Christine Milcarek

hnRNP F influences binding of a 64-kilodalton subunit of cleavage stimulation factor to mRNA precursors in mouse B cells.

ABSTRACT Previous studies on the regulation of polyadenylation of the immunoglobulin (Ig) heavy-chain pre-mRNA argued fortrans-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.

A comparison of apparent mRNA half-life using kinetic labeling techniques vs decay following administration of transcriptional inhibitors.

Several different techniques were used to determine the apparent half-lives of immunoglobulin gamma 2b heavy chain and kappa light chain mRNAs in mouse myeloma 4T001 and a mutant derived from 4T001, i.e., mutant I17. The mutant I17 Ig heavy chain mRNA lacks CH1 and has fused CH2 and CH3 domains resulting in a truncated protein. By all four techniques the Ig heavy chain mRNA from mutant I17 displays a half-life that is approximately 70% the half-life of Ig mRNA in 4T001 cells. However, the absolute values of apparent half-life varied by greater than twofold for both lines among several of the techniques employed. The half-life of Ig gamma 2b mRNA in 4T001 cells was found to be 6.4 h by measuring decay following administration of the adenosine analog DRB to block new mRNA synthesis and 5.7 hr by measuring accumulation in an approach to steady-state labeling protocol. In contrast, the observed Ig mRNA half-lives determined by measuring decay following administration of actinomycin D to block new mRNA synthesis, or in a pulse-chase analysis were 2.9 and 3.8 h, respectively. The apparent half-life for Ig kappa light chain mRNA was the same in the 4T001 and I17 lines using any one technique but the value varied depending on the technique from a high value of 5.9 h following DRB to a low value of 2.4 h with actinomycin decay. Approach to steady-state is theoretically the most accurate method to measure mRNA half-life when that value is less than the doubling time of the cells. Pulse-chase analyses are accurate for measuring mRNA half-life when that value is longer than the effective chase period. Measuring preformed message decay following administration of drugs to block new mRNA synthesis is adaptable over a range of half-lives, but the cells must be shown to retain correct RNA metabolism over the time frame of the experiment. Determining a correct half-life for a particular mRNA may not be feasible using only one method and may, in fact, require several different approaches until a consensus value emerges.

Regulation of poly(A) site use during mouse B-cell development involves a change in the binding of a general polyadenylation factor in a B-cell stage-specific manner.

During the development of mouse B cells there is a regulated shift from the production of membrane to the secretion-specific forms of immunoglobulin (Ig) mRNA, which predominate in the late-stage or plasma B cells. By DNA transfection experiments we have previously shown that there is an increase in polyadenylation efficiency accompanying the shift to secretion-specific forms of Ig mRNA (C. R. Lassman, S. Matis, B. L. Hall, D. L. Toppmeyer, and C. Milcarek, J. Immunol. 148:1251-1260, 1992). When we look in vitro at nuclear extracts prepared from early or memory versus late-stage or plasma B cells, we see cell stage-specific differences in the proteins which are UV cross-linked to the input RNAs. We have characterized one of these proteins as the 64-kDa subunit of the general polyadenylation factor cleavage-stimulatory factor (CstF) by immunoprecipitation of UV-cross-linked material. The amount of 64-kDa protein and its mobility on two-dimensional gels do not vary between the B-cell stages. However, the activity of binding of the protein to both Ig and non-Ig substrates increases four- to eightfold in the late-stage or plasma cell lines relative to the binding seen in the early or memory B-cell lines. Therefore, the binding activity of a constitutive factor required for polyadenylation is altered in a B-cell-specific fashion. The increased binding of the 64-kDa protein may lead to a generalized increase in polyadenylation efficiency in plasma cells versus early or memory B cells which may be responsible for the increased use of the secretory poly(A) site seen in vivo.

Transcription elongation factor ELL2 directs immunoglobulin secretion in plasma cells by stimulating altered RNA processing

Immunoglobulin secretion is modulated by competition between the use of a weak promoter-proximal poly(A) site and a nonconsensus splice site in the final secretory-specific exon of the heavy chain pre-mRNA. The RNA polymerase II transcription elongation factor ELL2, which is induced in plasma cells, enhanced both polyadenylation and exon skipping with the gene encoding the immunoglobulin heavy-chain complex (Igh) and reporter constructs. Lowering ELL2 expression by transfection of heterogenous ribonucleoprotein F (hnRNP F) or small interfering RNA resulted in lower abundance of secretory-specific forms of immunoglobulin heavy-chain mRNA. ELL2 and the polyadenylation factor CstF-64 tracked together with RNA polymerase II across the Igh μ- and γ-gene segments; the association of both factors was blocked by ELL2-specific small interfering RNA. Thus, loading of ELL2 and CstF-64 on RNA polymerase II was linked, caused enhanced use of the proximal poly(A) site and was necessary for processing of immunoglobulin heavy-chain mRNA.

Elevated levels of the 64-kDa cleavage stimulatory factor (CstF-64) in lipopolysaccharide-stimulated macrophages influence gene expression and induce alternative poly(A) site selection.

Lipopolysaccharide (LPS) activation of murine RAW 264.7 macrophages influences the expression of multiple genes through transcriptional and post-transcriptional mechanisms. We observed a 5-fold increase in CstF-64 expression following LPS treatment of RAW macrophages. The increase in CstF-64 protein was specific in that several other factors involved in 3′-end processing were not affected by LPS stimulation. Activation of RAW macrophages with LPS caused an increase in proximal poly(A) site selection within a reporter mini-gene containing two linked poly(A) sites that occurred concomitant with the increase in CstF-64 expression. Furthermore, forced overexpression of the CstF-64 protein also induced alternative poly(A) site selection on the reporter minigene. Microarray analysis performed on CstF-64 overexpressing RAW macrophages revealed that elevated levels of CstF-64 altered the expression of 51 genes, 14 of which showed similar changes in gene expression with LPS stimulation. Sequence analysis of the 3′-untranslated regions of these 51 genes revealed that over 45% possess multiple putative poly(A) sites. Two of these 51 genes demonstrated alternative polyadenylation under both LPS-stimulating and CstF-64-overexpressing conditions. We concluded that the physiologically increased levels of CstF-64 observed in LPS-stimulated RAW macrophages contribute to the changes in expression and alternative polyadenylation of a number of genes, thus identifying another level of gene regulation that occurs in macrophages activated with LPS.

E47 controls the developmental integrity and cell cycle quiescence of multipotential hematopoietic progenitors.

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.

C/EBPβ regulates transcription factors critical for proliferation and survival of multiple myeloma cells

CCAAT/enhancer-binding protein beta (C/EBPbeta), also known as nuclear factor-interleukin-6 (NF-IL6), is a transcription factor that plays an important role in the regulation of growth and differentiation of myeloid and lymphoid cells. Mice deficient in C/EBPbeta show impaired generation of B lymphocytes. We show that C/EBPbeta regulates transcription factors critical for proliferation and survival in multiple myeloma. Multiple myeloma cell lines and primary multiple myeloma cells strongly expressed C/EBPbeta, whereas normal B cells and plasma cells had little or no detectable levels of C/EBPbeta. Silencing of C/EBPbeta led to down-regulation of transcription factors such as IRF4, XBP1, and BLIMP1 accompanied by a strong inhibition of proliferation. Further, silencing of C/EBPbeta led to a complete down-regulation of antiapoptotic B-cell lymphoma 2 (BCL2) expression. In chromatin immunoprecipitation assays, C/EBPbeta directly bound to the promoter region of IRF4, BLIMP1, and BCL2. Our data indicate that C/EBPbeta is involved in the regulatory network of transcription factors that are critical for plasma cell differentiation and survival. Targeting C/EBPbeta may provide a novel therapeutic strategy in the treatment of multiple myeloma.

The hnRNPs F and H2 bind to similar sequences to influence gene expression.

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.

Sequences near the 3' secretion-specific polyadenylation site influence levels of secretion-specific and membrane-specific IgG2b mRNA in myeloma cells.

The expressed immunoglobulin gamma 2b (IgG2b) heavy-chain gene of 4T001 was cloned into the shuttle vector pSV2-gpt and transfected into myeloma J558L and lymphoma A20.2J. Northern blots indicated that the transfected gamma 2b gene was processed in a manner similar to the endogenous heavy chain in both lymphoma and myeloma cells. To identify sequences important for immunoglobulin mRNA processing, we constructed deletions around the secretion-specific polyadenylation site and introduced the deleted genes into J558L cells. The BAL deletion lacked 670 base pairs of intervening sequence between secreted and membrane regions; the Kpn deletion lacked 830 base pairs in this region. J558L cells transfected with either the entire gamma 2b gene or the delta BAL vector produced predominantly secretion-specific gamma 2b mRNA and protein. J558L cells transfected with the delta Kpn vector produced approximately equimolar amounts of secretion-specific and membrane-specific gamma 2b mRNA. Both 55,000-dalton secreted and 62,000-dalton putative surface IgG2b proteins were detected in the delta Kpn transfectants. We conclude that sequences absent in the Kpn deletion but present in the BAL deletion exert an important role in the production of secretion-specific mRNA. The Kpn deletion removes the normal site of cleavage and poly(A) addition, and it is possible that it is the absence of this site which changes the processing pattern. Alternatively, it is possible that sequences absent in the Kpn deletion but present in the BAL deletion function in regulating the production of predominantly secretion-specific mRNA in myeloma cells. The possible role of a highly conserved sequence found in this region is discussed.

From B cell to plasma cell: regulation of V(D)J recombination and antibody secretion.

B cell development culminates in the formation of plasma cells, potent secretors of the immunoglobulins (Ig), proteins crucial for the health of the organism. Two distinctive and crucial steps are required during B cell differentiation. First, variable gene segments encoding the antigen-binding region of Ig undergo directed rearrangement through a process known as V(D)J recombination. Second, alternative processing of the Ig heavy chain mRNA transcript enables plasma cells to secrete high levels of Ig protein. This review focuses on the molecular mechanisms that control V(D)J recombination in B cell progenitors and alternative RNA processing in plasma cells.