, in addition to NF-I, bind to the NF-I consensus sequence and may have their binding altered by acetaldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)

Three subtypes of alpha 2-adrenergic receptors (alpha 2A, alpha 2B and alpha 2C) have been described that differ in their primary sequence and tissue-specific expression and are encoded by three distinct genes. Previous work has shown that the human alpha 2A-adrenergic receptor gene promoter consists of a TATA-box (TATAAA), palindromic sequence (CCCACGTGGG) and GC-box (GGGGCGG) motif. Sequence analysis of the putative promoter region of the rat alpha 2A-adrenergic receptor gene showed that these promoter regions are conserved in their sequence and relative location. We analysed the transcriptional activity of these regions using RINm5F, a rat insulinoma cell line that expresses the endogenous alpha 2A-adrenergic receptor gene. These results showed that the region from -484 to -92 has a negative effect on transcription, as deletion of this region in alpha 2A-adrenergic receptor gene-chloramphenicol acetyltransferase reporter constructs increased reporter gene activity. This region included the GC-box sequence which is a consensus binding site for the nuclear factor SP1, which is a positive activator of transcription. Gel-mobility-shift assays and supershift assays with an antibody that recognizes SP1 showed binding of the SP1 nuclear factor as well as other nuclear factors to this GC-box region. Additional nuclear factors bind to the downstream palindromic region. We suggest that positive- and negative-acting nuclear factors contribute to the activity of the alpha 2-adrenergic receptor promoter.