To identify new gene products that participate in ribosome biogenesis, we carried out a screen for mutations that result in lethality in combination with mutations in DRS1, a Saccharomyces cerevisiae nucleolar DEAD-box protein required for synthesis of 60S ribosomal subunits. We identified the gene NOP7that encodes an essential protein. The temperature-sensitive nop7-1 mutation or metabolic depletion of Nop7p results in a deficiency of 60S ribosomal subunits and accumulation of halfmer polyribosomes. Analysis of pre-rRNA processing indicates that nop7 mutants exhibit a delay in processing of 27S pre-rRNA to mature 25S rRNA and decreased accumulation of 25S rRNA. Thus Nop7p, like Drs1p, is required for essential steps leading to synthesis of 60S ribosomal subunits. In addition, inactivation or depletion of Nop7p also affects processing at the A0, A1, and A2 sites, which may result from the association of Nop7p with 35S pre-rRNA in 90S pre-rRNPs. Nop7p is localized primarily in the nucleolus, where most steps in ribosome assembly occur. Nop7p is homologous to the zebrafish pescadillo protein necessary for embryonic development. The Nop7 protein contains the BRCT motif, a protein-protein interaction domain through which, for example, the human BRCA1 protein interacts with RNA helicase A.

To investigate the role of mRNA 3' inverted repeats (IRs) in stabilizing plant chloroplast mRNAs, we have measured the processing and stability of wild-type and mutant RNAs corresponding to the 3' end of the spinach chloroplast psbA mRNA. wild-type and mutant 3' IR-RNA precursors were processed at similar rates in a homologous in vitro system, but RNAs with either a mutant loop sequence CUUCGG or a specific base substitution in the IR exhibited an enhanced accumulation of mature product. Incubation of mature products in the in vitro system demonstrated that this was due to an increased stability of the product. These mutant RNAs displayed the same order of stabilities when their decay was measured following electroporation into intact chloroplasts. We found that the in vitro system contains an endonuclease activity that cleaves the wild-type 3' IR-RNA within the loop and also in single-stranded regions, suggesting a possible role for the loop sequence in determining RNA longevity in vitro. Interestingly, the altered loop sequence CUUCGG, which enhances RNA stability in bacteria (1), prolonged the half-life of psbA 3' IR-RNA in vitro and also resulted in an altered endonuclease cleavage pattern. Such nucleases could potentially play an important role in plastid mRNA decay in vivo.

In chloroplasts, the 3 untranslated regions of most mRNAs contain a stem-loop-forming inverted repeat (IR) sequence that is required for mRNA stability and correct 3-end formation. The IR regions of several mRNAs are also known to bind chloroplast proteins, as judged from in vitro gel mobility shift and UV cross-linking assays, and these RNA-protein interactions may be involved in the regulation of chloroplast mRNA processing and/or stability. Here we describe in detail the RNA and protein components that are involved in 3 IR-containing RNA (3 IR-RNA)-protein complex formation for the spinach chloroplast petD gene, which encodes subunit IV of the cytochrome b 6 /f complex. We show that the complex contains 55-, 41-, and 29-kDa RNA-binding proteins (ribonucleoproteins [RNPs]). These proteins together protect a 90-nucleotide segment of RNA from RNase T 1 digestion; this RNA contains the IR and downstream flanking sequences. Competition experiments using 3 IR-RNAs from the psbA or rbcL gene demonstrate that the RNPs have a strong specificity for the petD sequence. Site-directed mutagenesis was carried out to define the RNA sequence elements required for complex formation. These studies identified an 8-nucleotide AU-rich sequence downstream of the IR; mutations within this sequence had moderate to severe effects on RNA-protein complex formation. Although other similar sequences are present in the petD 3 untranslated region, only a single copy, which we have termed box II, appears to be essential for in vitro protein binding. In addition, the IR itself is necessary for optimal complex formation. These two sequence elements together with an RNP complex may direct correct 3-end processing and/or influence the stability of petD mRNA in chloroplasts.The roles of mRNA 3Ј untranslated regions (UTRs) in regulating gene expression have become prominent (1,18,30,44). In chloroplasts, the 3Ј UTRs of most mRNAs are flanked by stem-loop structure-forming inverted repeat (IR) sequences that participate in 3Ј-end formation and are also thought to impede the progress of processive exoribonucleases (32,36,39,40). Both the 3Ј IR and the 5Ј UTR have been shown by in vitro assays to associate with proteins which may regulate RNA processing, RNA stability, and/or translation initiation (10,28,32,38,46).The mechanism of chloroplast mRNA 3Ј-end formation has been studied by using both in vitro assays and chloroplast transformation. In vitro transcription experiments were used to show that although the chloroplast RNA polymerase is capable of recognizing rho-independent prokaryotic transcription termination signals (8, 36), most chloroplast 3Ј IRs have little or no transcription termination activity (36). Data obtained by comparing in vivo transcription rates upstream and downstream of the Chlamydomonas atpB 3Ј IR are consistent with the in vitro observations (39). This finding suggests that most chloroplast mRNAs are synthesized as precursors, which are subsequently processed to yield the uniform 3Ј termini found in vivo.Both RNases and R...

The aims of this study are to examine the constructs of transformational leadership as they played out for one nurse who steered significant change in the care of the dying in the United States and to provide deeper insights into how nursing leaders can design and direct meaningful changes in the delivery of health care in turbulent times. A significant problem was identified in how the terminally ill were treated in this country post World War II. The introduction of hospice care in the United States represented a paradigm shift in how the health care community viewed and treated dying patients. Critical to this transformation was the work of Florence Wald, who organized with community leaders, clergy, and other health care providers to create a vision and synergy around palliative care. She was instrumental in opening the first American hospice in 1971 in Connecticut. Within 15 years, there were more than 1,000 hospices in the United States. A single case study design was chosen for this qualitative research grounded in the theory of transformational leadership (J.M. Burns, 1978). The study used narrative inquiry to conduct an in-depth exploration of Florence Wald's transformational leadership and the perceptions of the group of founders she organized to conceptualize, build, and open the first hospice in the United States. The participants chosen for interview were involved directly in the designing, planning, and beginning of the first American hospice. In addition to the seven in-depth interviews conducted in 2007 in Connecticut, this research examined three groups of documents from The Florence and Henry Wald Archives in the Yale University Library. The findings from both interviews and the Yale Archives showed that Florence Wald based her leadership on the strong values of reverence for life and social justice for all. To direct meaningful change, Florence Wald elevated the consciousness of her hospice team by conducting a 2-year research study on the needs of dying patients to ensure interventions were based on evidence. To encourage a high level of participation, Florence Wald demonstrated a caring component in her leadership with a strong commitment to mentoring. Wald worked to transform the quality of end-of-life care by assessing the readiness for change prior to acting and by working to provide supports for success. Finally, the findings showed that Florence Wald built consensus on vision before executing purposeful change by collaborating with the Founders and asking the hard questions to examine standards of care. Florence Wald provided transformational leadership in creating a value-driven culture of inquiry among the Founders where decision making was evidence-based and significantly improved the quality of palliative care in the United States. Nursing leaders who build upon the shared values to provide direction and promote momentum critical to the change will have more success in reaching strategic outcomes of transformational efforts. Transformational nursing leaders who build consensus on vis...

The PRP4 gene encodes a protein that is a component of the U4/U6 small nuclear ribonucleoprotein particle and is necessary for both spliceosome assembly and pre-mRNA splicing. To identify genes whose products interact with the PRP4 gene or gene product, we isolated second-site suppressors of temperature-sensitive prp4 mutations. We limited ourselves to suppressors with a distinct phenotype, cold sensitivity, to facilitate analysis of mutants. Ten independent recessive suppressors were obtained that identified four complementation groups, spp41, spp42, spp43 and spp44 (suppressor of prp4, numbers 1-4). spp41-spp44 suppress the pre-mRNA splicing defect as well as the temperature-sensitive phenotype of prp4 strains. Each of these spp mutations also suppresses prp3; spp41 and spp42 suppress prp11 as well. Neither spp41 nor spp42 suppressors null alleles of prp3 or prp4, indicating that the suppression does not occur via a bypass mechanism. The spp41 and spp42 mutations are neither allele- nor gene-specific in their pattern of suppression and do not result in a defect in pre-mRNA splicing. Thus the SPP41 and SPP42 gene products are unlikely to participate directly in mRNA splicing or interact directly with Prp3p or Prp4p. Expression of PRP3-lacZ and PRP4-lacZ gene fusions is increased in spp41 strains, suggesting that wild-type Spp41p represses expression of PRP3 and PRP4. SPP41 was cloned and sequenced and found to be essential. spp43 is allelic to the previously identified suppressor srn1, which encodes a negative regulator of gene expression.