A p50-like Y-box protein with a putative translational role becomes associated with pre-mRNA concomitant with transcription

Abstract

In vertebrates free messenger ribonucleoprotein (RNP) particles and polysomes contain an abundant Y-box protein called p50 (YB-1), which regulates translation, presumably by affecting the packaging of the RNA. Here, we have identified a p50-like protein in the dipteran Chironomus tentans and studied its relation with the biogenesis of mRNA in larval salivary glands. The salivary gland cells contain polytene chromosomes with the transcriptionally active regions blown up as puffs. A few giant puffs, called Balbiani rings (BRs), generate a transcription product, a large RNP particle,which can be visualised (with the electron microscope) during its assembly on the gene and during its transport to and through the nuclear pores. The p50-like protein studied, designated Ct-p40/50 (or p40/50 for short), was shown to contain a central cold-shock domain, an alanine- and proline-rich N-terminal domain, and a C-terminal domain with alternating acidic and basic regions, an organisation that is characteristic of p50 (YB-1). The p40/50 protein appears in two isoforms, p40 and p50, which contain 264 and 317 amino acids, respectively. The two isoforms share the first 258 amino acids and thus differ in amino-acid sequence only in the region close to the C-terminus. When a polyclonal antibody was raised against p40/50, western blot analysis and immunocytology showed that p40/50 is not only abundant in the cytoplasm but is also present in the nucleus. Immunolabelling of isolated polytene chromosomes showed that p40/50 appears in transcriptionally active regions, including the BRs. Using immunoelectron microscopy we revealed that p40/50 is added along the nascent transcripts and is also present in the released BR RNP particles in the nucleoplasm. Finally, by UV crosslinking in vivo we showed that p40/50 is bound to both nuclear and cytoplasmic poly(A) RNA. We conclude that p40/50 is being added cotranscriptionally along the growing BR pre-mRNA, is released with the processed mRNA into the nucleoplasm and probably remains associated with the mRNA both during nucleocytoplasmic transport and protein synthesis. Given that the p40/p50 protein, presumably with a role in translation, is loaded onto the primary transcript concomitant with transcription, an early programming of the cytoplasmic fate of mRNA is indicated.

The Phosphoinositide-Dependent Kinase-1 Inhibitor 2-Amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide (OSU-03012) Prevents Y-Box Binding Protein-1 from Inducing Epidermal Growth Factor Receptor

“…For example, YB-1 has been characterized previously for its ability to stabilize interleukin-2 mRNA by binding to AU-rich elements (ARE) on the 3Ј-untranslated region of interleukin-2 (Chen et al, 2000). This was a rather novel finding because YB-1 is more commonly believed to regulate translation through cap-dependent binding (Soop et al, 2003). Whereas the ability of YB-1 to regulate AREs is a relatively new concept, it is reminiscent of the way in which the widely characterized translation factor HuR stabilizes the mRNA of proto-oncogenes such as urokinase plasminogen activator (Tran et al, 2003).…”

An Acidic Protein, YBAP1, Mediates the Release of YB-1 from mRNA and Relieves the Translational Repression Activity of YB-1

“…Immunostaining with the same antibodies detected FRGY2 or mRNP3 in lampbrush chromosomes of oocytes (64). Moreover, immunoelectron microscopy revealed that, in the salivary gland cells of the dipteran Chironomus tentans, Y-box proteins became associated with nascent transcripts and also were detected in nuclear pre-mRNPs (65). These studies suggested that Y-box proteins associate with pre-mRNA cotranscriptionally and accompany mRNA into the cytoplasm.…”

Targeting the Y/CCAAT box in cancer: YB-1 (YBX1) or NF-Y?

“…One finding consistent with this interpretation is that chromatin association of YB-1 is apparently lost upon treatment with ribonucleases, which destroys pre-mRNAs. 138 It is well established that coactivators, which do not bind to DNA directly, can be crosslinked efficiently to DNA, resulting in peaks in ChIPs and ChIP-Seq. It is also known that promoter structures and specific TF combinations have an impact on the loading and composition of the mRNA splicing apparatus.…”