Gretchen Edwalds-Gilbert

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.

Yeast Ntr1/Spp382 Mediates Prp43 Function in Postspliceosomes

ABSTRACT The Ntr1 and Ntr2 proteins of Saccharomyces cerevisiae have been reported to interact with proteins involved in pre-mRNA splicing, but their roles in the splicing process are unknown. We show here that they associate with a postsplicing complex containing the excised intron and the spliceosomal U2, U5, and U6 snRNAs, supporting a link with a late stage in the pre-mRNA splicing process. Extract from cells that had been metabolically depleted of Ntr1 has low splicing activity and accumulates the excised intron. Also, the level of U4/U6 di-snRNP is increased but those of the free U5 and U6 snRNPs are decreased in Ntr1-depleted extract, and increased levels of U2 and decreased levels of U4 are found associated with the U5 snRNP protein Prp8. These results suggest a requirement for Ntr1 for turnover of the excised intron complex and recycling of snRNPs. Ntr1 interacts directly or indirectly with the intron release factor Prp43 and is required for its association with the excised intron. We propose that Ntr1 promotes release of excised introns from splicing complexes by acting as a spliceosome receptor or RNA-targeting factor for Prp43, possibly assisted by the Ntr2 protein.

Dominant negative mutants of the yeast splicing factor Prp2 map to a putative cleft region in the helicase domain of DExD/H-box proteins

The Prp2 protein of Saccharomyces cerevisiae is an RNA-dependent ATPase required before the first transesterification reaction in pre-mRNA splicing. Prp2 binds to the spliceosome in the absence of ATP and is released following ATP hydrolysis. We determined what regions in Prp2 are essential for release from the spliceosome by analyzing dominant negative mutants in vivo and in vitro. We made mutations in conserved motif II (DExH) and motif VI (QRxGR) of the helicase (H) domain. Mutations that inactivated PRP2 had a dominant negative phenotype when overexpressed in vivo. To test whether mutations outside of the H domain could confer a dominant negative phenotype, we mutagenized a GAL1-PRP2 construct and screened for mutants unable to grow on galactose-containing media. Five dominant negative mutants were characterized; three mapped within the H domain and two mapped downstream of motif VI, indicating that an extended helicase domain is required for release of Prp2 from the spliceosome. Most mutants stalled in the spliceosome in vitro. However, not all mutants that were dominant negative in vivo were dominant negative in vitro, indicating that multiple mechanisms may cause a dominant negative phenotype. Structural modeling of the H domain of Prp2 suggests that mutants map to a cleft region found in helicases of known structure.

Isolation and characterization of polyadenylation complexes assembled in vitro.

We developed a two-step purification of mammalian polyadenylation complexes assembled in vitro. Biotinylated pre-mRNAs containing viral or immunoglobulin poly(A) sites were incubated with nuclear extracts prepared from mouse myeloma cells under conditions permissive for in vitro cleavage and polyadenylation and the mixture was fractionated by gel filtration; complexes containing biotinylated pre-mRNA and bound proteins were affinity purified on avidin-agarose resin. Western analysis of known components of the polyadenylation complex demonstrated copurification of polyadenylation factors with poly(A) site-containing RNA but not with control RNA substrates containing either no polyadenylation signals or a point mutation of the AAUAAA polyadenylation signal. Polyadenylation complexes that were assembled on exogenous RNA eluted from the Sephacryl column in fractions consistent with their size range extending from 2 to 4 x 10(6) Mr. Complexes endogenous to the extract were of approximately the same apparent size, but more heterogeneous in distribution. This method can be used to study polyadenylation/cleavage complexes that may form upon a number of different RNA sequences, an important step towards defining which factors might differentially associate with specific RNAs.