Genes Important in the U. V. Survival of Herpes Simplex Virus

The den V gene from bacteriophage T4 codes for a small, pyrimidine dimer specific, endonuclease. Recent studies have shown that transfection of the gene into DNA excision repair deficient, Xeroderma Pigmentosum cells, can partially restore the excision repair ability of the cells and results in an increased resistance to UV light. In this study the den V gene has been inserted into Herpes Simplex Virus type 1 (HSV 1) in order to determine if HSV 1 can be used as a suitable vector for studying DNA repair genes. A 1.9 kb cartridge containing the den V gene, the 3' LTR of Rous Sarcoma Virus as the promoter, and the SV40 polyadenylation signals was inserted as the thymidine kinase locus of the virus. Properly initiated transcription form the construct, HDV 1, was verified by primer extension analysis. The Host cell reactivation of this virus and several other strains of HSV 1 were examined in normal and Xeroderma Pigmentosum cells. The results from these experiments suggest that both the viral DNA polymerase and thymidine kinase genes play important roles for the survival of UV irradiated HSV 1.ThesisMaster of Science (MS

Maturation of the 90S pre-ribosome requires Mrd1 dependent U3 snoRNA and 35S pre-rRNA structural rearrangements

In eukaryotes, ribosome biogenesis requires folding and assembly of the precursor rRNA (pre-rRNA) with a large number of proteins and snoRNPs into huge RNA-protein complexes. In spite of intense genetic, biochemical and high-resolution cryo-EM studies in Saccharomyces cerevisiae, information about the structure of the 35S pre-rRNA is limited. To overcome this, we performed high-throughput SHAPE chemical probing on the 35S pre-rRNA within 90S pre-ribosomes. We focused our analyses on external (5' ETS) and internal (ITS1) transcribed spacers as well as the 18S rRNA region. We show that in the 35S pre-rRNA, the central pseudoknot is not formed and the central core of the 18S rRNA is in an open configuration but becomes more constrained in 20S pre-rRNA. The essential ribosome biogenesis protein Mrd1 influences the structure of the 18S rRNA region locally and is involved in organizing the central pseudoknot and surrounding structures. We demonstrate that U3 snoRNA dynamically interacts with the 35S pre-rRNA and that Mrd1 is required for disrupting U3 snoRNA base pairing interactions in the 5' ETS. We propose that the dynamic U3 snoRNA interactions and Mrd1 are essential for establishing the structure of the central core of 18S rRNA that is required for processing and 40S subunit function.</p

First Report of Circulating MicroRNAs in Tumour Necrosis Factor Receptor-Associated Periodic Syndrome (TRAPS)

Tumor necrosis factor-receptor associated periodic syndrome (TRAPS) is a rare autosomal dominant autoinflammatory disorder characterized by recurrent episodes of long-lasting fever and inflammation in different regions of the body, such as the musculo-skeletal system, skin, gastrointestinal tract, serosal membranes and eye. Our aims were to evaluate circulating microRNAs (miRNAs) levels in patients with TRAPS, in comparison to controls without inflammatory diseases, and to correlate their levels with parameters of disease activity and/or disease severity. Expression levels of circulating miRNAs were measured by Agilent microarrays in 29 serum samples from 15 TRAPS patients carrying mutations known to be associated with high disease penetrance and from 8 controls without inflammatory diseases. Differentially expressed and clinically relevant miRNAs were detected using GeneSpring GX software. We identified a 6 miRNAs signature able to discriminate TRAPS from controls. Moreover, 4 miRNAs were differentially expressed between patients treated with the interleukin (IL)-1 receptor antagonist, anakinra, and untreated patients. Of these, miR-92a-3p and miR-150-3p expression was found to be significantly reduced in untreated patients, while their expression levels were similar to controls in samples obtained during anakinra treatment. MiR-92b levels were inversely correlated with the number of fever attacks/year during the 1st year from the index attack of TRAPS, while miR-377-5p levels were positively correlated with serum amyloid A (SAA) circulating levels. Our data suggest that serum miRNA levels show a baseline pattern in TRAPS, and may serve as potential markers of response to therapeutic intervention

Point mutations in the Rpb9-homologous domain of Rpc11 that impair transcription termination by RNA polymerase III

RNA polymerase III recognizes and pauses at its terminator, an oligo(dT) tract in non-template DNA, terminates 3′ oligo(rU) synthesis within this sequence, and releases the RNA. The pol III subunit Rpc11p (C11) mediates RNA 3′–5′ cleavage in the catalytic center of pol III during pausing. The amino and carboxyl regions of C11 are homologous to domains of the pol II subunit Rpb9p, and the pol II elongation and RNA cleavage factor, TFIIS, respectively. We isolated C11 mutants from Schizosaccharomyces pombe that cause pol III to readthrough terminators in vivo. Mutant RNA confirmed the presence of terminator readthrough transcripts. A predominant mutation site, F32, resides in the C11 Rpb9-like domain. Another mutagenic approach confirmed the F32 mutation and also isolated I34 and Y30 mutants. Modeling Y30, F32 and I34 of C11 in available cryoEM pol III structures predicts a hydrophobic patch that may interface with C53/37. Another termination mutant, Rpc2-T455I, appears to reside internally, near the RNA–DNA hybrid. We show that the Rpb9 and TFIIS homologous mutants of C11 reflect distinct activities, that differentially affect terminator recognition and RNA 3′ cleavage. We propose that these C11 domains integrate action at the upper jaw and center of pol III during termination

An evolutionary intra-molecular shift in the preferred U3 snoRNA binding site on pre-ribosomal RNA

Correct docking of U3 small nucleolar RNA (snoRNA) on pre-ribosomal RNA (pre-rRNA) is essential for rRNA processing to produce 18S rRNA. In this report, we have used Xenopus oocytes to characterize the structural requirements of the U3 snoRNA 3′-hinge interaction with region E1 of the external transcribed spacer (ETS) of pre-rRNA. This interaction is crucial for docking to initiate rRNA processing. 18S rRNA production was inhibited when fewer than 6 of the 8 bp of the U3 3′–hinge complex with the ETS could form; moreover, base pairing involving the right side of the 3′-hinge was more important than the left. Increasing the length of the U3 hinge–ETS interaction by 9 bp impaired rRNA processing. Formation of 18S rRNA was also inhibited by swapping the U3 5′- and 3′-hinge interactions with the ETS or by shifting the base pairing of the U3 3′-hinge to the sequence directly adjacent to ETS region E1. However, 18S rRNA production was partially restored by a compensatory shift that allowed the sequence adjacent to the U3 3′-hinge to pair with the eight bases directly adjacent to ETS region E1. The results suggest that the geometry of the U3 snoRNA interaction with the ETS is critical for rRNA processing

Comparative whole genome sequencing reveals phenotypic tRNA gene duplication in spontaneous Schizosaccharomyces pombe La mutants

We used a genetic screen based on tRNA-mediated suppression (TMS) in a Schizosaccharomyces pombe La protein (Sla1p) mutant. Suppressor pre-tRNASerUCA-C47:6U with a debilitating substitution in its variable arm fails to produce tRNA in a sla1-rrm mutant deficient for RNA chaperone-like activity. The parent strain and spontaneous mutant were analyzed using Solexa sequencing. One synonymous single-nucleotide polymorphism (SNP), unrelated to the phenotype, was identified. Further sequence analyses found a duplication of the tRNASerUCA-C47:6U gene, which was shown to cause the phenotype. Ninety percent of 28 isolated mutants contain duplicated tRNASerUCA-C47:6U genes. The tRNA gene duplication led to a disproportionately large increase in tRNASerUCA-C47:6U levels in sla1-rrm but not sla1-null cells, consistent with non-specific low-affinity interactions contributing to the RNA chaperone-like activity of La, similar to other RNA chaperones. Our analysis also identified 24 SNPs between ours and S. pombe 972h- strain yFS101 that was recently sequenced using Solexa. By including mitochondrial (mt) DNA in our analysis, overall coverage increased from 52% to 96%. mtDNA from our strain and yFS101 shared 14 mtSNPs relative to a ‘reference’ mtDNA, providing the first identification of these S. pombe mtDNA discrepancies. Thus, strain-specific and spontaneous phenotypic mutations can be mapped in S. pombe by Solexa sequencing

A La Autoantigen Homologue Is Required for the Internal Ribosome Entry Site Mediated Translation of Giardiavirus

Translation of Giardiavirus (GLV) mRNA is initiated at an internal ribosome entry site (IRES) in the viral transcript. The IRES localizes to a downstream portion of 5′ untranslated region (UTR) and a part of the early downstream coding region of the transcript. Recent studies indicated that the IRES does not require a pre-initiation complex to initiate translation but may directly recruit the small ribosome subunit with the help of a number of trans-activating protein factors. A La autoantigen homologue in the viral host Giardia lamblia, GlLa, was proposed as one of the potential trans-activating factors based on its specific binding to GLV-IRES in vitro. In this study, we further elucidated the functional role of GlLa in GLV-IRES mediated translation in Giardia by knocking down GlLa with antisense morpholino oligo, which resulted in a reduction of GLV-IRES activity by 40%. An over-expression of GlLa in Giardia moderately stimulated GLV-IRES activity by 20%. A yeast inhibitory RNA (IRNA), known to bind mammalian and yeast La autoantigen and inhibit Poliovirus and Hepatitis C virus IRES activities in vitro and in vivo, was also found to bind to GlLa protein in vitro and inhibited GLV-IRES function in vivo. The C-terminal domain of La autoantigen interferes with the dimerization of La and inhibits its function. An over-expression of the C-terminal domain (200–348aa) of GlLa in Giardia showed a dominant-negative effect on GLV-IRES activity, suggesting a potential inhibition of GlLa dimerization. HA tagged GlLa protein was detected mainly in the cytoplasm of Giardia, thus supporting a primary role of GlLa in translation initiation in Giardiavirus

The RNA binding protein HuR differentially regulates unique subsets of mRNAs in estrogen receptor negative and estrogen receptor positive breast cancer

<p>Abstract</p> <p>Background</p> <p>The discordance between steady-state levels of mRNAs and protein has been attributed to posttranscriptional control mechanisms affecting mRNA stability and translation. Traditional methods of genome wide microarray analysis, profiling steady-state levels of mRNA, may miss important mRNA targets owing to significant posttranscriptional gene regulation by RNA binding proteins (RBPs).</p> <p>Methods</p> <p>The ribonomic approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip), provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich elements (ARE) of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR has been described to control genes in several of the acquired capabilities of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established.</p> <p>Results</p> <p>We used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+) and MDA-MB-231 (estrogen receptor negative, ER-) breast cancer cell lines. We identified unique subsets of HuR-associated mRNAs found individually or in both cell types. Two novel HuR targets, <it>CD9 </it>and <it>CALM2 </it>mRNAs, were identified and validated by quantitative RT-PCR and biotin pull-down analysis.</p> <p>Conclusion</p> <p>This is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer. We found distinct, differentially expressed subsets of cancer related genes in ER+ and ER- breast cancer cell lines, and noted that the differential regulation of two cancer-related genes by HuR was contingent upon the cellular environment.</p

Getting to the end of RNA: Structural analysis of protein recognition of 5′ and 3′ termini

Accepted versio

Integrity of SRP RNA is ensured by La and the nuclear RNA quality control machinery

The RNA component of signal recognition particle (SRP) is transcribed by RNA polymerase III, and most steps in SRP biogenesis occur in the nucleolus. Here, we examine processing and quality control of the yeast SRP RNA (scR1). In common with other pol III transcripts, scR1 terminates in a U-tract, and ma-ture scR1 retains a U4–5 sequence at its 3 ′ end. In cells lacking the exonuclease Rex1, scR1 terminates in a longer U5–6 tail that presumably represents the primary transcript. The 3 ′ U-tract of scR1 is protected from aberrant processing by the La homologue, Lhp1 and overexpressed Lhp1 apparently competes with both the RNA surveillance system and SRP assem-bly factors. Unexpectedly, the TRAMP and exosome nuclear RNA surveillance complexes are also impli-cated in protecting the 3 ′ end of scR1, which accu-mulates in the nucleolus of cells lacking the activities of these complexes. Misassembled scR1 has a pri-mary degradation pathway in which Rrp6 acts early, followed by TRAMP-stimulated exonuclease degra-dation by the exosome. We conclude that the RNA surveillance machinery has key roles in both SRP biogenesis and quality control of the RNA, poten-tially facilitating the decision between these alterna-tive fates