To characterize sequences in the RNA helicase‐like PRP2 protein of Saccharomyces cerevisiae that are essential for its function in pre‐mRNA splicing, a pool of random PRP2 mutants was generated. A dominant negative allele was isolated which, when overexpressed in a wild‐type yeast strain, inhibited cell growth by causing a defect in pre‐mRNA splicing. This defect was partially alleviated by simultaneous co‐overexpression of wild‐type PRP2. The dominant negative PRP2 protein inhibited splicing in vitro and caused the accumulation of stalled splicing complexes. Immunoprecipitation with anti‐PRP2 antibodies confirmed that dominant negative PRP2 protein competed with its wild‐type counterpart for interaction with spliceosomes, with which the mutant protein remained associated. The PRP2‐dn1 mutation led to a single amino acid change within the conserved SAT motif that in the prototype helicase eIF‐4A is required for RNA unwinding. Purified dominant negative PRP2 protein had approximately 40% of the wild‐type level of RNA‐stimulated ATPase activity. As ATPase activity was reduced only slightly, but splicing activity was abolished, we propose that the dominant negative phenotype is due primarily to a defect in the putative RNA helicase activity of PRP2 protein.

The RNA helicase‐like splicing factor PRP2 interacts only transiently with spliceosomes. To facilitate analysis of interactions of PRP2 with spliceosomal components, PRP2 protein was stalled in splicing complexes by two different methods. A dominant negative mutant form of PRP2 protein, which associates stably with spliceosomes, was found to interact directly with pre‐mRNAs, as demonstrated by UV‐crosslinking experiments. The use of various mutant and truncated pre‐mRNAs revealed that this interaction requires a spliceable pre‐mRNA and an assembled spliceosome; a 3′ splice site is not required. To extend these observations to the wild‐type PRP2 protein, spliceosomes were depleted of ATP; PRP2 protein interacts with pre‐mRNA in these spliceosomes in an ATP‐independent fashion. Comparison of RNA binding by PRP2 protein in the presence of ATP or gamma S‐ATP showed that ATP hydrolysis rather than mere ATP binding is required to release PRP2 protein from pre‐mRNA. As PRP2 is an RNA‐stimulated ATPase, these experiments strongly suggest that the pre‐mRNA is the native co‐factor stimulating ATP hydrolysis by PRP2 protein in spliceosomes. Since PRP2 is a putative RNA helicase, we propose that the pre‐mRNA is the target of RNA displacement activity of PRP2 protein, promoting the first step of splicing.

No abstract

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Δ allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.

Defensins are major components of a peptide-based, antimicrobial system in human neutrophils. While packed with peptide, circulating cells contain no defensin-1 (def1) transcripts, except in some leukemia patients and in derivative promyelocytic leukemia cell lines. Expression is modulated by serum factors, mediators of inflammation, and kinase activators and inhibitors, but the underlying mechanisms are not fully understood. A minimal def1 promoter drives transcription in HL-60 cells under control of PU.1 and a def1-binding protein (“D1BP”), acting through, respectively, proximal (−22/−19) and distal (−62/−59) GGAA elements. In this study, we identify D1BP, biochemically and functionally, as GA-binding protein (GABP)α/GABPβ. Whereas GABP operates as an essential upstream activator, PU.1 assists the flanking “TTTAAA” element (−32/−27), a “weak” but essential TATA box, to bring TBP/TFIID to the transcription start site. PU.1 thus imparts a degree of cell specificity to the minimal promoter and provides a potential link between a number of signaling pathways and TFIID. However, a “strong” TATA box (“TATAAA”) eliminates the need for the PU.1 binding site and for PU.1, but not for GABP. As GABP is widely expressed, a strong TATA box thus alleviates promyelocytic cell specificity of the def1 promoter. These findings suggest how the myeloid def1 promoter may have evolutionarily acquired its current properties.