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SF2, an activity necessary for 5' splice site cleavage and lariat formation during pre-mRNA splicing in v/tro, has been purified to near homogeneity from HeLa cells. The purest fraction contains only two related polypeptides of 33 kD. This fraction is sufficient to complement an SI00 fraction, which contains the remaining splicing factors, to splice several pre-mRNAs. The optimal amount of SF2 required for efficient splicing depends on the pre-mRNA substrate. SF2 is distinct from the hnRNP A1 and U1 snRNP A polypeptides, which are similar in size. Endogenous hnRNA copurifies with SF2, but this activity does not appear to have an essential RNA component. SF2 appears to be necessary for the assembly or stabilization of the earliest specific prespliceosome complex, although in the absence of other components, it can bind RNA in a nonspecific manner. SF2 copurifies with an activity that promotes the annealing of complementary RNAs. Thus, SF2 may promote specific RNA-RNA interactions between snRNAs and pre-mRNA, between complementary snRNA regions, and/or involving intramolecular pre-mRNA helices. Other purified proteins with RNA annealing activity cannot substitute for SF2 in the splicing reaction. Splicing of individual introns in eukaryotic pre-mRNAs takes place by a sequential two-step cleavage-ligation reaction, which has been reproduced m vitro {for reviews, see Green 1986;Padgett et al. 1986; Krainer and Maniatis 19881. In mammalian cell-free systems premRNA splicing has been shown to require multiple nucleoprotein and protein factors {Kr/imer et al.

An assay for the in vitro assembly of HeLa cell 40S nuclear ribonucleoprotein particles (hnRNP particles) has been developed. The substrates were single-stranded nucleic acid polymers of defined length and sequence prepared in vitro and the six major core particle proteins from isolated 40S hnRNP. The fidelity of in vitro assembly was evaluated on various physical parameters, including sedimentation, salt dissociation, polypeptide stoichiometry, UV-activated protein-RNA cross-linking, and overall morphology. Correct particle assembly depended on RNA length and on the input protein/RNA ratio but not on the concentration of the reactant mixture nor on the presence or absence of internal RNA processing signals, a 5'-cap structure, a 3'-poly(A) moiety, or ATP as energy source. RNA lengths between 685 and 726 nucleotides supported correct particle assembly. Dimers and oligomeric complexes that possessed the same polypeptide stoichiometry as native hnRNP assembled on RNA chains that were integral multiples of 700 nucleotides. Intermediate-length RNA supported the assembly of nonstoichiometric complexes lacking structural homogeneity. An analysis of these complexes indicates that proteins Al and A2 may be the first proteins to bind RNA during particle assembly. We conclude that the major proteins of 40S hnRNP particles contain the necessary information for packaging nascent transcripts into a repeating "ribonucleosomal" structure possessing a defined RNA length and protein composition but do not themselves contain the information for modulating packaging that may be required for RNA splicing.The biochemical events of pre-mRNA processing are known largely through studies on the RNA intermediates produced during splicing and studies on the nucleotide sequences required for RNA maturation (for reviews, see references 17 and 42). Nascent transcripts, however, are packaged by a specific subset of major nuclear proteins during transcription to form a ribonucleoprotein (RNP) complex in which the events of RNA splicing occur (see references 8 and 10 for reviews). Historically, these complexes were termed heterogeneous nuclear ribonucleoprotein particles (hnRNP) because the packaged moiety was heterogeneous nuclear RNA and not because the individual particles were known to be heterogeneous in composition and structure (10,38,41). Several observations indicate that nascent transcripts are in fact packaged into a regular repeating structure composed of a contiguous array of 40S hnRNP complexes. These observations suggest that the complexes may play only a passive role in RNA splicing. Other observations suggest that packaging may be transcript specific and thus imply that hnRNP may play a more direct role in the events of RNA processing.Observations in support of a fundamental packaging function for hnRNP are the following. (i) The intranuclear concentration of the major hnRNP is high. For example, in actively growing mammalian cells there is 20 to 30% as much individual core particle hnRNP as individual core particle histone (...

Signal transducers and transcription factors are used in common for developmental cell migration, vasculogenesis, branching morphogenesis, as well as neuronal pathfinding. STAT3, a transcription factor, has been shown to function in all of these processes except neuronal pathfinding. Here, it is shown that STAT3 also facilitates this process. Elimination of STAT3 signaling results in half of zebrafish CaP motoneurons stalling along their ventral pathfinding trajectory. Conversely, constitutive activation leads to precocious branching and redefines CaP axons as a responding population to dorsal guidance cues, resulting in bifurcated axons innervating normal ventral targets as well as additional dorsal muscle groups. These results are consistent with and highlight a fundamental role for STAT3 as a factor promoting cellular responses to guidance cues, not only in nonneural cells but also in pathfinding neurons.

Establishment of the vertebrate body plan requires a variety of signaling molecules. In a search for tyrosine kinases expressed in early zebrafish embryos, a model system for the study of vertebrate development, we discovered Jak1 kinase to be maternally encoded and the mRNA evenly distributed among the cells of blastula-stage embryos. Injection of RNA-encoding dominant-negative Jak1 kinases reduces a specific cell migration, epiboly, and results in the reduction of goosecoid expression and of anterior structures. This work establishes that, in addition to its role in signal transduction of cytokines in adult tissues, Jak1 kinase has a role in early vertebrate development.One of the earliest events of vertebrate development is the specification of the primordial germ layers, ectoderm, endoderm, and mesoderm, and the extensive cell migrations that immediately follow. These processes are likely to require a variety of signal transduction cascades, and several peptide growth factors have been implicated in these processes (1-3). In this study, we show that Jak1 kinase also is required for these processes.In a model vertebrate, the zebrafish, developmental events are easily visualized and studied. In a search for signal transducers that might regulate early development, we used a degenerate PCR approach to identify tyrosine kinases expressed during zebrafish gastrulation. Other than the FGF receptor, no other tyrosine kinase has been implicated in the earliest events of vertebrate development. Using degenerate primers to conserved residues in the catalytic domain of tyrosine kinases, we have isolated a homologue of the Jak1 kinase from early gastrula embryos. This is surprising, as Jak1 kinase has been implicated thus far only in signaling for adult tissues in vertebrates.Jak1 is one of four members of a tyrosine kinase family that possesses several features in common (4, 5). The Jak family members, Jak1, Jak2, Jak3, and Tyk2, lack SH2 and SH3 domains, are cytoplasmic, and possess two kinase domains. The N-terminal kinase domain has only limited homology to the phosphotransferase domains of other tyrosine kinases and the isolated domain, expressed as a glutathione S-transferase (GST) fusion, does not possess kinase activity (6). The Cterminal domain, however, possesses all the motifs found in tyrosine kinase domains and this domain, fused to GST, has phosphotransferase activity (6).Jak1 kinase is ubiquitously expressed in adult mouse tissues (6) and has been shown to transduce signals from a variety of cytokines (4, 5). Its developmental expression, however, has never been analyzed. Jak1 has been thought to function exclusively in adults, primarily in the modulation of immune function. We show that during early development, Jak1 kinase is exclusively of maternal origin, and through the use of dominant negative Jak1 kinases, that this kinase is required for cell migrations, goosecoid expression, and anterior shield formation. In agreement with our findings, recent studies have shown that Jak kinase plays a ro...