The conserved 5'-untranslated leader of Spi-1 (PU.1) mRNA is highly structured and potently inhibits translation in vitro but not in vivo.

The transcription factor Spi-1 (PU.1) has a central role in regulating myeloid gene expression during hematopoietic development and its overexpression has been implicated in erythroleukemic transformation. Thus regulation of Spi-1 expression has broad significance for hematopoietic development. A comparison of human and murine cDNA sequences demonstrates that the 5'-untranslated region (5'-UTR) of Spi-1 mRNA is as highly conserved as the coding region (87% identical), suggesting that this sequence may be involved in regulating expression of this protein. The experiments presented in this manuscript provide evidence that the 5'-UTR of Spi-1 contains extensive secondary structure, including three stem-loops that precede the AUG codon. Analysis of the in vitro transcribed Spi-1 5'-UTR by partial nuclease digestion sensitivity is consistent with the existence of two of these stem-loops. The 5'-UTR decreased translation of Spi-1 transcripts in reticuloctye lysates 8- to 10-fold. A series of partial deletions of the 5'-UTR identified the sequence corresponding to the stem-loop most proximal to the initiating AUG codon as sufficient for inhibition of translation. However, the effect of the 5'-UTR on translation in vivo was negligible and resulted in only a slight reduction in the number of ribosomes that became associated with the mRNA. Further, this sequence had no affect on expression of luciferase. The disparity between in vivo and in vitro effects, coupled with the observation that endogenous Spi-1 mRNA is wholly associated with polysomes in MEL cells, suggests that additional cellular mechanisms contribute to regulation of Spi-1 expression in these cells or that conservation of these sequences serves a function that is independent of translation.

Inducers of erythroleukemic differentiation cause messenger RNAs that lack poly(A)-binding protein to accumulate in translationally inactive, salt-labile 80 S ribosomal complexes.

Translation has an established role in the regulation of cell growth. Posttranslational modification of translation initiation and elongation factors or regulation of mRNA polyadenylation represent common means of regulating translation in response to mitogenic or developmental signals. Induced differentiation of Friend virus-transformed erythroleukemia cells is accompanied by a rapid decrease in the translation rate of these cells. Although inducers do not alter initiation factor modifications, characterization of their effect on mRNA translation provides evidence that this is mediated by the poly(A)-binding protein (PABP). Inducer exposure results in an increase in the amount of mRNA that sediments at 80 S and a decrease in the amount in polysomes. Although these 80 S ribosomes have characteristics previously attributed to "vacant ribosomal couples," including lability in 500 mM KCl and an inability to incorporate amino acids into protein, we provide evidence that these 80 S complexes are not vacant but contain mRNA that is stably bound to the 40 S subunit, whereas the 60 S subunit is dissociated from the complex by high salt. The absence of eukaryotic initiation factor 2 from these complexes suggests that translation has proceeded through subunit joining. Immunoblotting demonstrates that the mRNAs in these 80 S ribosomal complexes do not contain bound PABP and that this protein is found to be almost exclusively associated with translating polysomes. These data suggest that the PABP plays a role in the accumulation of these 80 S ribosomal.mRNA complexes and may facilitate the formation of translationally active salt-stable ribosomes.

RNA polymerase II inhibitor, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) causes erythroleukemic differentiation and transcriptional activation of erythroid genes.

Friend virus-transformed murine erythroleukemia (MEL) cells are a useful system for studying the regulation of erythroid growth and differentiation. As a manifestation of the leukemic process, these erythroblasts are blocked in their ability to terminally differentiate. However, this block is reversible as a variety of different agents are capable of inducing differentiation of these malignant erythroblasts. The mechanisms by which these agents cause differentiation remains unknown. We report here that 5,6-dichlorobenzimidazole (DRB), which inhibits RNA polymerase II by causing premature termination of transcription, induces differentiation of these cells, including the transcriptional activation of erythroid genes. The effects of DRB on nonerythroid gene expression and on cell growth are substantially different than that of the commonly used inducer, dimethyl sulfoxide (DMSO). The shared ability of DMSO, DRB, and other unrelated agents to induce erythroid gene expression in MEL cells while having differing effects on nonerythroid gene expression and on cell growth suggests that expression of the terminally differentiated phenotype represents a common pathway that can be triggered by different mechanisms.

Expression of the transcription factor, Spi-1 (PU.1), in differentiating murine erythroleukemia cells Is regulated post-transcriptionally. Evidence for differential stability of transcription factor mRNAs following inducer exposure.

Increased expression of the transcription factor Spi-1 (PU.1) results from retroviral insertion in nearly all Friend spleen focus-forming virus-transformed murine erythroleukemia cell lines and exposure of these cells to Me2SO, induces their differentiation and decreases Spi-1 mRNA level by 4-5-fold. While these results suggest that alterations in Spi-1 expression have significant effects on erythroblast growth and differentiation, neither the cause nor the effect of the decrease in Spi-1 expression that follows Me2SO exposure has been established. The experiments described here demonstrate that the effect of inducers on Spi-1 expression is regulated post-transcriptionally. Nuclear run-off transcriptions demonstrated that Spi-1 transcription was not decreased following Me2SO exposure. Additionally, expression of a recombinant Spi-1 mRNA under transcriptional control of a constitutively active Rous sarcoma virus promoter was regulated identically to endogenous Spi-1 mRNA. The ability of Me2SO to destabilize Spi-1 mRNA was selective, as the stability of the erythroid transcription factors GATA-1 and NF-E2 were not similarly effected. The effect of Me2SO on the stability of Spi-1 mRNA provides a novel means of altering gene expression in these cells and is likely to have significance for the differentiation of these cells.

Identification of nuclear beta II protein kinase C as a mitotic lamin kinase.

Multisite phosphorylation of the nuclear lamins is thought to regulate the process of mitotic nuclear envelope breakdown in vivo. Here we investigate the involvement of two proposed human mitotic lamin kinases, beta II protein kinase C (PKC) and p34cdc2/cyclin B kinase, in human lamin B1 phosphorylation in vitro and in intact cells. We find that both kinases can phosphorylate purified soluble lamin B at similar rates. However, beta II PKC phosphorylates interphase nuclear envelope lamin B at more than 200 times the rate of human p34cdc2/cyclin B kinase. beta II PKC-mediated phosphorylation of lamin B is confined to two sites, Ser395 and Ser405, within the carboxyl-terminal domain, whereas human p34cdc2/cyclin B kinase phosphorylates a single site, Ser23, in the amino-terminal domain. A second potential p34cdc2/cyclin B kinase site within the carboxyl-terminal domain, Ser393, is not phosphorylated by human p34cdc2/cyclin B kinase. However, invertebrate p34cdc2/cyclin B kinase from sea star exhibits a different specificity, phosphorylating both amino- and carboxyl-terminal sites. Mitotic human lamin B from intact cells is phosphorylated predominantly in its carboxyl-terminal domain. Comparative tryptic phosphopeptide mapping demonstrates that the beta II PKC site, Ser405, is a prominent target of mitotic lamin B phosphorylation in vivo. beta II PKC translocates to the nucleus during the G2/M phase of cell cycle concomitant with phosphorylation of Ser405, indicating a physiologic role for nuclear beta II PKC activation in mitotic lamin B phosphorylation in vivo. The presence of phosphorylation sites within the carboxyl-terminal domain of mitotic lamin B which are not phosphorylated by either beta II PKC or p34cdc2/cyclin B kinase suggests the involvement of other lamin kinase(s) in G2/M phase lamin B phosphorylation.