An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly.

Splicing of nuclear precursor messenger RNA (pre-mRNA) occurs on a large ribonucleoprotein complex, the spliceosome. Several small nuclear ribonucleoproteins (snRNP's) are subunits of this complex that assembles on the pre-mRNA. Although the U1 snRNP is known to recognize the 5' splice site, its roles in spliceosome formation and splice site alignment have been unclear. A new affinity purification method for the spliceosome is described which has provided insight into the very early stages of spliceosome formation in a yeast in vitro splicing system. Surprisingly, the U1 snRNP initially recognizes sequences at or near both splice junctions in the intron. This interaction must occur before the other snRNP's (U2, U4, U5, and U6) can join the complex. The results suggest that interaction of the two splice site regions occurs at an early stage of spliceosome formation and is probably mediated by U1 snRNP and perhaps other factors.

Pre-mRNA splicing in yeast.

Splicing of introns from nuclear precursor messenger RNAs (pre-mRNAs) occurs in all eukaryotes. Two aspects of the splicing mechanism need to be understood: how intron sequences are recognized and aligned and how splicing is catalysed. Recent genetic and biochemical studies in the simple eukaryote Saccharomyces cerevisiae are revealing some of the features of the splicing mechanism.

Specific Saccharomyces cerevisiae genes are expressed in response to DNA-damaging agents.

When exposed to DNA-damaging agents, the yeast Saccharomyces cerevisiae induces the expression of at least six specific genes. We have previously identified one damage inducible (DIN) gene as a gene fusion (din-lacZ fusion) whose expression increases in response to DNA-damaging treatments. We describe here the identification of five additional DIN genes as din-lacZ fusions and the responses of all six DIN genes to DNA-damaging agents. Northern blot analyses of the transcripts of two of the DIN genes show that their levels increase after exposure to DNA-damaging agents. Five of the din-lacZ fusions are induced in S. cerevisiae cells exposed to UV light, gamma rays, methotrexate, or alkylating agents. One of the din-lacZ fusions is induced by either UV or methotrexate but not by the other agents. This finding suggests that there are sets of DIN genes that are regulated differently.

Affinity chromatography with biotinylated RNAs.

Small, reversibly biotinylated RNAs as described here are versatile ligands for affinity chromatography of RNA-binding components. These RNAs can be attached to a solid support by binding to avidin and used as ligands, or they may be hybridized to another RNA which acts as the ligand. The incorporation of a disulfide bond in the linker arm connecting biotin to the RNA makes it possible to dissociate the RNA from avidin under mild conditions. Our results regarding the binding and elution of the biotinylated RNA may be applied to other, reversibly biotinylated molecules.

Dynamics of the U1 small nuclear ribonucleoprotein during yeast spliceosome assembly.

U1 small nuclear ribonucleoprotein (snRNP) may function during several steps of spliceosome assembly. Most spliceosome assembly assays, however, fail to detect the U1 snRNP. Here, I used a new native gel electrophoretic assay to find the yeast U1 snRNP in three pre-splicing complexes (delta, beta1, alpha2) formed in vitro. The order of complex formation is deduced to be delta --> beta1 --> alpha2 --> alpha1 --> beta2, the active spliceosome. The delta complex is formed when U1 snRNP binds to pre-mRNA in the absence of ATP. There are two forms of delta: a major one, deltaun, unstable to competitor RNA; and a minor one, deltacommit, committed to the splicing pathway. The other complexes are formed in the presence of ATP and contain the following snRNPs: beta1, the pre-spliceosome, has both U1 and U2; alpha2 has all five, however, U1 is reduced compared with the others; and alpha1 and beta2 have U2, U5, and U6. Prior work by others suggests that U1 is "handing off" the 5' splice site region to the U5 and U6 snRNPs before splicing begins. The reduced levels of U1 snRNP in the alpha2 complex suggests that the handoff occurs during formation of this complex.

Four yeast spliceosomal proteins (PRP5, PRP9, PRP11, and PRP21) interact to promote U2 snRNP binding to pre-mRNA.

We have analyzed the functions of several pre-mRNA processing (PRP) proteins in yeast spliceosome formation. Here, we show that PRP5 (a DEAD box helicase-like protein), PRP9, and PRP11 are each required for the U2 snRNP to bind to the pre-spliceosome during spliceosome assembly in vitro. Genetic analyses of their functions suggest that they and another protein, PRP21, act concertedly and/or interact physically with each other and with the stem-loop IIa of U2 snRNA to bind U2 snRNP to the pre-mRNA. Biochemical complementation experiments also indicate that the PRP9 and PRP11 proteins interact. The PRP9 and PRP11 proteins may be functioning similarly in yeast and mammalian cells. The requirement for ATP and the helicase-like PRP5 protein suggests that these factors might promote a conformational change (involving either the U1 or U2 snRNP) that is required for the association of U2 snRNP with the pre-mRNA.

New roles for the Snp1 and Exo84 proteins in yeast pre-mRNA splicing.

The mammalian 70K protein, a component of the U1 small nuclear ribonucleoprotein involved in pre-mRNA splicing, interacts with a number of proteins important for regulating constitutive and alternative splicing. Similar proteins that interact with the yeast homolog of the 70K protein, Snp1p, have yet to be identified. We used the two-hybrid system to find four U1-Snp1 associating (Usa) proteins. Two of these proteins physically associate with Snp1p as assayed by coimmunoprecipitation. One is Prp8p, a known, essential spliceosomal component. This interaction suggests some novel functions for Snp1p and the U1 small nuclear ribonucleoprotein late in spliceosome development. The other, Exo84p, is a conserved subunit of the exocyst, an eight-protein complex functioning in secretion. We show here that Exo84p is also involved in pre-mRNA splicing. A temperature-sensitive exo84 mutation caused increased ratios of pre-mRNA to mRNA for the Rpl30 and actin transcripts in cells incubated at the non-permissive temperature. The mutation also led to a defect in splicing and prespliceosome formation in vitro; an indication that Exo84p has a direct role in splicing. The results elucidate a surprising link between splicing and secretion.

Translation and developmental regulation of RNA encoded by the eukaryotic transposable element copia.

copia-specific RNA was isolated from Drosophila melanogaster tissue culture cells by hybridization of cytoplasmic polyadenylylated RNA to copia DNA immobilized on cellulose. The purified RNA was translated in reticulocyte lysates. One major polypeptide of approximately 51,000 daltons was synthesized in addition to several others between 18,000 and 38,000 daltons. The 51,000-dalton polypeptide and several of the others are encoded by mRNAs of about 2000 nucleotides. The approximate locations on the copia element of the coding sequences for the 51,000-dalton polypeptide and several other proteins were determined by hybrid-arrested translation with copia restriction fragments. The relative abundance of copia-specific RNA was determined at various stages of the Drosophila life cycle. The level of copia-specific RNA is modulated during development of the organism, with the highest level occurring during the larval stages.