Expression, purification and structural analysis of the Pyrococcus abyssi RNA binding protein PAB1135

Abstract Background The gene coding for the uncharacterized protein PAB1135 in the archaeon Pyrococcus abyssi is in the same operon as the ribonuclease P (RNase P) subunit Rpp30. Findings Here we report the expression, purification and structural analysis of PAB1135. We analyzed the interaction of PAB1135 with RNA and show that it binds efficiently double-stranded RNAs in a non-sequence specific manner. We also performed molecular modeling of the PAB1135 structure using the crystal structure of the protein Af2318 from Archaeoglobus fulgidus (2OGK) as the template. Conclusions Comparison of this model has lead to the identification of a region in PAB1135 that could be involved in recognizing double-stranded RNA.

Transforming growth factor alpha immunoreactivity. A study in hepatocellular carcinoma and in non-neoplastic liver tissue

Background. Transforming growth factor alpha (TGF alpha) is an important mitogen that binds to epidermal growth factor receptor and is associated with the development of several tumors. Aims. Assessment of the immunoexpression of TGF alpha in hepatocellular carcinoma (HCC) and in non-neoplastic liver tissue and its relationship to morphological patterns of HCC. Material and methods. The immunohistochemical expression of TGF alpha was studied in 47 cases of HCC (27 multinodular, 20 nodular lesions). Five lesions measured up to 5 cm and 15 lesions above 5 cm. Thirty-two cases were graded as I or II and 15 as III or IV. The non-neoplastic tissue was examined in 40 cases, of which 22 had cirrhosis. HBsAg and anti-HCV were positive in 5/38 and 15/37 patients, respectively. The statistical analysis for possible association of immunostaining of TGF alpha and pathological features was performed through chi-square test. Results. TGF alpha was detected in 31.9% of the HCC and in 42.5% of the non-neoplastic. There was a statistically significant association between the expression of TGF alpha and cirrhosis (OR = 8.75, 95% CI = [1.93, 39.75]). The TGF alpha was detected more frequently in patients anti-HCV(+) than in those HBsAg(+). The immunoexpression of TGF alpha was not found related to tumor size or differentiation. In conclusion the TGF alpha is present in hepatocarcinogenesis in HBV negative patients. Further analysis is needed to examine the involvement of TGF alpha in the carcinogenesis associated with HCV and other possible agents. In addition, TGF alpha has an higher expression in hepatocyte regeneration and proliferation in cirrhotic livers than in HCC.

Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5' ETS molecule using three distinct methods and located the acceptor site between two known 5' ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5' ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5' ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5' ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

Re-treatment of previous non-responders and relapsers to interferon plus ribavirin with peginterferon alfa-2a (40KD), ribavirin +/- amantadine in patients with chronic hepatitis C: randomized multicentre clinical trial

Introduction. A large number of patients with chronic hepatitis C have not been cured with interferon-based therapy. Therefore, we evaluated the efficacy of amantadine combined with the standard of care (pegylated interferon plus ribavirin) in patients who had not responded to or had relapsed after 24 weeks of treatment with conventional interferon plus ribavirin. Material and methods. Patients stratified by previous response (i.e., non-response or relapse) were randomized to 48 weeks of open-label treatment with peginterferon alfa-2a (401(D) 180 pg/week plus ribavirin 1,000/1,200 mg/day plus amantadine 200 mg/day (triple therapy), or the standard of care (peginterferon alfa-2a [40KD] plus ribavirin). Results. The primary outcome was sustained virological response (SVR), defined as undetectable hepatitis C virus RNA in serum (< 50 IU/mL) at end of follow-up (week 72). Among patients with a previous non-response, 12/53 (22.6%; 95% confidence interval [CI] 12.3-36.2%) randomized to triple therapy achieved an SVR compared with 16/52 (30.8%; 95% CI 18.7-45.1%) randomized to the standard of care. Among patients with a previous relapse 22/39 (56.4%; 95% CI 39.6-72.2%) randomized to triple therapy achieved an SVR compared with 23/38 (60.5%; 95% CI 43.4-76.0%) randomized to the standard of care. Undetectable HCV RNA (< 50 IU/mL) at week 12 had a high positive predictive value for SVR. A substantial proportion of non-responders and relapsers to conventional interferon plus ribavirin achieve an SVR when re-treated with peginterferon alfa-2a (40KD) plus ribavirin. Conclusion. Amantadine does not enhance SVR rates in previously treated patients with chronic hepatitis C and cannot be recommended in this setting.

Identical sequence patterns in the ends of exons and introns of human protein-coding genes

Intron splicing is one of the most important steps involved in the maturation process of a pre-mRNA. Although the sequence profiles around the splice sites have been studied extensively, the levels of sequence identity between the exonic sequences preceding the donor sites and the intronic sequences preceding the acceptor sites has not been examined as thoroughly. In this study we investigated identity patterns between the last 15 nucleotides of the exonic sequence preceding the 5' splice site and the intronic sequence preceding the 3' splice site in a set of human protein-coding genes that do not exhibit intron retention. We found that almost 60% of consecutive exons and introns in human protein-coding genes share at least two identical nucleotides at their 3' ends and, on average, the sequence identity length is 2.47 nucleotides. Based on our findings we conclude that the 3' ends of exons and introns tend to have longer identical sequences within a gene than when being taken from different genes. Our results hold even if the pairs are non-consecutive in the transcription order. (C) 2012 Elsevier Ltd. All rights reserved.

Trypanosoma cruzi Gene Expression in Response to Gamma Radiation

Trypanosoma cruzi is an organism highly resistant to ionizing radiation. Following a dose of 500 Gy of gamma radiation, the fragmented genomic DNA is gradually reconstructed and the pattern of chromosomal bands is restored in less than 48 hours. Cell growth arrests after irradiation but, while DNA is completely fragmented, RNA maintains its integrity. In this work we compared the transcriptional profiles of irradiated and non-irradiated epimastigotes at different time points after irradiation using microarray. In total, 273 genes were differentially expressed; from these, 160 were up-regulated and 113 down-regulated. We found that genes with predicted functions are the most prevalent in the down-regulated gene category. Translation and protein metabolic processes, as well as generation of precursor of metabolites and energy pathways were affected. In contrast, the up-regulated category was mainly composed of obsolete sequences (which included some genes of the kinetoplast DNA), genes coding for hypothetical proteins, and Retrotransposon Hot Spot genes. Finally, the tyrosyl-DNA phosphodiesterase 1, a gene involved in double-strand DNA break repair process, was up-regulated. Our study demonstrated the peculiar response to ionizing radiation, raising questions about how this organism changes its gene expression to manage such a harmful stress.

RNA interference-mediated knockdown of CD49e (α5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion

Abstract Background The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin α5β1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. Results Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. Conclusion Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation.

Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA

Abstract Background The ability to manipulate the genetic networks underlying the physiological and behavioural repertoires of the adult honeybee worker (Apis mellifera) is likely to deepen our understanding of issues such as learning and memory generation, ageing, and the regulatory anatomy of social systems in proximate as well as evolutionary terms. Here we assess two methods for probing gene function by RNA interference (RNAi) in adult honeybees. Results The vitellogenin gene was chosen as target because its expression is unlikely to have a phenotypic effect until the adult stage in bees. This allowed us to introduce dsRNA in preblastoderm eggs without affecting gene function during development. Of workers reared from eggs injected with dsRNA derived from a 504 bp stretch of the vitellogenin coding sequence, 15% had strongly reduced levels of vitellogenin mRNA. When dsRNA was introduced by intra-abdominal injection in newly emerged bees, almost all individuals (96 %) showed the mutant phenotype. An RNA-fragment with an apparent size similar to the template dsRNA was still present in this group after 15 days. Conclusion Injection of dsRNA in eggs at the preblastoderm stage seems to allow disruption of gene function in all developmental stages. To dissect gene function in the adult stage, the intra-abdominal injection technique seems superior to egg injection as it gives a much higher penetrance, it is much simpler, and it makes it possible to address genes that are also expressed in the embryonic, larval or pupal stages.

The intronic long noncoding RNA ANRASSF1 recruits PRC2 to the RASSF1A promoter, reducing the expression of RASSF1A and increasing cell proliferation

The down-regulation of the tumor-suppressor gene RASSF1A has been shown to increase cell proliferation in several tumors. RASSF1A expression is regulated through epigenetic events involving the polycomb repressive complex 2 (PRC2); however, the molecular mechanisms modulating the recruitment of this epigenetic modifier to the RASSF1 locus remain largely unknown. Here, we identify and characterize ANRASSF1, an endogenous unspliced long noncoding RNA (lncRNA) that is transcribed from the opposite strand on the RASSF1 gene locus in several cell lines and tissues and binds PRC2. ANRASSF1 is transcribed through RNA polymerase II and is 5'-capped and polyadenylated; it exhibits nuclear localization and has a shorter half-life compared with other lncRNAs that bind PRC2. ANRASSF1 endogenous expression is higher in breast and prostate tumor cell lines compared with non-tumor, and an opposite pattern is observed for RASSF1A. ANRASSF1 ectopic overexpression reduces RASSF1A abundance and increases the proliferation of HeLa cells, whereas ANRASSF1 silencing causes the opposite effects. These changes in ANRASSF1 levels do not affect the RASSF1C isoform abundance. ANRASSF1 overexpression causes a marked increase in both PRC2 occupancy and histone H3K27me3 repressive marks, specifically at the RASSF1A promoter region. No effect of ANRASSF1 overexpression was detected on PRC2 occupancy and histone H3K27me3 at the promoter regions of RASSF1C and the four other neighboring genes, including two well-characterized tumor suppressor genes. Additionally, we demonstrated that ANRASSF1 forms an RNA/DNA hybrid and recruits PRC2 to the RASSF1A promoter. Together, these results demonstrate a novel mechanism of epigenetic repression of the RASSF1A tumor suppressor gene involving antisense unspliced lncRNA, in which ANRASSF1 selectively represses the expression of the RASSF1 isoform overlapping the antisense transcript in a location-specific manner. In a broader perspective, our findings suggest that other non-characterized unspliced intronic lncRNAs transcribed in the human genome might contribute to a location-specific epigenetic modulation of genes.

The dengue virus non-structural 1 protein: risks and benefits

The dengue virus (DENV) non-structural 1 (NS1) protein plays a critical role in viral RNA replication and has a central position in DENV pathogenesis. DENV NS1 is a glycoprotein expressed in infected mammalian cells as soluble monomers that dimerize in the lumen of the endoplasmic reticulum; NS1 is subsequently transported to the cell surface, where it remains membrane associated or is secreted into the extracellular milieu as a hexameric complex. During the last three decades, the DENV NS1 protein has also been intensively investigated as a potential target for vaccines and antiviral drugs. In addition, NS1 is the major diagnostic marker for dengue infection. This review highlights some important issues regarding the role of NS1 in DENV pathogenesis and its biotechnological applications, both as a target for the development of safe and effective vaccines and antiviral drugs and as a tool for the generation of accurate diagnostic methods