Prp8 in a Reduced Spliceosome Lacks a Conserved Toggle that Correlates with Splicing Complexity across Diverse Taxa.

Conformational rearrangements are critical to regulating the assembly and activity of the spliceosome. The spliceosomal protein Prp8 undergoes multiple conformational changes during the course of spliceosome assembly, activation, and catalytic activity. Most of these rearrangements of Prp8 involve the disposition of the C-terminal Jab-MPN and RH domains with respect to the core of Prp8. Here we use x-ray structural analysis to show that a previously characterized and highly conserved ß-hairpin structure in the RH domain that acts as a toggle in the spliceosome is absent in Prp8 from the reduced spliceosome of the red alga Cyanidioschyzon merolae. Using comparative sequence analysis, we show that the presence or absence of this hairpin corresponds to the presence or absence of protein partners that interact with this hairpin as observed by x-ray and cryo-EM studies. The presence of the toggle correlates with increasing intron number suggesting a role in the regulation of splicing.

A type XI collagen mutation leads to increased degradation of type II collagen in articular cartilage.

OBJECTIVE: To determine in articular cartilage whether degraded type II collagen is more abundant in Col11a1 mutant cho/+ than in age-matched +/+ mice and whether collagen degradation occurs in a generalized or localized fashion. DESIGN: Knee joints from cho/+ and +/+ mice at 6, 9, 12 and 15 months of age were dissected, fixed, cryosectioned, and stained with antibody COL2-3/4m against denatured type II collagen using a FITC-conjugated secondary antibody. Sections were viewed and photographed under a fluorescence microscope and areas of staining were quantified. RESULTS: Before 12 months of age, little degraded collagen staining was detectable in +/+ or cho/+ mice. By 15 months, however, cho/+ mice showed significantly more degraded type II collagen than age-matched controls. Degraded collagen staining was localized at the articular surface, not distributed generally throughout the articular cartilage. CONCLUSIONS: The results suggest a model in which cumulative biomechanical stresses trigger increased collagen synthesis and degradation in both +/+ and cho/+ mice at around 12 months of age. Cho/+ mice, however, are less able to synthesize and assemble normal replacement collagen fibrils because of the Col11a1 mutation. Degradation is further activated, resulting in the accumulation of degraded type II collagen in the articular cartilage extracellular matrix. Similar mutations that do not overtly affect skeletal development may likewise predispose humans to increased collagen degradation and resultant osteoarthritis.

frizzled 9 is expressed in neural precursor cells in the developing neural tube.

The wnt signaling pathway has important functions in nervous system development. To better understand this process we have cloned and analyzed the expression of the wnt receptor, frizzled 9, in the developing nervous system in mouse, chick and zebrafish. The earliest expression of mouse frizzled 9 mRNA expression begins at E8.5 with expression throughout the entire rostral-caudal neuraxis. This early expression pattern within the neural tube appears to be conserved between chick and zebrafish. Expression becomes restricted to a ventral domain in the mouse ventricular zone at E11.5, a region specified to give rise to neurons and glia. Using a polyclonal antibody to MFZ9 further shows expression limited to neural restricted precursors cells.

Frizzled-4 expression during chick kidney development.

Wnts have been implicated in metanephric kidney development. To determine whether Frizzleds, the genes that encode Wnt receptors, are present at early stages of nephrogenesis, we examined the expression of several recently identified Frizzled genes in the chick by in situ hybridization. Here we report the cloning and characterization of chick Frizzled-4 (cFz-4), which we found to be expressed in the developing chick kidney. cFz-4 was first expressed in the pronephros caudal to the third somite at Hamburger and Hamilton stage 10. Its expression increased with maturation, becoming restricted to the newly induced glomeruli and tubules in the mesonephros and metanephros. Within the metanephros, cFz-4 and Wnt-4 expression patterns were similar, whereas Wnt-11 was expressed solely in the tips of the branching ureteric bud. cFz-4 expression was compared with that of known kidney markers. It preceded that of Lmx-1, but was similarly restricted to developing glomeruli and tubules. In contrast, Pax-2 expression and Lim 1/2 antibody labeling occurred in intermediate mesoderm caudal to the fifth somite in the early pronephros, and each persisted in both the tubules and nephric ducts throughout further development.

Characterization of avian frizzled genes in cranial placode development.

To determine the possible role of Wnt signaling in cranial placode development, we have cloned several chick frizzled genes, a family of putative Wnt receptor molecules, and analyzed their expression during chick embryogenesis. Chick frizzled-2 (cFz-2) and frizzled-7 (cFz-7) are expressed broadly in cranial ectoderm, tissue that is competent to express markers of the trigeminal placode (Stark et al., 1997. Development 124, 4287-4295; Baker et al., 1999. Development 126, 147-156). In addition, cFz-2 and cFz-7 are uniquely expressed in other cranial placodes, including the olfactory, lens, and otic placodes. Chick frizzled-1 (cFz-1) is expressed in the lens, otic placode and, along with cFz-7, in epibranchial placodes. Each frizzled gene expressed in the otic placode displays a unique domain of expression: cFz-1 transcripts are detected in the medial wall of the vesicle, cFz-2 in the rostral rim of the vesicle, and cFz-7 in the lateral half of the vesicle. Other chick frizzled family members cloned that do not show striking expression in cranial placodes include frizzled-4 (cFz-4), frizzled-8 (cFz-8), frizzled-9 (cFz-9), and frizzled-10 (cFz-10). A brief summary of their expression is given, along with a brief summary of non-placodal expression of cFz-1, cFz-2, and cFz-7. In all, frizzled genes show dynamic expression at key times during embryonic development, particularly in the cranial placodes.

Timing and cell interactions underlying neural induction in the chick embryo.

Previous studies on neural induction have identified regionally localized inducing activities, signaling molecules, potential competence factors and various other features of this important, early differentiation event. In this paper, we have developed an improved model system for analyzing neural induction and patterning using transverse blastoderm isolates obtained from gastrulating chick embryos. We use this model to establish the timing of neural specification and the spatial distribution of perinodal cells having organizer activity. We show that a tissue that acts either as an organizer or as an inducer of an organizer is spatially co-localized with the prospective neuroectoderm immediately rostral to the primitive streak in the early gastrula. As the primitive streak elongates, this tissue with organizing activity and the prospective neuroectoderm rostral to the streak separate. Furthermore, we show that up to and through the mid-primitive streak stage (i.e., stage 3c/3+), the prospective neuroectoderm cannot self-differentiate (i.e. , express neural markers and acquire neural plate morphology) in isolation from tissue with organizer activity. Signals from the organizer and from other more caudal regions of the primitive streak act on the rostral prospective neuroectoderm and the latter gains potency (i.e., is specified) by the fully elongated primitive streak stage (i.e., stage 3d). Transverse blastoderm isolates containing non-specified, prospective neuroectoderm provide an improved model system for analyzing early signaling events involved in neuraxis initiation and patterning.

A trans-acting peptide activates the yeast a1 repressor by raising its DNA-binding affinity.

The cooperative binding of gene regulatory proteins to DNA is a common feature of transcriptional control in both prokaryotes and eukaryotes. It is generally viewed as a simple energy coupling, through protein-protein interactions, of two or more DNA-binding proteins. In this paper, we show that the simple view does not account for the cooperative DNA binding of a1 and alpha2, two homeodomain proteins from budding yeast. Rather, we show through the use of chimeric proteins and synthetic peptides that, upon heterodimerization, alpha2 instructs a1 to bind DNA. This change is induced by contact with a peptide contributed by alpha2, and this contact converts a1 from a weak to a strong DNA-binding protein. This explains, in part, how high DNA-binding specificity is achieved only when the two gene regulatory proteins conjoin. We also provide evidence that features of the a1-alpha2 interaction can serve as a model for other examples of protein-protein interactions, including that between the herpes virus transcriptional activator VP16 and the mammalian homeodomain-containing protein Oct-l.

Competence, specification and induction of Pax-3 in the trigeminal placode.

Placodes are discrete regions of thickened ectoderm that contribute extensively to the peripheral nervous system in the vertebrate head. The paired-domain transcription factor Pax-3 is an early molecular marker for the avian ophthalmic trigeminal (opV) placode, which forms sensory neurons in the ophthalmic lobe of the trigeminal ganglion. Here, we use collagen gel cultures and heterotopic quail-chick grafts to examine the competence, specification and induction of Pax-3 in the opV placode. At the 3-somite stage, the whole head ectoderm rostral to the first somite is competent to express Pax-3 when grafted to the opV placode region, though competence is rapidly lost thereafter in otic-level ectoderm. Pax-3 specification in presumptive opV placode ectoderm occurs by the 8-somite stage, concomitant with robust Pax-3 expression. From the 8-somite stage onwards, significant numbers of cells are committed to express Pax-3. The entire length of the neural tube has the ability to induce Pax-3 expression in competent head ectoderm and the inductive interaction is direct. We propose a detailed model for Pax-3 induction in the opV placode.

Coordinate actions of BMPs, Wnts, Shh and noggin mediate patterning of the dorsal somite.

Shortly after their formation, somites of vertebrate embryos differentiate along the dorsoventral axis into sclerotome, myotome and dermomyotome. The dermomyotome is then patterned along its mediolateral axis into medial, central and lateral compartments, which contain progenitors of epaxial muscle, dermis and hypaxial muscle, respectively. Here, we used Wnt-11 as a molecular marker for the medial compartment of dermomyotome (the 'medial lip') to demonstrate that BMP in the dorsal neural tube indirectly induces formation of the medial lip by up-regulating Wnt-1 and Wnt-3a (but not Wnt-4) expression in the neural tube. Noggin in the dorsal somite may inhibit the direct action of BMP on this tissue. Wnt-11 induction is antagonized by Sonic Hedgehog, secreted by the notochord and the floor plate. Together, our results show that the coordinated actions of the dorsal neural tube (via BMP and Wnts), the ventral neural tube/notochord (via Shh) and the somite itself (via noggin) mediates patterning of the dorsal compartment of the somite.

Neural tube-ectoderm interactions are required for trigeminal placode formation.

Cranial sensory ganglia in vertebrates develop from the ectodermal placodes, the neural crest, or both. Although much is known about the neural crest contribution to cranial ganglia, relatively little is known about how placode cells form, invaginate and migrate to their targets. Here, we identify Pax-3 as a molecular marker for placode cells that contribute to the ophthalmic branch of the trigeminal ganglion and use it, in conjunction with DiI labeling of the surface ectoderm, to analyze some of the mechanisms underlying placode development. Pax-3 expression in the ophthalmic placode is observed as early as the 4-somite stage in a narrow band of ectoderm contiguous to the midbrain neural folds. Its expression broadens to a patch of ectoderm adjacent to the midbrain and the rostral hindbrain at the 8- to 10-somite stage. Invagination of the first Pax-3-positive cells begins at the 13-somite stage. Placodal invagination continues through the 35-somite stage, by which time condensation of the trigeminal ganglion has begun. To challenge the normal tissue interactions leading to placode formation, we ablated the cranial neural crest cells or implanted barriers between the neural tube and the ectoderm. Our results demonstrate that, although the presence of neural crest cells is not mandatory for Pax-3 expression in the forming placode, a diffusible signal from the neuroectoderm is required for induction and/or maintenance of the ophthalmic placode.

Lability of the presumptive pectoral and pelvic girdle territories compared using celomic grafts.

The concept of "limbness" usually implies "wingness" or "legness". We present evidence in this study that in the case of the proximal forelimb, limbness may exist independent of wingness. Presumptive wing and leg regions were grafted into the opposite limb (wing into leg or leg into wing) of stage 18 hosts. Several stage 10+ to 11+ presumptive wing regions grafted into host legs developed pelvic girdles associated with distal wing elements. This phenomenon was not seen in presumptive wing region grafts older than stage 12-. Presumptive leg regions grafted into host wings developed leg structures only, including pelvic girdle and leg digits. The pectoral girdle appears to be labile in early stages, whereas the pelvic girdle is not labile even during early stages.

Accessibility of alpha 2-repressed promoters to the activator Gal4.

It has been proposed that eukaryotic repressors of transcription can act by organizing chromatin, thereby preventing the accessibility of nearby DNA to activator proteins required for transcription initiation. In this study, we test this idea for the yeast alpha 2 repressor using a simple, artificial promoter that contains a single binding site for the activator protein Gal4 and a single binding site for the repressor alpha 2. When both the repressor and the activator are expressed in the same cell, the artificial promoter is efficiently repressed. In vivo footprinting experiments demonstrate that Gal4 can occupy its binding site even when the promoter is repressed. This result indicates that alpha 2-directed repression must result from interference with some stage in transcription initiation other than activator binding to DNA.

Crystal structure of the MATa1/MAT alpha 2 homeodomain heterodimer bound to DNA.

The Saccharomyces cerevisiae MATa1 and MAT alpha 2 homeodomain proteins, which play a role in determining yeast cell type, form a heterodimer that binds DNA and represses transcription in a cell type-specific manner. Whereas the alpha 2 and a1 proteins on their own have only modest affinity for DNA, the a1/alpha 2 heterodimer binds DNA with high specificity and affinity. The three-dimensional crystal structure of the a1/alpha 2 homeodomain heterodimer bound to DNA was determined at a resolution of 2.5 A. The a1 and alpha 2 homeodomains bind in a head-to-tail orientation, with heterodimer contacts mediated by a 16-residue tail located carboxyl-terminal to the alpha 2 homeodomain. This tail becomes ordered in the presence of a1, part of it forming a short amphipathic helix that packs against the a1 homeodomain between helices 1 and 2. A pronounced 60 degree bend is induced in the DNA, which makes possible protein-protein and protein-DNA contacts that could not take place in a straight DNA fragment. Complex formation mediated by flexible protein-recognition peptides attached to stably folded DNA binding domains may prove to be a general feature of the architecture of other classes of eukaryotic transcriptional regulators.

Interaction between two homeodomain proteins is specified by a short C-terminal tail.

Two yeast homeodomain proteins, a1 and alpha 2, interact and cooperatively bind the haploid-specific gene (hsg) operator, resulting in the repression of a set of genes involved in the determination of cell type. The cooperative binding of a1 and alpha 2 to DNA can be reconstituted in vitro using purified fragments of a1 and alpha 2. Only the homeodomain is needed for a1, but for alpha 2 a C-terminal 22-amino-acid tail is required as well. As most of the specificity of DNA binding appears to derive from a1, we proposed that alpha 2 functions in the a1/alpha 2 heterodimer to contact a1 with its tail. By construction and analysis of several chimaeric proteins, we investigate how two DNA-binding proteins, one with low intrinsic specificity (alpha 2) and one with no apparent intrinsic DNA-binding ability (a1), can together create a highly specific DNA-binding activity. We show that the 22-amino-acid region of alpha 2 immediately C-terminal to the homeodomain, when grafted onto the a1 homeodomain, converts a1 to a strong DNA-binding protein. This alpha 2 tail can also be attached to the Drosophila engrailed homeodomain, and the chimaeric protein now binds cooperatively to DNA with a1, showing how a simple change can create a new homeodomain combination that specifically recognizes a new DNA operator.

Heterodimerization of the yeast homeodomain transcriptional regulators alpha 2 and a1 induces an interfacial helix in alpha 2.

The homeodomain proteins a1 and alpha 2 act cooperatively to regulate cell type specific genes in yeast. The basis of the cooperativity is a weak interaction between the two proteins which forms heterodimers that bind DNA tightly and specifically. In this paper, we examine the mechanism of heterodimerization. We show that two relatively small fragments of a1 and alpha 2 are capable of heterodimerization and tight DNA binding. The alpha 2 fragment contains the homeodomain followed by the natural 22 C-terminal amino acids of the protein; these 22 amino acids are unstructured in the alpha 2 fragment. The a1 fragment contains only the homeodomain, indicating that the a1 homeodomain mediates both DNA binding and protein-protein interactions with alpha 2. We used isotope-edited NMR spectroscopy to study the interaction in solution of these two fragments. Samples in which only the alpha 2 fragment was uniformly labeled with 15N allowed us to visualize changes in the NMR spectra of the alpha 2 fragment produced by heterodimerization. We found that the a1 homeodomain perturbs the resonances of only the C-terminal tail of alpha 2; moreover, contact with a1 converts a portion of this tail (residues 193-203) from its unstructured state to an alpha-helix, as determined by J coupling and NOE measurements. Thus the heterodimerization of two homeodomain proteins involves the specific interaction between a tail of one protein and the homeodomain of the other. This interaction is accompanied by the acquisition of secondary structure in the tail.

Molecular cloning of gp42, a cell-surface molecule that is selectively induced on rat natural killer cells by interleukin 2: glycolipid membrane anchoring and capacity for transmembrane signaling.

We have previously shown that in vitro culture of rat natural killer (NK) cells in high concentrations of recombinant interleukin 2 (rIL-2) leads to the expression of a surface glycoprotein with a molecular mass of approximately 42 kD. This glycoprotein, gp42, is not induced on other lymphocytes and thus provides a lineage-specific marker for rIL-2-activated NK cells. We here present the nucleotide sequence for gp42 cDNA. The open reading frame encodes 233 amino acids with three potential sites for N-linked glycosylation. The deduced amino acid sequence lacks an apparent transmembrane domain and instead contains a hydrophobic COOH terminus that is characteristic of glycosylphosphatidylinositol (GPI)-anchored surface proteins. Consistent with this, gp42 is cleaved from the NK-like cell line, RNK-16, by phosphatidylinositol-specific phospholipase C (PI-PLC), as is gp42 expressed on CHO cells that have been transformed with gp42 cDNA. On rIL-2-activated NK cells, gp42 is resistant to PI-PLC, though our studies suggest that gp42 on these cells is still expressed as a GPI-anchored molecule. Antibody to gp42 stimulates in RNK-16 cells an increase in inositol phosphates and in intracellular calciu, signals that are associated with the activation of lymphocytes, including NK cells. rIL-2-activated NK cells, however, lack this response to gp42 as well as to other stimuli. Thus, gp42, the only NK-specific activation antigen, is a GPI-anchored surface molecule with the capacity to stimulate transmembrane signaling.