The hnRNPs (heterogeneous nuclear ribonucleoproteins) F and H2 share a similar protein structure. Both have been implicated as regulating polyadenylation, but hnRNP H2 had a positive effect, whereas hnRNP F acted negatively. We therefore carried out side-by-side comparisons of their RNA-binding and in vivo actions. The binding of the CstF2 (64 kDa cleavage stimulatory factor) to SV40 (simian virus 40) late pre-mRNA substrates containing a downstream GRS (guanine-rich sequence) was reduced by hnRNP F, but not by hnRNP H2, in a UV-cross-linking assay. Point mutations of the 14-nt GRS influenced the binding of purified hnRNP F or H2 in parallel. Co-operative binding of the individual proteins to RNA was lost with mutations of the GRS in the G1−5 or G12−14 regions; both regions seem to be necessary for optimal interactions. Using a reporter green fluorescent protein assay with the GRS inserted downstream of the poly(A) (polyadenine) signal, expression in vivo was diminished by a mutant G1−5 sequence which decreased binding of both hnRNPs (SAA20) and was enhanced by a 12–14-nt mutant that showed enhanced hnRNP F or H2 binding (SAA10). Using small interfering RNA, down-regulation of hnRNP H2 levels diminished reporter expression, confirming that hnRNP H2 confers a positive influence; in contrast, decreasing hnRNP F levels had a negligible influence on reporter expression with the intact GRS. A pronounced diminution in reporter expression was seen with the SAA20 mutant for both. Thus the relative levels of hnRNP F and H2 in cells, as well as the target sequences in the downstream GRS on pre-mRNA, influence gene expression.
The head assembly pathway of bacteriophage HK97 shares many features with head assembly pathways determined for other dsDNA phages, and it also provides examples of novel variations on the basic theme. We describe aspects of two specific steps in the assembly pathway, the covalent cross-linking among the assembled head protein subunits and the cleavage of those subunits that takes place earlier in the pathway. Comparisons of head assembly pathways among different phages, as well as comparisons of the organization of the genes that specify those pathways, suggest the range of different solutions phages have found to common assembly problems and give insight into the evolutionary histories of these assembly processes.
Abstract Little is known about the transcriptional regulators that control the proliferation of multipotent bone marrow progenitors. Understanding the mechanisms that restrict proliferation is of significant interest since the loss of cell cycle integrity can be associated with hematopoietic exhaustion, bone marrow failure, or even oncogenic transformation. Herein, we show that multipotent LSKs (lineage−Scahighc-kit+) from E47-deficient mice exhibit a striking hyperproliferation associated with a loss of cell cycle quiescence and increased susceptibility to in vivo challenge with a mitotoxic drug. Total LSKs contain long-term self-renewing hematopoietic stem cells and downstream multipotential progenitors (MPPs) that possess very limited or no self-renewal ability. Within total LSKs, we found specific developmental and functional deficits in the MPP subset. E47 knockout mice have grossly normal numbers of self-renewing hematopoietic stem cells but a 50–70% reduction in nonrenewing MPPs and downstream lineage-restricted populations. The residual MPPs in E47 knockout mice fail to fully up-regulate flk2 or initiate V(D)J recombination, hallmarks of normal lymphoid lineage progression. Consistent with the loss of normal cell cycle restraints, we show that E47-deficient LSKs have a 50% decrease in p21, a cell cycle inhibitor and known regulator of LSK proliferation. Moreover, enforced expression studies identify p21 as an E47 target gene in primary bone marrow LSKs. Thus, E47 appears to regulate the developmental and functional integrity of early hematopoietic subsets in part through effects on p21-mediated cell cycle quiescence.
Previous studies on the regulation of polyadenylation of the immunoglobulin (Ig) heavy-chain pre-mRNA argued for trans-acting modifiers of the cleavage-polyadenylation reaction operating differentially during B-cell developmental stages. Using four complementary approaches, we demonstrate that a change in the level of hnRNP F is an important determinant in the regulated use of alternative polyadenylation sites between memory and plasma stage B cells. First, by Western analyses of cellular proteins, the ratio of hnRNP F to H or H' was found to be higher in memory B cells than in plasma cells. In memory B cells the activity of CstF-64 binding to pre-mRNA, but not its amount, was reduced. Second, examination of the complexes formed on input pre-mRNA in nuclear extracts revealed large assemblages containing hnRNP H, H', and F but deficient in CstF-64 in memory B-cell extracts but not in plasma cells. Formation of these large complexes is dependent on the region downstream of the AAUAAA in pre-mRNA, suggesting that CstF-64 and the hnRNPs compete for a similar region. Third, using a recombinant protein we showed that hnRNP F could bind to the region downstream of a poly(A) site, block CstF-64 association with RNA, and inhibit the cleavage reaction. Fourth, overexpression of recombinant hnRNP F in plasma cells resulted in a decrease in the endogenous Ig heavy-chain mRNA secretory form-to-membrane ratio. These results demonstrate that mammalian hnRNP F can act as a negative regulator in the pre-mRNA cleavage reaction and that increased expression of F in memory B cells contributes to the suppression of the Ig heavy-chain secretory poly(A) site.
The amount of the 64-kDa subunit of polyadenylation/cleavage stimulatory factor (CstF-64) increases 5-fold during the G 0 to S phase transition and concomitant proliferation induced by serum in 3T6 fibroblasts. Higher levels of CstF-64 result in an increase in CstF trimer. The rise in CstF-64 occurs at a time when the amount of poly(A)-containing RNA rose at least 5–8 fold in the cytoplasm. Primary human splenic B cells, resting in G 0 , show a similar 5-fold increase in CstF-64 when cultured under conditions inducing proliferation (CD40 ligand exposure). Therefore, the increase in CstF-64 is associated with the G 0 to S phase transition. As B cell development progresses, RNA processing changes occur at the Ig heavy chain locus resulting in a switch from the membrane- to the upstream secretory-specific poly(A) site. Treating resting B cells with agents triggering this switch in Ig mRNA production along with proliferation (CD40 ligand plus lymphokines or Stapylococcus aureus protein A) induces no further increase in CstF-64 above that seen for proliferation alone. The rise in CstF-64 is therefore insufficient to induce secretion. After stimulation of a continuously growing B cell line with lymphokines, a switch to Ig μ secretory mRNA and protein occurs but without a change in the CstF-64 level. Therefore, an increase in CstF-64 levels is not necessary to mediate the differentiation-induced switch to secreted forms of Ig-μ heavy chain. Because augmentation of CstF-64 levels is neither necessary nor sufficient for Ig secretory mRNA production, we conclude that other lymphokine-induced factors play a role.
The erbAα gene encodes two α-thyroid hormone receptor isoforms, TRα1 and TRα2, which arise from alternatively processed mRNAs, erbAα1 (α1) and erb α2 (α2). The splicing and alternative polyadenylation patterns of these mRNAs resemble that of mRNAs encoding different forms of immunoglobulin heavy chains, which are regulated at the level of alternative processing during B cell differentiation. This study examines the levels of erbAα mRNA in eight B cell lines representing four stages of differentiation in order to determine whether regulation of the alternatively processed α1 and α2 mRNAs parallels the processing of immunoglobulin heavy chain mRNAs. Results show that the pattern of α1 and α2 mRNA expression is clearly different from that observed for immunoglobulin heavy chain mRNAs. B cell lines display characteristic ratios of α1/α2 mRNA at distinct stages of differentiation. Furthermore, expression of an overlapping gene, Rev-ErbAα (RevErb), was found to correlate strongly with an increase in the ratio of α1/α2 mRNA. These results suggest that alternative processing of erbAα mRNAs is regulated by a mechanism which is distinct from that regulating immunoglobulin mRNA. The correlation between RevErb and erbAα mRNA is consistent with negative regulation of α2 via antisense interactions with the complementary RevErb mRNA.
Abstract B cells produce Ig H chain (IgH) mRNA and protein, primarily of the membrane-bound specific form. Plasma cells produce 20- to 50-fold higher amounts of IgH mRNA, most processed to the secretory specific form; this shift is mediated by substantial changes in RNA processing but only a small increase in IgH transcription rate. We investigated RNA polymerase II (RNAP-II) loading and phosphorylation of its C-terminal domain (CTD) on the IgG2a H chain gene, comparing two mouse cell lines representing B (A20) and plasma cells (AxJ) that express the identical H chain gene whose RNA is processed in different ways. Using chromatin immunoprecipitation and real-time PCR, we detected increased RNAP-II and Ser-2 and Ser-5 phosphorylation of RNAP-II CTD close to the IgH promoter in plasma cells. We detected increased association of several 3′ end-processing factors, ELL2 and PC4, at the 5′ end of the IgH gene in AxJ as compared with A20 cells. Polymerase progress and factor associations were inhibited by 5,6-dichlorobenzimidazole riboside, a drug that interferes with the addition of the Ser-2 to the CTD of RNAP-II. Taken together, these data indicate a role for CTD phosphorylation and polyadenylation/ELL2/PC4 factor loading on the polymerase in the choice of the secretory poly(A) site for the IgH gene.
