Glioma-specific alternative RNA splicing: A role for polypyrimidine tract binding protein-1

Alternative RNA splicing plays an integral role in cell fate determination and function, especially in the cells of the brain. Errors in RNA processing contribute to diseases such as cancer, where it leads to the production of oncogenic proteins or the loss of tumor suppressors. In silica mining suggests that hundreds of splice isoforms are misexpressed in the glial cell-derived glioma. However, there is little experimental evidence of the prevalence and contribution of these changes and whether they contribute to the formation and progression of this devastating malignancy. To determine the frequency of these aberrant events, global profiling of alternative RNA splice patterns in glioma and nontumor brain was conducted using an exon array. Most splicing changes were less than 5-fold in magnitude and 14 cassette exon events were validated, including 7 previously published events. To determine the possible causes of missplicing, the differential expression levels of splicing factors in these two tissues were also analyzed. Six RNA splicing factors had greater than 2-fold changes in expression. The highest differentially expressed factor was polypyrimidine tract binding protein-1 (PTB). Evaluation by immunohistochemistry determined that this factor was elevated in both early and late stages of glioma. Glial cell-specific PTB expression in the adult brain led me to examine the role of PTB in gliomagenesis. Downregulation of PTB slowed glioma cell proliferation and migration and enhanced cell adhesion to fibronectin and vitronectin. To determine whether PTB was affecting these processes through splicing, genome-wide exon expression levels were correlated with PTB levels. Surprisingly, previously reported PTB target transcripts were insensitive to changes in PTB levels in both patient samples and PTB-depleted glioma cells. Only one validated glioma-specific splice target, RTN4/Nogo, had a significant PTB-mediated splicing change. Downregulation of PTB enhanced inclusion of its alternative exon 3, which encodes an auxiliary domain within a neurite inhibitor protein. Overexpression of this splice isoform in glioma cells slowed proliferation in a manner similar to that observed in PTB knockdown cells. In summary, aberrant expression of splicing factors such as PTB in glioma may elicit changes in splicing patterns that enhance tumorigenesis. ^

The Role of the Schizosaccharomyces pombe gar2 Protein in Nucleolar Structure and Function Depends on the Concerted Action of its Highly Charged N Terminus and its RNA-binding Domains

Nonribosomal nucleolar protein gar2 is required for 18S rRNA and 40S ribosomal subunit production in Schizosaccharomyces pombe. We have investigated the consequences of the absence of each structural domain of gar2 on cell growth, 18S rRNA production, and nucleolar structure. Deletion of gar2 RNA-binding domains (RBDs) causes stronger inhibition of growth and 18S rRNA accumulation than the absence of the whole protein, suggesting that other factors may be titrated by its remaining N-terminal basic/acidic serine-rich domain. These drastic functional defects correlate with striking nucleolar hypertrophy. Point mutations in the conserved RNP1 motifs of gar2 RBDs supposed to inhibit RNA–protein interactions are sufficient to induce severe nucleolar modifications but only in the presence of the N-terminal domain of the protein. Gar2 and its mutants also distribute differently in glycerol gradients: gar2 lacking its RBDs is found either free or assembled into significantly larger complexes than the wild-type protein. We propose that gar2 helps the assembly on rRNA of factors necessary for 40S subunit synthesis by providing a physical link between them. These factors may be recruited by the N-terminal domain of gar2 and may not be released if interaction of gar2 with rRNA is impaired.

An initiation element in the yeast CUP1 promoter is recognized by RNA polymerase II in the absence of TATA box-binding protein if the DNA is negatively supercoiled

Purified RNA polymerase II initiated transcription from the yeast CUP1 promoter fused to a C-less cassette if the DNA was negatively supercoiled. Relaxed plasmid was not transcribed. Transcription did not require addition of any other transcription factors. TATA box-binding protein (TBP) was not detectable in the polymerase preparation and the TATA box was not required. Deletion analysis of the CUP1 promoter revealed that a 25-bp element containing the initiation region was sufficient for recognition by polymerase. Two transcription start sites were mapped, one of which is identical to one of the two major start sites observed in vivo. Our observations can be accounted for by using a theoretical analysis of the probability of DNA melting within the plasmid as a function of superhelix density: the CUP1 initiation element is intrinsically unstable to superhelical stress, permitting entry of the polymerase, which then scans the DNA to locate the start site. In support of this analysis, the CUP1 promoter was sensitive to mung bean nuclease. These observations and a previous theoretical analysis of yeast genes support the idea that promoters are stress points within the DNA superhelix. The role of transcription factors might be to mark the promoter and to regulate specific melting of promoter DNA.

The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein.

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.

High-affinity RNA-binding domains of alfalfa mosaic virus coat protein are not required for coat protein-mediated resistance.

A virus-based vector was used for the transient expression of the alfalfa mosaic virus coat protein (CP) gene in protoplasts and plants. The accumulation of wild-type CP conferred strong protection against subsequent alfalfa mosaic virus infection, enabling the efficacy of CP mutants to be determined without developing transgenic plants. Expression of the CP mRNA alone without CP accumulation conferred weaker protection against infection. The activity of the N-terminal mutant CPs in protection did not correlate with their activities in genome activation. The activity of a C-terminal mutant suggested that encapsidation did not have a role in protection. Our results indicate that interaction of the CP with alfalfa mosaic virus RNA is not important in protection, thereby leaving open the possibility that interactions with host factors lead to protection.

TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, is a toroid-shaped molecule that binds transcripts containing GAG or UAG repeats separated by two nucleotides.

The trp RNA-binding attenuation protein of Bacillus subtilis, TRAP, regulates both transcription and translation by binding to specific transcript sequences. The optimal transcript sequences required for TRAP binding were determined by measuring complex formation between purified TRAP protein and synthetic RNAs. RNAs were tested that contained repeats of different trinucleotide sequences, with differing spacing between the repeats. A transcript containing GAG repeats separated by two-nucleotide spacers was bound most tightly. In addition, transmission electron microscopy was used to examine the structure of TRAP and the TRAP-transcript complex. TRAP was observed to be a toroid-shaped oligomer when free or when bound to either a natural or a synthetic RNA.

The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation

The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plant's RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0PE, in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0PE has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from poleroviruses, P0PE destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery. © 2012 Elsevier Inc.

The Mycobacterial MsDps2 Protein Is a Nucleoid-Forming DNA Binding Protein Regulated by Sigma Factors sigma(A) and sigma(B)

The Dps (DNA-binding protein from starved cells) proteins from Mycobacterium smegmatis MsDps1 and MsDps2 are both DNA-binding proteins with some differences. While MsDps1 has two oligomeric states, with one of them responsible for DNA binding, MsDps2 has only one DNA-binding oligomeric state. Both the proteins however, show iron-binding activity. The MsDps1 protein has been shown previously to be induced under conditions of starvation and osmotic stress and is regulated by the extra cellular sigma factors sigma(H) and sigma(F). We show here, that the second Dps homologue in M. smegmatis, namely MsDps2, is purified in a DNA-bound form and exhibits nucleoid-like structures under the atomic force microscope. It appears that the N-terminal sequence of Dps2 plays a role in nucleoid formation. MsDps2, unlike MsDps1, does not show elevated expression in nutritionally starved or stationary phase conditions; rather its promoter is recognized by RNA polymerase containing sigma(A) or sigma(B), under in vitro conditions. We propose that due to the nucleoid-condensing ability, the expression of MsDps2 is tightly regulated inside the cells.

mRNA-binding protein HuR in breast carcinogenesis

Breast cancer is the most common cancer among women. Although its prognosis has improved nowadays, methods to predict the progression of the disease or to treat it are not comprehensive. This thesis work was initiated to elucidate in breast carcinogenesis the role of HuR, a ubiquitously expressed mRNA-binding protein that regulates gene expression posttranscriptionally. HuR is predominantly nuclear, but it shuttles between the nucleus and the cytoplasm, and this nucleocytoplasmic translocation is important for its function as a RNA-stabilizing and translational regulator. HuR has been associated with diverse cellular processes, for example carcinogenesis. The specific aims of my thesis work were to study the prognostic value of HuR in breast cancer and to clarify the mechanisms by which HuR contributes to breast carcinogenesis. My ultimate goal is, by better understanding the role of HuR in breast carcinogenesis, to aid in the discovery of novel targets for cancer therapies. HuR expression and localization was studied in paraffin-embedded preinvasive (atypical ductal hyperplasia, ADH, and ductal carcinoma in situ, DCIS) specimens as well in sporadic and familial breast cancer specimens. Our results show that cytoplasmic HuR expression was already elevated in ADH and remained elevated in DCIS as well as in cancer specimens. Clinicopathological analysis showed that cytoplasmic HuR expression associated with the more aggressive form of the disease in DCIS, and in cancer specimens it proved an independent marker for poor prognosis. Importantly, cytoplasmic HuR expression was significantly associated with poor outcome in the subgroups of small (2 cm) and axillary lymph node-negative breast cancers. HuR proved to be the first mRNA stability protein the expression of which is associated in breast cancer with poor outcome. To explore the mechanisms of HuR in breast carcinogenesis, lentiviral constructs were developed to inhibit and to overexpress the HuR expression in a breast epithelial cell line (184B5Me). Our results suggest that HuR mediates breast carcinogenesis by participating in processes important in cell transformation, in programmed cell death, and in cell invasion. Global gene expression analysis shows that HuR regulates genes participating in diverse cellular processes, and affects several pathways important in cancer development. In addition, we identified two novel target transcripts (connective tissue growth factor, CTGF, and Ras oncogene family member 31, RAB31) for HuR. In conclusion, because cytoplasmic HuR expression in breast cancer can predict the outcome of the disease it could serve in clinics as a prognostic marker. HuR accumulates in the cytoplasm even at its non-invasive stage (ADH and DCIS) of the carcinogenic process and supports functions essential in cell alteration. These data suggest that HuR contributes to carcinogenesis of the breast epithelium.

Mycobacterium tuberculosis Rho Is an NTPase with Distinct Kinetic Properties and a Novel RNA-Binding Subdomain

Two mechanisms - factor independent and dependent termination - ensure the completion of RNA synthesis in eubacteria. Factor-dependent mechanism relies on the Rho protein to terminate transcription by interacting with RNA polymerase. Although well studied in Escherichia coli, the properties of the Rho homologs from most bacteria are not known. The rho gene is unusually large in genus Mycobacterium and other members of actinobacteria, having,150 additional residues towards the amino terminal end. We describe the distinct properties of Rho from Mycobacterium tuberculosis. It is an NTPase with a preference for purine nucleoside triphosphates with kinetic properties different from E. coli homolog and an ability to use various RNA substrates. The N-terminal subdomain of MtbRho can bind to RNA by itself, and appears to contribute to the interaction of the termination factor with RNAs. Furthermore, the interaction with RNA induces changes in conformation and oligomerization of MtbRho.

Functional domains and the antiviral effect of the double-stranded RNA-dependent protein kinase PKR from Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase PKR is thought to mediate a conserved antiviral pathway by inhibiting viral protein synthesis via the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2 alpha). However, little is known about the data related to the lower vertebrates, including fish. Recently, the identification of PKR-like, or PKZ, has addressed the question of whether there is an orthologous PKR in fish. Here, we identify the first fish PKR gene from the Japanese flounder Paralichthys olivaceus (PoPKR). PoPKR encodes a protein that shows a conserved structure that is characteristic of mammalian PKRs, having both the N-terminal region for dsRNA binding and the C-terminal region for the inhibition of protein translation. The catalytic activity of PoPKR is further evidence that it is required for protein translation inhibition in vitro. PoPKR is constitutively transcribed at low levels and is highly induced after virus infection. Strikingly, PoPKR overexpression increases eIF2 alpha phosphorylation and inhibits the replication of Scophthalmus maximus rhabdovirus (SMRV) in flounder embryonic cells, whereas phosphorylation and antiviral effects are impaired in transfected cells expressing the catalytically inactive PKR-K421R variant, indicating that PoPKR inhibits virus replication by phosphorylating substrate eIF2 alpha. The interaction between PoPKR and eIF2 alpha is demonstrated by coimmunoprecipitation assays, and the transfection of PoPKR-specific short interfering RNA further reveals that the enhanced eIF2 alpha phosphorylation is catalyzed by PoPKR during SMRV infection. The current data provide significant evidence for the existence of a PKR-mediated antiviral pathway in fish and reveal considerable conservation in the functional domains and the antiviral effect of PKR proteins between fish and mammals.

Rubisco small subunit, chlorophyll a/b-binding protein and sucrose : fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light

Chungui Lu, Olga A. Koroleva, John F. Farrar, Joe Gallagher, Chris J. Pollock, and A. Deri Tomos (2002). Rubisco small subunit, chlorophyll a/b-binding protein and sucrose : fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light. Plant Physiology, 130 (3) pp.1335-1348 Sponsorship: BBSRC RAE2008