Yoshio Takagaki

The Polyadenylation Factor CstF-64 Regulates Alternative Processing of IgM Heavy Chain Pre-mRNA during B Cell Differentiation

The switch from membrane-bound to secreted-form IgM that occurs during differentiation of B lymphocytes has long been known to involve regulated processing of the heavy chain pre-mRNA. Here, we show that accumulation of one subunit of an essential polyadenylation factor (CstF-64) is specifically repressed in mouse primary B cells and that overexpression of CstF-64 is sufficient to switch heavy chain expression from membrane-bound (microm) to secreted form (micros). We further show that CstF-64 is limiting for formation of intact CstF, that CstF has a higher affinity for the microm poly(A) site than for the micros site, and that the microm site is stronger in a reconstituted in vitro processing reaction. Our results indicate that CstF-64 plays a key role in regulating IgM heavy chain expression during B cell differentiation.

Complex protein interactions within the human polyadenylation machinery identify a novel component.

ABSTRACT Polyadenylation of mRNA precursors is a two-step reaction requiring multiple protein factors. Cleavage stimulation factor (CstF) is a heterotrimer necessary for the first step, endonucleolytic cleavage, and it plays an important role in determining the efficiency of polyadenylation. Although a considerable amount is known about the RNA binding properties of CstF, the protein-protein interactions required for its assembly and function are poorly understood. We therefore first identified regions of the CstF subunits, CstF-77, CstF-64, and CstF-50, required for interaction with each other. Unexpectedly, small regions of two of the subunits participate in multiple interactions. In CstF-77, a proline-rich domain is necessary not only for binding both other subunits but also for self-association, an interaction consistent with genetic studies in Drosophila. In CstF-64, a small region, highly conserved in metazoa, is responsible for interactions with two proteins, CstF-77 and symplekin, a nuclear protein of previously unknown function. Intriguingly, symplekin has significant similarity to a yeast protein, PTA1, that is a component of the yeast polyadenylation machinery. We show that multiple factors, including CstF, cleavage-polyadenylation specificity factor, and symplekin, can be isolated from cells as part of a large complex. These and other data suggest that symplekin may function in assembly of the polyadenylation machinery.

Levels of Polyadenylation Factor CstF-64 Control IgM Heavy Chain mRNA Accumulation and Other Events Associated with B Cell Differentiation

Cleavage stimulation factor (CstF) is one of the multiple factors required for mRNA polyadenylation. The concentration of one CstF subunit (CstF-64) increases during activation of B cells, and this is sufficient to switch IgM heavy chain mRNA expression from membrane-bound form to secreted form. To extend this observation, we disrupted the endogenous CstF-64 gene in the B cell line DT40 and replaced it with a regulatable transgene. Strikingly, a 10-fold decrease in CstF-64 concentration did not markedly affect cell growth but specifically and dramatically reduced accumulation of IgM heavy chain mRNA. Further reduction caused reversible cell cycle arrest in G0/G1 phase, while depletion resulted in apoptotic cell death. Our results indicate that CstF-64 plays unexpected roles in regulating gene expression and cell growth in B cells.

Multiple forms of poly(A) polymerases purified from HeLa cells function in specific mRNA 3'-end formation.

Poly(A) polymerases (PAPs) from HeLa cell cytoplasmic and nuclear fractions were extensively purified by using a combination of fast protein liquid chromatography and standard chromatographic methods. Several forms of the enzyme were identified, two from the nuclear fraction (NE PAPs I and II) and one from the cytoplasmic fraction (S100 PAP). NE PAP I had chromatographic properties similar to those of S100 PAP, and both enzymes displayed higher activities in the presence of Mn2+ than in the presence of Mg2+, whereas NE PAP II was chromatographically distinct and had approximately equal levels of activity in the presence of Mn2+ and Mg2+. Each of the enzymes, when mixed with other nuclear fractions containing cleavage or specificity factors, was able to reconstitute efficient cleavage and polyadenylation of pre-mRNAs containing an AAUAAA sequence element. The PAPs alone, however, showed no preference for precursors containing an intact AAUAAA sequence over a mutated one, providing further evidence that the PAPs have no intrinsic ability to recognize poly(A) addition sites. Two additional properties of the three enzymes suggest that they are related: sedimentation in glycerol density gradients indicated that the native size of each enzyme is approximately 50 to 60 kilodaltons, and antibodies against a rat hepatoma PAP inhibited the ability of each enzyme to function in AAUAAA-dependent polyadenylation.