Isolated growth hormone deficiency type II caused by a point mutation that alters both splice site strength and splicing enhancer function.

A heterozygous single base mutation in the human growth hormone (GH) gene (GH-1) was identified in a family presenting with isolated GH deficiency type II (IGHD II). Affected individuals have a guanine to adenine transition at the first nucleotide of exon 3 (E3+1 G-->A) that results in exon skipping and production of a dominant-negative 17.5-kDa isoform. We show that the mechanistic basis for exon skipping is due to the unique position of this mutation because it weakens the 3' splice site and simultaneously disrupts a splicing enhancer located within the first seven bases of exon 3. A G-->T mutation at this same position not only affects splicing but also results in a premature stop codon for those transcripts that include exon 3. Thus, mutations that alter the first nucleotide of exon 3 illustrate the various mechanisms by which changes in sequence can cause disease: splice site selection, splicing enhancer function, messenger RNA decay, missense mutations, and nonsense mutations. For IGHD II, only exon skipping leads to production of the dominant-negative isoform, with increasing skipping correlating with increasing disease severity.

siRNA therapeutics: big potential from small RNAs.

RNA interference (RNAi) is now an umbrella term referring to post-transcriptional gene silencing mediated by either degradation or translation arrest of target RNA. This process is initiated by double-stranded RNA with sequence homology driving specificity. The discovery that 21-23 nucleotide RNA duplexes (small-interfering RNAs, siRNAs) mediate RNAi in mammalian cells opened the door to the therapeutic use of siRNAs. While much work remains to optimize delivery and maintain specificity, the therapeutic advantages of siRNAs for treatment of viral infection, dominant disorders, cancer, and neurological disorders show great promise.

Nuclear relocalization of the pre-mRNA splicing factor PSF during apoptosis involves hyperphosphorylation, masking of antigenic epitopes, and changes in protein interactions.

The spatial nuclear organization of regulatory proteins often reflects their functional state. PSF, a factor essential for pre-mRNA splicing, is visualized by the B92 mAb as discrete nuclear foci, which disappeared during apoptosis. Because this mode of cell death entails protein degradation, it was considered that PSF, which like other splicing factors is sensitive to proteolysis, might be degraded. Nonetheless, during the apoptotic process, PSF remained intact and was N-terminally hyperphosphorylated on serine and threonine residues. Retarded gel migration profiles suggested differential phosphorylation of the molecule in mitosis vs. apoptosis and under-phosphorylation during blockage of cells at G1/S. Experiments with the use of recombinant GFP-tagged PSF provided evidence that in the course of apoptosis the antigenic epitopes of PSF are masked and that PSF reorganizes into globular nuclear structures. In apoptotic cells, PSF dissociated from PTB and bound new partners, including the U1--70K and SR proteins and therefore may acquire new functions.

An RNA recognition motif (RRM) is required for the localization of PTB-associated splicing factor (PSF) to subnuclear speckles.

Using fusions with green fluorescent protein (GFP), we have identified sequences in the polypyrimidine tract binding protein-associated splicing factor (PSF) that are involved in nuclear and subnuclear localization. Like other splicing factors, PSF localizes to the nucleus, is absent from nucleoli, and accumulates in punctate structures within the nucleus referred to as speckles. However, PSF lacks the known speckle localization domains that have been identified in other proteins. Instead, the localization of PSF to speckles is dependent on an RNA recognition motif (RRM). PSF comprises an N-terminal proline- and glutamine-rich domain, two RRMs (RRM1 and RRM2), and a C-terminal region that contains two nuclear localization signals, both of which are required for complete nuclear localization. Deletion of RRM2 led to a complete loss of speckle localization and resulted in diffuse accumulation of PSF in the nucleus, indicating that RRM2 is required for subnuclear localization. Thus, PSF appears to localize to speckles through a novel pathway that is dependent on its second RRM. Consistent with the use of a novel subnuclear targeting pathway, PSF redistributes to perinucleolar clusters upon the addition of a transcription inhibitor whereas other splicing factors display increased localization to speckles in the absence of transcription. A yeast two-hybrid screen identified four-and-a-half LIM-only protein 2 (FHL2) as a potential RRM2 interaction partner, indicating a possible role for zinc-finger or LIM domains in the localization of splicing factors to subnuclear speckles.

Identification and characterization of a novel serine-arginine-rich splicing regulatory protein.

We have identified an 86-kDa protein containing a single amino-terminal RNA recognition motif and two carboxy-terminal domains rich in serine-arginine (SR) dipeptides. Despite structural similarity to members of the SR protein family, p86 is clearly unique. It is not found in standard SR protein preparations, does not precipitate in the presence of high magnesium concentrations, is not recognized by antibodies specific for SR proteins, and cannot complement splicing-defective S100 extracts. However, we have found that p86 can inhibit the ability of purified SR proteins to activate splicing in S100 extracts and can even inhibit the in vitro and in vivo activation of specific splice sites by a subset of SR proteins, including ASF/SF2, SC35, and SRp55. In contrast, p86 activates splicing in the presence of SRp20. Thus, it appears that pairwise combination of p86 with specific SR proteins leads to altered splicing efficiency and differential splice site selection. In all cases, such regulation requires the presence of the two RS domains and a unique intervening EK-rich region, which appear to mediate direct protein-protein contact between these family members. Full-length p86, but not a mutant lacking the RS-EK-RS domains, was found to preferentially interact with itself, SRp20, ASF/SF2, SRp55, and, to a slightly lesser extent, SC35. Because of the primary sequence and unique properties of p86, we have named this protein SRrp86 for SR-related protein of 86 kDa.

A new double-stranded RNA-binding protein that interacts with PKR.

We have identified a 74 kDa double-stranded (ds)RNA-binding protein that shares extensive homology with the mouse spermatid perinuclear RNA-binding (Spnr) protein. p74 contains two dsRNA-binding motifs (dsRBMs) that are essential for preferential binding to dsRNA. Previously, dsRNA-binding proteins were shown to undergo homo- and heterodimerization, raising the possibility that regulation of activity could be controlled by interactions between different family members. Homodimerization is required to activate the dsRNA-dependent protein kinase PKR, whereas hetero-dimerization between PKR and other dsRNA-binding proteins can inhibit kinase activity. We have found that p74 also interacts with PKR, both the wild-type enzyme and a catalytically defective mutant (K296R). While co-expression of p74 and wild-type PKR in the yeast Saccharomyces cerevisiae did not alter PKR activity, co-expression of p74 and the catalytically defective K296R mutant surprisingly resulted in abnormal morphology and cell death in transformants that maintained a high level of p74 expression. These transformants could be rescued by overexpression of the alpha-subunit of wild-type eukaryotic translation initiation factor 2 (eIF2alpha), one of the known substrates for PKR. We hypothesize that competing heterodimers between p74-K296R PKR and eIF2alpha-K296R PKR may control cell growth such that stabilization of the p74-K296R PKR heterodimer induces abnormal morphology and cell death.

Identification of PSF, the polypyrimidine tract-binding protein-associated splicing factor, as a developmentally regulated neuronal protein.

The polypyrimidine tract-binding protein-associated splicing factor (PSF), which plays an essential role in mammalian spliceosomes, has been found to be expressed by differentiating neurons in developing mouse brain. The sequence of a fragment of mouse PSF was found to be remarkably similar to that of human PSF. Both the expression of PSF mRNA in cortex and cerebellum and PSF immunoreactivity in all brain areas were high during embryonic and early postnatal life and almost disappeared in adult tissue, except in the hippocampus and olfactory bulb where various neuronal populations remained PSF-immunopositive. Double-labeling experiments with anti-PSF antibody and anti-neurofilaments or anti-glial fibrillary acidic protein antibodies on sections of cortex, hippocampus, and cerebellum indicate that PSF is expressed by differentiating neurons but not by astrocytic cells. In vitro, mouse PSF was found to be expressed by differentiating cortical and cerebellar neurons. Radial glia or astrocyte nuclei were not immunopositive; however, oligodendrocytes differentiating in vitro were found to express PSF. The restricted expression of PSF suggests that this splicing factor could be involved in the control of neuronal-specific splicing events occurring at particular stages of neuronal differentiation and maturation.

Enhancer elements activate the weak 3' splice site of alpha-tropomyosin exon 2.

We have identified four purine-rich sequences that act as splicing enhancer elements to activate the weak 3' splice site of alpha-tropomyosin exon 2. These elements also activate the splicing of heterologous substrates containing weak 3' splice sites or mutated 5' splice sites. However, they are unique in that they can activate splicing whether they are placed in an upstream or downstream exon, and the two central elements can function regardless of their position relative to one another. The presence of excess RNAs containing these enhancers could effectively inhibit in vitro pre-mRNA splicing reactions in a substrate-dependent manner and, at lower concentrations of competitor RNA, the addition of SR proteins could relieve the inhibition. However, when extracts were depleted by incubation with biotinylated exon 2 RNAs followed by passage over streptavidin agarose, SR proteins were not sufficient to restore splicing. Instead, both SR proteins and fractions containing a 110-kD protein were necessary to rescue splicing. Using gel mobility shift assays, we show that formation of stable enhancer-specific complexes on alpha-tropomyosin exon 2 requires the presence of both SR proteins and the 110-kD protein. By analogy to the doublesex exon enhancer elements in Drosophila, our results suggest that assembly of mammalian exon enhancer complexes requires both SR and non-SR proteins to activate selection of weak splice sites.

Multiple RRMs contribute to RNA binding specificity and affinity for polypyrimidine tract binding protein.

Polypyrimidine tract binding protein (PTB) is a 57 kD hnRNP protein (hnRNP I) that binds to the pyrimidine tract typically found near the 3' end of introns. Primary sequence analysis suggests that PTB contains four RNA recognition motifs (RRMs). Data from comparative structural and deletional analysis of PTB are consistent with the presence of a four reiterated domain structure. Since PTB exists in solution as a homodimer, it contains an oligomeric array of eight RRMs. Though the function of RRMs in a monomeric context has been addressed, the significance of their presence in an oligomeric context has not been investigated. To correlate structural motifs with function, we have analyzed the RNA binding properties of wild-type and deletion constructs of PTB that contain RRMs in both an oligomeric and monomeric context. These studies indicate that there is not a strong correlation between the RNA binding affinity and specificity upon oligomerization. However, the mode of RNA interaction and dimerization is linked. We have also found that the primary contributor to the free energy of PTB binding and the primary determinant for RNA binding specificity resides in RRM 3, while the primary contributor to dimer stabilization coincides with residues in RRM 2.

Mutation of PTB binding sites causes misregulation of alternative 3' splice site selection in vivo.

Alternative splicing of pre-mRNA is a commonly used mechanism to regulate gene expression in higher eukaryotes. However, with the exception of regulated cascades in Drosophila, the cis-acting elements and the trans-acting factors that control tissue- and/or developmentally regulated splicing remain largely unidentified. Cis-acting elements that control smooth muscle-specific repression of exon 3 of alpha-tropomyosin (alpha-TM) have been identified recently and consist of two regions that flank this exon. Deletion of either element causes misregulated splicing of alpha-TM in transfected smooth muscle cells. In experiments designed to characterize essential sequences within each element and the factors that interact with these sequences, we have identified two overlapping sequences within the downstream regulatory element (DRE) that are identical to binding sites for polypyrimidine tract binding protein (PTB) that were identified using iterative selection techniques. Mutation of these sites caused aberrant splicing regulation in transfected smooth muscle cells. In addition, sequences identical to high-affinity PTB binding sites were also detected upstream of exon 3 and mutation of these sites also resulted in misregulation of splicing in vivo, suggesting that PTB binding to specific sequences flanking exon 3 is responsible, in part, for the repression of exon 3. Consistent with this hypothesis, UV crosslinking and equilibrium binding assays confirm that the same mutations that cause misregulated splicing also disrupt PTB binding to RNA.

Enhancing the clinical practicum experience through journal writing.

Although journaling is commonplace, its usefulness or effectiveness has been rarely supported by empirical data or examples. The purpose of this article is to highlight integration of classroom and clinical learning, decision-making skills and level of skill acquisition in the Senior Practicum experiences through student journal entry exemplars. Critical thinking, observation and description, and empathy and release of feelings will be discussed.

Functional analysis of the polypyrimidine tract in pre-mRNA splicing.

The polypyrimidine tract is one of the important cis-acting sequence elements directing intron removal in pre-mRNA splicing. Progressive deletions of the polypyrimidine tract have been found to abolish correct lariat formation, spliceosome assembly and splicing. In addition, the polypyrimidine tract can alter 3'-splice site selection by promoting alternative branch site selection. However, there appears to be great flexibility in the specific sequence of a given tract. Not only the optimal composition of the polypyrimidine tract, but also the role of the tract in introns with no apparent polypyrimidine tracts or where changes in the tract are apparently harmless are uncertain. Accordingly, we have designed a series of cis-competition splicing constructs to test the functional competitive efficiency of a variety of systematically mutated polypyrimidine tracts. An RT/PCR assay was used to detect spliced product formation as a result of differential branch point selection dependent on direct competition between two opposing polypyrimidine tracts. We found that pyrimidine tracts containing 11 continuous uridines are the strongest pyrimidine tracts. In such cases, the position of the uridine stretch between the branch point and 3'-splice site AG is unimportant. In contrast, decreasing the continuous uridine stretch to five or six residues requires that the tract be located immediately adjacent to the AG for optimal competitive efficiency. The block to splicing with decreasing polypyrimidine tract strength is primarily prior to the first step of splicing. While lengthy continuous uridine tracts are the most competitive, tracts with decreased numbers of consecutive uridines and even tracts with alternating purine/pyrimidine residues can still function to promote branch point selection, but are far less effective competitors in 3'-splice site selection assays.

Functional crosstalk between exon enhancers, polypyrimidine tracts and branchpoint sequences.

We recently identified enhancer elements that activate the weak 3' splice site of alpha-tropomyosin exon 2 as well as a variety of heterologous weak 3' splice sites. To understand their mechanism of action, we devised an iterative selection strategy to identify functional pyrimidine tracts and branchpoint sequences in the presence or absence of enhancer elements. Surprisingly, we found that strong pyrimidine tracts were selected regardless of the presence of enhancer elements. However, the presence of enhancer elements resulted in the selection of multiple, non-consensus branchpoint sequences. Thus, enhancer elements apparently activate weak 3' splice sites primarily by increasing the efficiency of splicing of introns containing branchpoint sequences with less than optimal U2-branchpoint pairing arrangements. Comparison of consensus sequences from both our selection strategy and compilations of published intron sequences suggests that exon enhancer elements could be widespread and play an important role in the selection of 3' splice sites.

Interaction of protein phosphatase type 1 with a splicing factor.

A gizzard cDNA library was screened by the two-hybrid system using as bait the delta isoform of the catalytic subunit of protein phosphatase 1 (PP1delta). Among the proteins identified was a fragment of the polypyrimidine tract-binding protein-associated splicing factor (PSF) and for 242 residues was 97.1% identical to the human isoforms. Binding of PSF and PP1delta was confirmed by inhibition of phosphatase activity and by an overlay technique. The PP1delta binding site was contained in the N-terminal 82 residues of the PSF fragment. PSF may therefore act as a PP1 target molecule in the spliceosome.

Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA.

The requirement of PTB, polypyrimidine tract binding protein, for internal initiation of translation has been tested using an RNA affinity column to deplete rabbit reticulocyte lysates of PTB. The affinity column was prepared by coupling CNBr-activated Sepharose with the segment of the 5'-untranslated region of encephalomyocarditis virus (EMCV) RNA previously shown to bind PTB. Lysates passed through this column were devoid of PTB, and were incapable of internal initiation of translation dependent on the EMCV 5'-untranslated region, while retaining the capacity for translation dependent on ribosome scanning. Full activity for internal initiation was restored by the addition of recombinant PTB at the physiologically relevant concentration of about 5 micrograms/mL. When various PTB deletion mutants were tested, it was found that this activity required virtually the full-length protein. Thus, PTB is an essential protein for internal initiation promoted by the EMCV 5'-untranslated region. However, the PTB-depleted lysate retained the capacity for internal initiation promoted by the 5'-untranslated regions of another cardiovirus, Theiler's murine encephalomyelitis virus, and of the unrelated hepatitis C virus, and in neither case did addition of recombinant PTB stimulate internal initiation. Therefore, PTB is not a universal internal initiation factor that is indispensable in every case of internal ribosome entry.

Regulation of alternative 3' splice site selection by constitutive splicing factors.

We have devised an in vitro splicing assay in which the mutually exclusive exons 2 and 3 of alpha-tropomyosin act as competing 3' splice sites for joining to exon 1. Splicing in normal HeLa cell nuclear extracts results in almost exclusive joining of exons 1 and 3. Splicing in decreased nuclear extract concentrations and decreased ionic strength results in increased 1-2 splicing. We have used this assay to determine the role of three constitutive pre-mRNA splicing factors on alternative 3' splice site selection. Polypyrimidine tract binding protein (PTB) was found to inhibit the splicing of introns containing a strong binding site for this factor. However, the inhibitory effect of PTB could be partially reversed if pre-mRNAs were preincubated with U2 auxiliary factor (U2AF) prior to splicing in PTB-supplemented extracts. For alpha-tropomyosin, regulation of splicing by PTB and U2AF primarily affected the joining of exons 1-3 with no dramatic increases in 1-2 splicing being detected. Preincubation of pre-mRNAs with SR proteins led to small increases in 1-2 splicing. However, if pre-mRNAs were preincubated with SR proteins followed by splicing in PTB-supplemented extracts, there was a nearly complete reversal of the normal 1-2 to 1-3 splicing ratios. Thus, multiple pairwise, and sometimes antagonizing, interactions between constitutive pre-mRNA splicing factors and the pre-mRNA can regulate 3' splice site selection.

Looking for guarantees.

The article attempts to capture some of the author's experience with the diagnosis and subsequent treatment of her son's cancer. David was 15 when he was diagnosed with Ewing's sarcoma of the left femur. He underwent chemotherapy within 3 weeks of the diagnosis, having undergone port insertion, a needle biopsy of the tumor, and numerous other diagnostic procedures. After four chemotherapy treatments and a series of radiation treatments, David underwent an 8-hour operation to reconstruct the left femur. Bank bone was used as well as the fibula from his lower leg and iliac crest bone from the same side. David continued to be on chemotherapy treatments for just under 2 more years. As a result of this experience, the author's priorities, ways of being with clients, relationships with family members and friends, and outlook on life have all been affected. She has a new appreciation of all that a family goes through when a loved one is in crisis and how it affects the entire family and network of friends.

Interdisciplinary faculty and student practice in a nurse-managed campus wellness program.

The concept of nurse-managed care in centers for nursing is a growing phenomenon in this era of health care reform. Nursing faculty are utilizing nurse-managed centers as alternative sites for clinical experiences and faculty practice. In this paper the authors highlight interdisciplinary faculty and student practice for a Campus Wellness Program at a midwestern university. Empowering faculty and students, and offering needed health promotion services to the university community, are addressed. Historical background, administrative, practice and educational challenges, and triumphs will be presented.

A novel set of spliceosome-associated proteins and the essential splicing factor PSF bind stably to pre-mRNA prior to catalytic step II of the splicing reaction.

We have isolated and determined the protein composition of the spliceosomal complex C. The pre-mRNA in this complex has undergone catalytic step I, but not step II, of the splicing reaction. We show that a novel set of 14 spliceosome-associated proteins (SAPs) and the essential splicing factor PSF are specifically associated with the C complex, implicating these proteins in catalytic step II. Significantly, immunodepletion and biochemical complementation studies demonstrate directly that PSF is essential for catalytic step II. Purified PSF is known to UV crosslink to pyrimidine tracts, and our data show that PSF UV crosslinks to pre-mRNA in purified C complex. Thus, PSF may replace the 3' splice site binding factor U2AF65 which is destabilized during spliceosome assembly. Finally, we show that SAPs 60 and 90, which are present in both the B and C complexes, are specifically associated with U4 and U6 snRNPs, and thus may have important roles in the functioning of these snRNPs during the splicing reaction.