Rebecca L. Seipelt

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.

ELEVATED LEVELS OF A U4/U6.U5 SNRNP-ASSOCIATED PROTEIN, SPP381P, RESCUE A MUTANT DEFECTIVE IN SPLICEOSOME MATURATION

ABSTRACT U4 snRNA release from the spliceosome occurs through an essential but ill-defined Prp38p-dependent step. Here we report the results of a dosage suppressor screen to identify genes that contribute toPRP38 function. Elevated expression of a previously uncharacterized gene, SPP381, efficiently suppresses the growth and splicing defects of a temperature-sensitive (Ts) mutantprp38-1. This suppression is specific in that enhancedSPP381 expression does not alter the abundance of intronless RNA transcripts or suppress the Ts phenotypes of otherprp mutants. Since SPP381 does not suppress aprp38::LEU2 null allele, it is clear that Spp381p assists Prp38p in splicing but does not substitute for it. YeastSPP381 disruptants are severely growth impaired and accumulate unspliced pre-mRNA. Immune precipitation studies show that, like Prp38p, Spp381p is present in the U4/U6.U5 tri-snRNP particle. Two-hybrid analyses support the view that the carboxyl half of Spp381p directly interacts with the Prp38p protein. A putative PEST proteolysis domain within Spp381p is dispensable for the Spp381p–Prp38p interaction and for prp38-1 suppression but contributes to Spp381p function in splicing. Curiously, in vitro, Spp381p may not be needed for the chemistry of pre-mRNA splicing. Based on the in vivo and in vitro results presented here, we propose that two small acidic proteins without obvious RNA binding domains, Spp381p and Prp38p, act in concert to promote U4/U5.U6 tri-snRNP function in the spliceosome cycle.

A Nonimmunoglobulin Transgene and the Endogenous Immunoglobulin μ Gene Are Coordinately Regulated by Alternative RNA Processing during B-Cell Maturation

ABSTRACT The immunoglobulin (Ig) genes have been extensively studied as model systems for developmentally regulated alternative RNA processing. Transcripts from these genes are alternatively processed at their 3′ ends to yield a transcript that is either cleaved and polyadenylated at a site within an intron or spliced to remove the poly(A) site and subsequently cleaved and polyadenylated at a downstream site. Results obtained from expressing modified genes in established tissue culture cell lines that represent different stages of B-lymphocyte maturation have suggested that the only requirement for regulation is that a pre-mRNA contain competing cleavage-polyadenylation and splice reactions whose efficiencies are balanced. Since several non-Ig genes modified to have an Ig gene-like structure are regulated in cell lines, Ig-specific sequences are not essential for this control. This strongly implies that changes in the amounts or activities of general RNA processing components mediate the processing regulation. Despite numerous studies in cell lines, this model of Ig gene regulation has never been tested in vivo during normal lymphocyte maturation. We have now introduced a non-Ig gene with an Ig gene-like structure into the mouse germ line and demonstrate that RNA from the transgene is alternatively processed and regulated in murine splenic B cells. This establishes that the balance and arrangement of competing cleavage-polyadenylation reactions are sufficient for RNA processing regulation during normal B-lymphocyte development. These experiments also validate the use of tissue culture cell lines for studies of Ig processing regulation. This is the first transgenic mouse produced to test a specific model for regulated mRNA processing.

Apolactoferrin inhibits the catalytic domain of matrix metalloproteinase-2 by zinc chelation

Lactoferrin (LTF) is a multifunctional iron-binding protein that is also capable of binding other divalent metal cations, especially Zn2+. Recent investigations indicate that lactoferrin levels are elevated in many disease conditions in which matrix metalloproteinases (MMPs), particularly MMP-2, are also elevated, suggesting that the 2 proteins may interact. This possibility was examined by determining the effect of LTF in its holo (metal-bound) and apo (metal-free) forms on the proteolytic activity of MMP-2 and other similar zinc metalloproteases. Pre-incubation with apolactoferrin, but not hololactoferrin, greatly reduced the hydrolysis of a peptide substrate by MMP-2, but not by MMP-1, -8, -9, or -13. This inhibition was specific for the 42 kDa catalytic domain fragment of MMP-2 lacking the hemopexin domain, since the 66 kDa form was poorly inhibited by apolactoferrin. The inhibition of the MMP-2 catalytic domain was strongly temperature sensitive, indicating that the conformation of one or both proteins is crucial to this interaction. To ascertain the mechanism of inhibition, increasing concentrations of ZnCl2 and FeCl2 were added to the reaction. While addition of Fe2+ did not reverse inhibition, the addition of Zn2+ resulted in a recovery of MMP-2 activity, and furthermore, zinc-saturated LTF did not inhibit MMP-2. Together, these data strongly suggest that apolactoferrin is capable of removing the catalytic zinc from the active site of MMP-2, although an exosite-based interaction between the 2 proteins cannot be fully ruled out. This inhibitory activity suggests a novel function for LTF and may represent a novel regulatory mechanism that regulates proteolysis by MMP-2 in vivo.

LIPID COMPOSITION OF CHLORARACHNIOPHYTES (CHLORARACHNIOPHYCEAE) FROM THE GENERA BIGELOWIELLA, GYMNOCHLORA, AND LOTHARELLA1

The Chlorarachniophyceae are unicellular eukaryotic algae characterized by an amoeboid morphology that may be the result of secondary endosymbiosis of a green alga by a nonphotosynthetic amoeba or amoeboflagellate. Whereas much is known about the phylogeny of chlorarachniophytes, little is known about their physiology, particularly that of their lipids. In an initial effort to characterize the lipids of this algal class, four organisms from three genera were examined for their fatty acid and sterol composition. Fatty acids from lipid fractions containing chloroplast‐associated glycolipids, storage triglycerides, and cytoplasmic membrane‐associated polar lipids were characterized. Glycolipid‐associated fatty acids were of limited composition, principally eicosapentaenoic acid [20:5(n‐3)] and hexadecanoic acid (16:0). Triglyceride‐associated fatty acids, although minor, were found to be similar in composition. The polar lipid fraction was dominated by lipids that did not contain phosphorus and had a more variable fatty acid composition with 16:0 and docosapentaenoic acid [22:5(n‐3)] dominant along with a number of minor C18 and C20 fatty acids. Crinosterol and one of the epimeric pair poriferasterol/stigmasterol were the sole sterols. Several genes required for synthesis of these sterols were computationally identified in Bigelowiella natans Moestrup. One sterol biosynthesis gene showed the greatest similarity to SMT1 of the green alga, Chlamydomonas reinhardtii. However, homologues to other species, mostly green plant species, were also found. Further, the method used for identification suggested that the sequences were transferred to a genetic compartment other than the likely original location, the nucleomorph nucleus.

Sterol Composition and Biosynthetic Genes of the Recently Discovered Photosynthetic Alveolate, Chromera velia (Chromerida), a Close Relative of Apicomplexans

Chromera velia is a recently discovered, photosynthetic, marine alveolate closely related to apicomplexan parasites, and more distantly to perkinsids and dinoflagellates. To date, there are no published studies on the sterols of C. velia. Because apicomplexans and perkinsids are not known to synthesize sterols de novo, but rather obtain them from their host organisms, our objective was to examine the composition of the sterols of C. velia to assess whether or not there is any commonality with dinoflagellates as the closest taxonomic group capable of synthesizing sterols de novo. Furthermore, knowledge of the sterols of C. velia may provide insight into the sterol biosynthetic capabilities of apicomplexans prior to loss of sterol biosynthesis. We have found that C. velia possesses two primary sterols, 24‐ethylcholesta‐5,22E‐dien‐3β‐ol, and 24‐ethylcholest‐5‐en‐3β‐ol, not common to dinoflagellates, but rather commonly found in other classes of algae and plants. In addition, we have identified computationally three genes, SMT1 (sterol‐24C‐methyltransferase), FDFT1 (farnesyl diphosphate farnesyl transferase, squalene synthase), and IDI1 (isopentenyl diphosphate Δ‐isomerase), predicted to be involved in sterol biosynthesis by their similarity to analogous genes in other sterol‐producing eukaryotes, including a number of algae.

A conserved tyrosine residue of Saccharomyces cerevisiae leukotriene A4 hydrolase stabilizes the transition state of the peptidase activity.

Saccharomyces cerevisiae leukotriene A4 hydrolase (LTA4H) is a bifunctional aminopeptidase/epoxide hydrolase and a member of the M1 family of metallopeptidases. In order to obtain a more thorough understanding of the aminopeptidase activity of the enzyme, two conserved tyrosine residues, Tyr244 and Tyr456, were altered to phenylalanine and the mutant proteins characterized by determining KM and kcat for various amino acid beta-naphthylamide substrates. While mutation of Tyr456 exhibited minimal effect on catalysis, mutation of Tyr244 caused an overall 25-100-fold reduction in catalytic activity for all substrates tested. Furthermore, LTA4H Y244F exhibited a 40-fold decrease in affinity for RB-3014, a transition state analog inhibitor, implicating Tyr244 in transition state stabilization.

Cookie-ases: Interactive Models for Teaching Genotype-Phenotype Relationships

intricate relationship are often initially presented simply as terms to be memorized. Many times the volume of material to be taught leaves little time to revisit their true meaning, and how an organism’s genetic makeup (genotype) generates its phenotype. The idea that a gene encodes a protein establishes the fundamental relationship, but it is often presented as an idea that the student memorizes. The student may also memorize that a mutant gene can generate a mutant phenotype. Further, the student may memorize that changing the sequence of the gene changes the amino acid sequence of the protein. The extra step of melding these memorized passages into a coherent model that incorporates the connection of protein activity and phenotype is rarely achieved without additional assistance. It is truly important that students grasp these abstract concepts, because they are core ideas that are important in a number of biological disciplines, including genetics, biochemistry, molecular and cellular biology, and physiology. It is because these concepts are so abstract that an active learning approach that is visual, concrete, and includes role-playing and modeling is most likely to be effective (Anderson, 1996; Rutledge, 2001). Several hands-on and wet laboratory activities have been proposed to model similar genetic concepts (Lanza & Cress, 2001; Heim, 1991; Guilfoile & Plum, 2000; Pigage, 1991; Bio-Rad Laboratories pGLO Bacterial Transformation Kit). The exercise presented here is a novel, time effective, student-centered, role-playing activity in which students learn about the intricate connection between genotype and phenotype by exploring the fundamental effect of mutation on protein function beginning with a very real and human phenotype, albinism. This exercise is based on a long established role-playing model of enzyme kinetics (author unknown) by allowing students to act out the role of enzyme (Oreo-ase). However, in this model exercise, instead of learning only about the enzyme, students learn about the genes and mutations, bringing this model to its full genetic extension.

Asparagine362 is essential for zinc binding and catalysis in the peptidase reaction of Saccharomyces cerevisiae leukotriene A4 hydrolase

Zinc metallopeptidases are ubiquitous enzymes with diverse cellular functions that can be found in most organisms. Leukotriene A₄ hydrolase (LTA4H; E.C. 3.3.2.6) is an unusual zinc metallopeptidase of the M1 family that also possesses an epoxide hydrolase activity; however, the role of its peptidase activity remains unknown. To further characterize the peptidase activity of LTA4H and other closely related metallopeptidases, a multiple sequence alignment and predicted structure were used to target three amino acid residues of yeast LTA4H for mutagenesis: Asn362, Trp365, and Asp399. Although mutating Trp365 and Asp399 had little effect on catalysis, altering Asn362 had varying effects on catalysis, depending on the replacement residue. Mutation of Asn362 to glutamine (N362Q) caused minor catalytic defects, while mutation to leucine (N362L) or glutamate (N362E) caused large reductions in activity. Both N362L and N362E also exhibited an altered pH dependence of catalysis, reduced chloride activation, and reduced zinc affinity and content, indicating that Asn362 may interact with the nearby zinc coordinating residue His344, and possibly with Glu363 as well, to polarize and/or orient these residues.