Effectors of a developmental mitogen-activated protein kinase cascade revealed by expression signatures of signaling mutants.

Despite the importance of mitogen-activated protein kinase (MAPK) signaling in eukaryotic biology, the mechanisms by which signaling yields phenotypic changes are poorly understood. We have combined transcriptional profiling with genetics to determine how the Kss1 MAPK signaling pathway controls dimorphic development in Saccharomyces cerevisiae. This analysis identified dozens of transcripts that are regulated by the pathway, whereas previous work had identified only a single downstream target, FLO11. One of the MAPK-regulated genes is PGU1, which encodes a secreted enzyme that hydrolyzes polygalacturonic acid, a structural barrier to microbial invasion present in the natural plant substrate of S. cerevisiae. A third key transcriptional target is the G(1) cyclin gene CLN1, a morphogenetic regulator that we show to be essential for pseudohyphal growth. In contrast, the homologous CLN2 cyclin gene is dispensable for development. Thus, the Kss1 MAPK cascade programs development by coordinately modulating a cell adhesion factor, a secreted host-destroying activity, and a specialized subunit of the Cdc28 cyclin-dependent kinase.

MAP kinase and cAMP filamentation signaling pathways converge on the unusually large promoter of the yeast FLO11 gene.

In Saccharomyces cerevisiae, two major signal transduction pathways, the Kss1 MAPK pathway and the cAMP-regulated pathway, are critical for the differentiation of round yeast form cells to multicellular, invasive pseudohyphae. Here we report that these parallel pathways converge on the promoter of a gene, FLO11, which encodes a cell surface protein required for pseudohyphal formation. The FLO11 promoter is unusually large, containing at least four upstream activation sequences (UASs) and nine repression elements which together span at least 2.8 kb. Several lines of evidence indicate that the MAPK and cAMP signals are received by distinct transcription factors and promoter elements. First, regulation via the MAPK pathway requires the transcription factors Ste12p/Tec1p, whereas cAMP-mediated activation requires a distinct factor, Flo8p. Secondly, mutations in either pathway block FLO11 transcription. Overexpression of STE12 can suppress the loss of FLO8, and overexpression of FLO8 can suppress the loss of STE12. Finally, multiple distinct promoter regions of the FLO11 promoter are required for its activation by either Flo8p or Ste12p/ Tec1p. Thus, like the promoters of the key developmental genes, HO and IME1, the FLO11 promoter is large and complex, endowing it with the ability to integrate multiple inputs.

The control of filamentous differentiation and virulence in fungi.

Many members of the fungal kingdom have a distinguishing feature, dimorphism, which is the ability to switch between two morphological forms: a cellular yeast form and a multicellular invasive filamentous form. At least three pathways are involved in regulating the transition between these two forms in the budding yeast Saccharomyces cerevisiae, and evidence is now emerging that homologous signalling modules are involved in regulating filament formation and virulence in a range of human and plant fungal pathogens. Strikingly, components used to signal sexual differentiation in the response to mating pheromones are often reutilized to regulate dimorphic development, suggesting an ancient link between these processes.

The riddle of MAP kinase signaling specificity.

Cells encounter an enormous variety of signals in their environments and must respond to each stimulus appropriately with changes in their genetic programs. Many of these external signals are transduced by a highly conserved eukaryotic signaling mechanism, the mitogen-activated protein kinase (MAPK) cascade. How are the myriad of inputs transduced accurately so that each evokes a specific response? One mechanism would be to have a distinct MAPK cascade for each signal; however, the situation appears to be more complicated.

MAP kinases with distinct inhibitory functions impart signaling specificity during yeast differentiation.

Filamentous invasive growth of S. cerevisiae requires multiple elements of the mitogen-activated protein kinase (MAPK) signaling cascade that are also components of the mating pheromone response pathway. Here we show that, despite sharing several constituents, the two pathways use different MAP kinases. The Fus3 MAPK regulates mating, whereas the Kss1 MAPK regulates filamentation and invasion. Remarkably, in addition to their kinase-dependent activation functions, Kss1 and Fus3 each have a distinct kinase-independent inhibitory function. Kss1 inhibits the filamentation pathway by interacting with its target transcription factor Ste12. Fus3 has a different inhibitory activity that prevents the inappropriate activation of invasion by the pheromone response pathway. In the absence of Fus3, there is erroneous crosstalk in which mating pheromone now activates filamentation-specific gene expression using the Kss1 MAPK.

Combinatorial control required for the specificity of yeast MAPK signaling.

In yeast, an overlapping set of mitogen-activated protein kinase (MAPK) signaling components controls mating, haploid invasion, and pseudohyphal development. Paradoxically, a single downstream transcription factor, Ste12, is necessary for the execution of these distinct programs. Developmental specificity was found to require a transcription factor of the TEA/ATTS family, Tec1, which cooperates with Ste12 during filamentous and invasive growth. Purified derivatives of Ste12 and Tec1 bind cooperatively to enhancer elements called filamentation and invasion response elements (FREs), which program transcription that is specifically responsive to the MAPK signaling components required for filamentous growth. An FRE in the TEC1 promoter functions in a positive feedback loop required for pseudohyphal development.

Genetic interactions between the yeast RNA helicase homolog Prp16 and spliceosomal snRNAs identify candidate ligands for the Prp16 RNA-dependent ATPase.

Pre-mRNA splicing occurs in a large and dynamic ribonucleoprotein complex, the spliceosome. Several protein factors involved in splicing are homologous to a family of RNA-dependent ATPases, the so-called DEAD/DEAH proteins. A subset of these factors exhibit RNA helicase activity in vitro. The DEAD/DEAH proteins involved in splicing are thought to mediate RNA conformational rearrangements during spliceosome assembly. However, the RNA ligands for these factors are currently unknown. Here, we present genetic evidence in Saccharomyces cerevisiae for a functional interaction between the DEAH protein Prp16, and the U6 and U2 spliceosomal snRNAs. Using a library of mutagenized U6 snRNA genes, we have identified 14 strong suppressors of the cold-sensitive (cs) allele, prp16-302. Remarkably, each suppressor contains a single nucleotide deletion of 1 of the 6 residues that lie immediately upstream of a sequence in U6 that interacts with the 5' splice site. Analysis of site-directed mutations revealed that nucleotide substitutions in the adjacent U2-U6 helix I structure also suppress prp16-302, albeit more weakly. The U6 suppressors tested also partially reverse the phenotype of two other cs alleles, prp16-1 and prp16-301, but not the four temperature-sensitive alleles tested. Finally, overexpression of each cs allele exacerbates its recessive growth phenotype and confers a dominant negative cs phenotype. We propose that the snRNA suppressors function by destabilizing an interaction between the U2-U6 complex and a hypothetical factor (X), which is trapped by cs mutants of PRP16. The phenotypes of overexpressed prp16 alleles are consistent with the model that this trapped interaction inhibits the dissociation of Prp16 from the spliceosome. We discuss the intriguing possibility that factor X is Prp16 itself.

Randomization-selection analysis of snRNAs in vivo: evidence for a tertiary interaction in the spliceosome.

Putative components of the spliceosomal active site include a bulged helix between U2 and U6 snRNAs (U2-U6 helix I) and the adjacent ACAGAG hexanucleotide in U6. We have developed an in vivo, bimolecular randomization-selection method to functionally dissect these elements. Although a portion of U2-U6 helix I resembles the G-binding site of group I introns, the data are inconsistent with an analogous functional role for this structure in the spliceosome. Instead, analysis of several novel covariants supports the existence of a structure in which the helix I bulge engages in a tertiary interaction with the terminal residue of the U6 hexanucleotide (ACAGAG). Such a higher order structure, together with other known interactions, would juxtapose the two clusters of residues of the U2-U6 complex that are specifically required for the second chemical step of pre-mRNA splicing with the 3' splice site. Indeed, mutations in the residues that participate in the tertiary interaction affect both the efficiency and fidelity of 3' splice site usage.

A novel base-pairing interaction between U2 and U6 snRNAs suggests a mechanism for the catalytic activation of the spliceosome.

Prior to the chemical steps of mRNA splicing, the extensive base-pairing interaction between the U4 and U6 spliceosomal snRNAs is disrupted. Here, we use a mutational analysis in yeast to demonstrate a conserved base-pairing interaction between the U6 and U2 snRNAs that is mutually exclusive with the U4-U6 interaction. In this novel pairing, conserved sequences in U6 interact with a sequence in U2 that is immediately upstream of the branch point recognition region. Remarkably, the residues in U6 that can be consequently juxtaposed with the intron substrate include those that have been proposed previously to be catalytic. Both the first and second steps of splicing are inhibited when this base-paired structure is mutated. These observations, together with the high conservation of the U2-U6 structure, lead us to propose that it might be a component of the spliceosomal active site.

Multiple roles for U6 snRNA in the splicing pathway.

U6 is the most highly conserved of the five spliceosomal RNAs. It is associated with U4 by an extensive base-pairing interaction, which is disrupted immediately prior to the first nucleolytic step of splicing. It has been proposed that this event activates catalysis by unmasking U6. Using a combination of doped synthesis and site-directed mutagenesis to generate point mutations in U6, we have now identified 12 positions, in three domains, at which single nucleotide substitutions or deletions result in lethal or temperature-sensitive phenotypes. Biochemical analysis demonstrates that most of these mutants retain the ability to assemble into U4/U6 and U4/U5/U6 snRNPs. Notably, although mutations at three positions in U6 that base-pair with U4 are lethal, mutations in the complementary residues in U4 are fully viable. Furthermore, compensatory mutations in U4 that restore base-pairing fail to suppress the phenotypes of the U6 mutations. This demonstrates a function for U6 independent of its role in base-pairing. Remarkably, two of the three essential regions in U6 identified genetically correspond to intron insertion points in two yeast species. A temperature-sensitive mutation at one of these sites is defective in the second step of splicing in vitro.

Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region.

The gag-pol protein of Rous sarcoma virus (RSV), the precursor to the enzymes responsible for reverse transcription and integration, is expressed from two genes that lie in different translational reading frames by ribosomal frameshifting. Here, we localize the site of frameshifting and show that the frameshifting reaction is mediated by slippage of two adjacent tRNAs by a single nucleotide in the 5' direction. The gag terminator, which immediately follows the frameshift site, is not required for frameshifting. Other suspected retroviral frameshift sites mediate frameshifting when placed at the end of RSV gag. Mutations in RSV pol also affect synthesis of the gag-pol protein in vitro. The effects of these mutations best correlate with the potential to form an RNA stem-loop structure adjacent to the frameshift site. A short sequence of RSV RNA, 147 nucleotides in length, containing the frameshift site and stem-loop structure, is sufficient to direct frameshifting in a novel genetic context.

Differential DNA repair in transcriptionally active and inactive proto-oncogenes: c-abl and c-mos.

DNA repair was examined in the c-abl and c-mos proto-oncogenes in UV-irradiated mouse 3T3 fibroblasts using an optimized assay for measuring pyrimidine dimers in single-copy nucleotide sequences. We found similar initial dimer frequencies in the two genes. Within 24 hr, 85% of the dimers were removed from the 20 kb intragenic BamHl restriction fragment of the expressed c-abl gene, while only 22% of the dimers were removed from the 15 kb EcoRl fragment that spans the transcriptionally inactive c-mos locus. Quiescent and actively growing cells showed similar relative efficiencies of repair in the two regions. This is the first demonstration of differential DNA repair in two different genes. These findings have important implications for the mechanisms of proto-oncogene activation.

alpha DNA in African green monkey cells is organized into extremely long tandem arrays.

We have determined the size of arrays formed by tandemly repeated monomers of alpha DNA in African green monkey cells. DNA fragments containing intact alpha DNA arrays were generated by digestion of genomic DNA with restriction endonuclease that do not have sites in the alpha DNA consensus sequence. Their size was determined by Southern analysis and by sedimentation through neutral sucrose gradients followed by probing of each fraction for alpha sequences. The restriction fragments varied in size with the most frequent being 78 kilobase pairs long. We have also shown that they contain very little non-alpha DNA sequences. This suggests a minimum array of 450 tandemly repeated alpha DNA monomers, which is more than an order of magnitude larger than previously supposed.