Ribonuclear Protein Complex Research Articles

Abstract PO5-25-09: rs2242652 POLYMORPHISM OF THE hTERT GENE IN WOMEN WITH BREAST CANCER

Abstract Introduction: Breast cancer is the most common neoplasm in women around the world. There are several risk factors for this cancer: sex, age, body mass index (BMI), hormone therapy, family history, genetic mutations, among several others. The vast majority of breast tumors are characterized as carcinomas and may originate from ducts or lobules. Immunohistochemistry is related to the expressions of the estrogen receptor (ER) and progesterone receptor (PR), as well as the expression of the human epidermal growth factor receptor 2 (HER2), cell proliferation or the absence of any expression from receptors on the tumor membrane. The telomeres protect the chromosomes natural endings from suffering DNA loss. Telomerase is a ribonuclear protein complex (RNA and protein) that neutralizes telomeres' shortening. Recurrent point mutations in the TERT telomerase promoter have been identified in several cancers, and the rs2242652 polymorphism is a single nucleotide change, and has been described as associated with breast cancer risk. Objective: The objective of this study is to analyze the TERT rs2242652 polymorphism in breast cancer women and to test the correlation of such data with the prognosis and various clinical variables. Methods: The study was conducted as an observational clinical study of women with breast cancer. A blood sample was collected from the patient and PCR was performed to extract the DNA and to analyze the telomerase polymorphisms in this population. To ensure a more descriptive analysis, the data was used in the construction of tables and graphs, that were then studied in a qualitative manner. The associations were analyzed through direct correlation tests and were followed by comparisons of categorization groups. Results and discussion: Fifty-four women presented polymorphism of the telomerase hTERT gene rs2242652. They were divided into two groups according to the A or G allele that confer the GA/AA and GG gene variants. It was found that the GA/AA group had a trend towards more aggressive tumors, presenting 33% triple negative and 22% of tumors with HER2 overexpression, different from the GG group: 19% triple negative and 10% HER2 overexpression. More aggressive T4 tumors, were more expressive in the GA/AA gene variant group representing 20% of the patients, whereas in the GG group they represented only 8%. Conclusion: The data found in this study on the telomerase TERT gene polymorphism rs2242652 with breast cancer refers to a tendency found in tumors that have a more severe prognosis, to associate with the gene variant linked to the A allele of this gene, however a larger sample number is required for statistical confirmation. Citation Format: Miriam Alvares, Elisa Mascarenhas, Joao Matos. rs2242652 POLYMORPHISM OF THE hTERT GENE IN WOMEN WITH BREAST CANCER [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-25-09.

Specific Signal Enhancement on an RNA-Protein Interface by Dynamic Nuclear Polarization.

Invited for the cover of this issue are the groups of Alexander Marchanka at the Leibniz University of Hannover and Björn Corzilius at the University of Rostock. The image depicts the local generation of nuclear spin hyperpolarization, which selectively "illuminates" the interaction surface of a ribonuclear protein complex for solid-state NMR spectroscopy. Read the full text of the article at 10.1002/chem.202203443.

RNA Modifiers Multitasking On Chromatin

It is becoming increasingly clear that in mammalian cells, RNA Polymerase II is regulated at the level of promoter proximal pause release and productive elongation prompted by P‐TEFb kinase activity. As a result, P‐TEFb recruitment and activation at particular gene promoters is a key regulatory switch, the understanding of which can open the way to new therapeutic opportunities for many diseases including cancer. P‐TEFb activity is inhibited by its interaction with the 7SK ribonuclear protein complex (RNPc), which sequesters unutilized P‐TEFb in the nucleoplasm; interestingly, recent studies, including ours, have shown that this RNPc often regulates P‐TEFb recruitment and activity directly on chromatin at the promoter of paused genes. Here, we will present our data showing how MePCE, an RNA methyltransferase of the 7SK RNPc, multitasks on chromatin to coordinate transcription and RNA processing with other DNA‐templated activities, including DNA repair.

Assessing the efficacy of CRISPR/Cas9 genome editing in the wheat pathogen Parastagonspora nodorum

BackgroundThe genome-editing tool CRISPR/Cas9 has revolutionized gene manipulation by providing an efficient method to generate targeted mutations. This technique deploys the Cas9 endonuclease and a guide RNA (sgRNA) which interact to form a Cas9-sgRNA complex that initiates gene editing through the introduction of double stranded DNA breaks. We tested the efficacy of the CRISPR/Cas9 approach as a means of facilitating a variety of reverse genetic approaches in the wheat pathogenic fungus Parastagonospora nodorum.ResultsParastagonospora nodorum protoplasts were transformed with the Cas9 protein and sgRNA in the form of a preassembled ribonuclear protein (RNP) complex targeting the Tox3 effector gene. Subsequent screening of the P. nodorum transformants revealed 100% editing of those mutants screened. We further tested the efficacy of RNP complex when co-transformed with a Tox3-Homology Directed Repair cassette harbouring 1 kb of homologous flanking DNA. Subsequent screening of resulting transformants demonstrated homologous recombination efficiencies exceeding 70%. A further transformation with a Tox3-Homology Directed Repair cassette harbouring a selectable marker with 50 bp micro-homology flanks was also achieved with 25% homologous recombination efficiency. The success of these homology directed repair approaches demonstrate that CRISPR/Cas9 is amenable to other in vivo DNA manipulation approaches such as the insertion of DNA and generating point mutations.ConclusionThese data highlight the significant potential that CRISPR/Cas9 has in expediting transgene-free gene knockouts in Parastagonospora nodorum and also in facilitating other gene manipulation approaches. Access to these tools will significantly decrease the time required to assess the requirement of gene for disease and to undertake functional studies to determine its role.

Improved Cas9 activity by specific modifications of the tracrRNA

CRISPR/Cas is a transformative gene editing tool, that offers a simple and effective way to target a catalytic Cas9, the most widely used is derived from Streptococcus pyogenes (SpCas9), with a complementary small guide RNA (sgRNA) to inactivate endogenous genes resulting from insertions and deletions (indels). CRISPR/Cas9 has been rapidly applied to basic research as well as expanded for potential clinical applications. Utilization of spCas9 as an ribonuclearprotein complex (RNP) is considered the most safe and effective method to apply Cas9 technology, and the efficacy of this system is critically dependent on the ability of Cas9 to generate high levels of indels. We find here that novel sequence changes to the tracrRNA significantly improves Cas9 activity when delivered as an RNP. We demonstrate that a dual-guide RNA (dgRNA) with a modified tracrRNA can improve reporter knockdown and indel formation at several targets within the long terminal repeat (LTR) of HIV. Furthermore, the sequence-modified tracrRNAs improved Cas9-mediated reduction of CCR5 surface receptor expression in cell lines, which correlated with higher levels of indel formation. It was demonstrated that a Cas9 RNP with a sequence modified tracrRNA enhanced indel formation at the CCR5 target site in primary CD4+ T-cells. Finally, we show improved activity at two additional targets within the HBB locus and the BCL11A GATA site. Overall, the data presented here suggests that novel facile tracrRNA sequence changes could potentially be integrated with current dgRNA technology, and open up the possibility for the development of sequence modified tracrRNAs to improve Cas9 RNP activity.

Distinct Functions of the Cap-Binding Complex in Stimulation of Nuclear mRNA Export.

Cap-binding complex (CBC) associates cotranscriptionally with the cap structure at the 5' end of nascent mRNA to protect it from exonucleolytic degradation. Here, we show that CBC promotes the targeting of an mRNA export adaptor, Yra1 (forming transcription export [TREX] complex with THO and Sub2), to the active genes and enhances mRNA export in Saccharomyces cerevisiae Likewise, recruitment of Npl3 (an hnRNP involved in mRNA export via formation of export-competent ribonuclear protein complex [RNP]) to the active genes is facilitated by CBC. Thus, CBC enhances targeting of the export factors and promotes mRNA export. Such function of CBC is not mediated via THO and Sub2 of TREX, cleavage and polyadenylation factors, or Sus1 (that regulates mRNA export via transcription export 2 [TREX-2]). However, CBC promotes splicing of SUS1 mRNA and, consequently, Sus1 protein level and mRNA export via TREX-2. Collectively, our results support the hypothesis that CBC promotes recruitment of Yra1 and Npl3 to the active genes, independently of THO, Sub2, or cleavage and polyadenylation factors, and enhances mRNA export via TREX and RNP, respectively, in addition to its role in facilitating SUS1 mRNA splicing to increase mRNA export through TREX-2, revealing distinct stimulatory functions of CBC in mRNA export.

Crosstalk between the RNA Methylation and Histone-Binding Activities of MePCE Regulates P-TEFb Activation on Chromatin

RNAP II switching from the paused to the productive transcription elongation state is a pivotal regulatory step that requires specific phosphorylations catalyzed by the P-TEFb kinase. Nucleosolic P-TEFb activity is inhibited by its interaction with the ribonuclear protein complex built around the 7SK small nuclear RNA (7SK snRNP). MePCE is the RNA methyltransferase that methylates and stabilizes 7SK in the nucleosol. Here, we report that MePCE also binds chromatin through the histone H4 tail to serve as a P-TEFb activator at specific genes important for cellular identity. Notably, this histone binding abolishes MePCE's RNA methyltransferase activity toward 7SK, which explains why MePCE-bound P-TEFb on chromatin may not be associated with the full 7SK snRNP and is competent for RNAP II activation. Overall, our results suggest that crosstalk between the histone-binding and RNA methylation activities of MePCE regulates P-TEFb activation on chromatin in a 7SK- and Brd4-independent manner.

Endosome Independent - Vesicular Transport and Targeting of the Leishmania RNA Import Complex to Mammalian Mitochondria in Vivo and in Vitro

The multi subunit RNA Import Complex (RIC) from the protozoal parasite Leishmania tropica is taken up by mammalian cells and trafficked to mitochondria, with functional RNA delivered to the matrix by an unknown mechanism. By live cell imaging of siRNA mediated knockdown cells we showed that intracellular vesicular transport of the ribonuclear protein complex is dependent on caveolin1, dynamin2 and filaminA but independent of components of the endosomal pathway such as EEA1, Rab5 or Rab7. Moreover, mitochondrial targeting was dependent on TOM20 and TOM22, two components of the outer membrane machinery. By immuno-selection we could distinguish two types of RNA loaded cytoplasmic vesicles, one containing caveolar components and the other lacking them. In vitro, transfer of RNP from the caveolin negative transport vesicles to mitochondria was dependent on ATP and on TOM22. RIC was transferred to TOM20 deficient mitochondrial membrane but the RNA was not imported into the matrix. These results imply that (1) endosomes are not involved in RIC trafficking and novel interactions between vesicle associated RIC and (2) TOM20 and TOM22 are required for transfer and positioning of RIC on the mitochondrial surface. Supported By: Project Bend of the Council of Scientific and Industrial Research. Ref: Mukherjee, J. (2014). Vesicular transport of a ribonucleoprotein to mitochondria. Biology Open, doi: 10.1242/​bio.20149076.

Using an Arabidopsis oligonucleotide microarray to analyze the secretory pathway genes' Response to TuMV infection in Brassica rapa

Previous evidence suggested the Turnip mosaic virus viral ribonuclear protein complex, comprising the viral RNA, moved to the plasmodesmata via the secretory pathway. We performed a global analysis of gene expression in Brassica rapa and identified a complex array of changes affecting pathogen response. Thirty-eight secretory pathway genes were identified. The quantitative real-time polymerase chain reaction results were consistent with microarray analysis. Combined with previous studies, we speculated that the secretory pathway participates in viral replication and movement. Exploring the precise function of the genes identified in this analysis will offer new insights into viral defense in B. rapa.

Regulation of CDK9 Activity by Phosphorylation and Dephosphorylation

HIV-1 transcription is regulated by CDK9/cyclin T1, which, unlike a typical cell cycle-dependent kinase, is regulated by associating with 7SK small nuclear ribonuclear protein complex (snRNP). While the protein components of this complex are well studied, the mechanism of the complex formation is still not fully understood. The association of CDK9/cyclin T1 with 7SK snRNP is, in part, regulated by a reversible CDK9 phosphorylation. Here, we present a comprehensive review of the kinases and phosphatases involved in CDK9 phosphorylation and discuss their role in regulation of HIV-1 replication and potential for being targeted for drug development. We propose a novel pathway of HIV-1 transcription regulation via CDK9 Ser-90 phosphorylation by CDK2 and CDK9 Ser-175 dephosphorylation by protein phosphatase-1.

Lost in translation

“Young” APE-type non-LTR retrotransposons (non-LTRs) typically encode two open reading frames (ORFs 1 and 2). The shorter ORF1 translation product (ORF1p) comprises an RNA binding activity, thought to bind to non-LTR transcript RNA, protect against nuclease degradation and specify nuclear import of the ribonuclear protein complex (RNP). ORF2 encodes a multifunctional protein (ORF2p) comprising apurinic/apyrimidinic endonuclease (APE) and reverse-transcriptase (RT) activities, responsible for genome replication and re-integration into chromosomal DNA. However, some clades of APE-type non-LTRs only encode a single ORF—corresponding to the multifunctional ORF2p outlined above (and for simplicity referred-to as ORF2 below). The absence of an ORF1 correlates with the acquisition of a 2A oligopeptide translational recoding element (some 18–30 amino acids) into the N-terminal region of ORF2p. In the case of non-LTRs encoding two ORFs, the presence of ORF1 would necessarily downregulate the translation of ORF2. We argue that in the absence of an ORF1, 2A could provide the corresponding translational downregulation of ORF2. While multiple molecules of ORF1p are required to decorate the non-LTR transcript RNA in the cytoplasm, conceivably only a single molecule of ORF2p is required for target-primed reverse transcription/integration in the nucleus. Why would the translation of ORF2 need to be controlled by such mechanisms? An “excess” of ORF2p could result in disadvantageous levels of genome instability by, for example, enhancing short, interspersed, element (SINE) retrotransposition and the generation of processed pseudogenes. If so, the acquisition of mechanisms—such as 2A—to control ORF2p biogenesis would be advantageous.

14-3-3ε Boosts Bleomycin-induced DNA Damage Response by Inhibiting the Drug-resistant Activity of MVP

Major vault protein (MVP) is the predominant constituent of the vault particle, the largest known ribonuclear protein complex. Although emerging evidence have been establishing the links between MVP (vault) and multidrug resistance (MDR), little is known regarding exactly how the MDR activity of MVP is modulated during cellular response to drug-induced DNA damage (DDR). Bleomycin (BLM), an anticancer drug, induces DNA double-stranded breaks (DSBs) and consequently triggers the cellular DDR. Due to its physiological implications in hepatocellular carcinoma (HCC) and cell fate decision, 14-3-3ε was chosen as the pathway-specific bait protein to identify the critical target(s) responsible for HCC MDR. By using an LC-MS/MS-based proteomic approach, MVP was first identified in the BLM-induced 14-3-3ε interactome formed in HCC cells. Biological characterization revealed that MVP possesses specific activity to promote the resistance to the BLM-induced DDR. On the other hand, 14-3-3ε enhances BLM-induced DDR by interacting with MVP. Mechanistic investigation further revealed that 14-3-3ε, in a phosphorylation-dependent manner, binds to the phosphorylated sites at both Thr52 and Ser864 of the monomer of MVP. Consequently, the phosphorylation-dependent binding between 14-3-3ε and MVP inhibits the drug-resistant activity of MVP for an enhanced DDR to BLM treatment. Our findings provide an insight into the mechanism underlying how the BLM-induced interaction between 14-3-3ε and MVP modulates MDR, implicating novel strategy to overcome the chemotherapeutic resistance through interfering specific protein-protein interactions.

Dyskerin Is Upregulated During Erythroid Differentiation of Human Hematopoietic Progenitor Cells and Hyperactivates Telomerase in Erythroid Precursor Cells

AbstractAbstract 980Telomerase is a ribonuclear protein complex that functions to maintain the integrity of chromosomal-end structures (telomeres), and thereby enables continued cell replication. The active telomerase holoenzyme includes telomerase reverse transcriptase (hTERT) as its catalytic core, an RNA component (hTR) that functions as a template for synthesis of telomere repeats and an RNA-modifying protein dyskerin, which stabilizes TERC within the telomerase holoenzyme. While telomerase activity is high in acute myeloid leukemia cells, low levels of telomerase enzyme activity are detected in human CD34+ progenitor cells (HPCs). Telomerase activity is increased by cytokine stimulation of HPCs, then downregulated during myelomonocytic cell differentiation. Data accumulated to date suggest that telomerase enzyme levels are principally determined by Myc-mediated transcriptional regulation of the gene encoding hTERT, although the dyskerin gene (DKC1) is also a known Myc target.Our studies of the regulation and function of telomerase in human myeloid cells utilize cord blood-derived HPCs subject to ex vivo expansion and differentiation along specific myeloid lineages. Our past results revealed for the first time, that in contrast to the repression of telomerase activity observed during granulocytic, monocytic and megakaryocytic differentiation, telomerase activity was upregulated during erythroid differentiation, to reach levels comparable to tumor cell lines in CD34-/Glycophorin A+ erythroid precursor cells (Schuller et al., Leukemia 21: 983–991. 2007). Here we show that the upregulation of telomerase during erythroid differentiation is accompanied by a parallel increase in dyskerin mRNA and protein expression. In contrast to dyskerin expression, but consistent with the pattern of hTERT expression in other myeloid lineages, TERT gene expression was downregulated to a minimally detectable level in erythroid precursor cells. Chromatin-immunoprecipitation revealed that while Myc-binding at the DKC1 promoter decreased, binding of the erythroid-specific transcription factor GATA-1 increased during erythroid differentiation. Overexpression of DKC1 in HPCs confirmed that the upregulation of dyskerin was sufficient to hyperactivate telomerase, while shRNA-mediated depletion of dyskerin from erythroid precursors cells suppressed telomerase activity and decreased expansion of erythroid precursor cells.These data are the first to demonstrate a cellular context in which the upregulation of dyskerin drives telomerase enzyme activity. Further, the results implicate GATA-1 in the regulation of DKC1 transcription and show that high levels of dyskerin are required to sustain proliferation of Glycophorin A+ erythroid precursors. These results have important implications in relation to the pathogenesis of the inherited syndrome dyskeratosis congenita in which the DKC1 gene is mutated, and illustrate a novel mechanism by which telomerase may be reactivated in myeloid leukemia. Disclosures:No relevant conflicts of interest to declare.

Down-regulation of viral replication by lentiviral-mediated expression of short-hairpin RNAs against vesicular stomatitis virus ribonuclear complex genes

Vesicular stomatitis virus (VSV) causes great economic impact to livestock industry and is a prototype for studying non-segmented negative-stranded RNA (NSNR) viruses. In this study, we evaluated the antiviral potential of unique short-hairpin RNA (shRNA) targeting genes that form the ribonuclear protein (RNP) complex of VSV serotype Indiana (VSIV). We used lentiviral vectors to construct cell lines that stably expressed one of seven shRNAs targeting the RNP genes of VSIV, namely nucleocapsid (N), phosphoprotein (P), or polymerase (L). We reported two N-shRNA sequences targeting the 5′ or 3′ end of N that significantly reduced N, P, and L viral transcripts (p<0.001), reduced viral protein expression, and reduced the viral particles shed in Vero cells (p<0.01). When we analyzed the sequence diversity in the target region of this N-shRNA from two field isolates, we detected a single base substitution outside the seed region. We also reported five other shRNA sequences targeting components of the viral RNA that significantly reduce N, P, and L viral transcripts (p<0.001) but failed to efficiently impair viral replication. The differences in the efficiency of the shRNAs tested were not due to mismatches within the target region in the genome of VSIV. Although partial silencing of viral transcripts by single shRNAs impaired but did not block VSIV replication, the combination of the shRNAs identified here into a multiple shRNA vector may result in inhibition of viral replication. These data contribute to ongoing development of RNAi-based technologies to combat viral diseases.

Abstract 2201: A novel androgen receptor regulatory loop: Posttranscriptional androgen receptor regulation through Midline 1 ribonuclear protein complex

Abstract Objective: Exploration of posttranscriptional regulation of androgen receptor (AR) protein through interaction with the midline 1/protein phosphatase 2A/Alpha4 (MID1/PP2A/α4) ribonuclear protein complex. Background: Deregulation of the androgen receptor (AR) signaling pathway is a hallmark of prostate cancer progression to an advanced, castration-resistant tumor stage. Several mechanisms like amplification of the AR gene, mutations, altered nuclear receptor cofactor expression, aberrant activation and AR overexpression have been proposed. In addition to gene amplification and enhanced transcription also posttranscriptional mechanisms contribute to regulation of AR protein in prostate cancer. The MID1/PP2A/ β4 complex which is a microtubule-associated ribonucleoprotein complex was shown to regulate the stability and intracellular localization of associated mRNAs, among them AR mRNA. Materials and Methods: RNA interference (siRNA) was used to knockdown proteins in several cellular prostate cancer models. For AR activitation or inhibition synthetic androgen analog R1881 and anti-androgen bicalutamide, respectively, were used. AR DNA binding sites in the MID1 gene were identified in DuCaP cells by chromatin immunoprecipitation (ChIP) followed by DNA sequencing. Reporter gene vectors were constructed with putative wild type and mutated androgen responsive elements (AREs) and their functional activities were determined in reporter gene assays. Results: Down regulation of MID1 or β4 proteins by specific siRNAs resulted in a reduced AR protein level in the AR positive cell lines. MID1 knockdown also affected proliferation and migration in both AR positive and negative prostate cancer cell lines. Androgen treatment decreased the MID1 protein as well as MID 1 mRNA level and bicalutamide abrogated this effect. Several AR binding sites were identified in the MID1 gene using ChIP sequencing. Their functional activities as androgen responsive elements (AREs) were analyzed and confirmed in reporter gene assays using reporter vectors with wild type and mutated AREs. Immunohistochemistry revealed a specific and unique expression pattern of MID1. Whereas immunoreactivity was absent or very weak in benign epithelium, there was a very heterogeneous expression in prostate tumors with expression especially in high grade tumors displaying a cribriform growth pattern. Very high immunoreactivity was also detected in some lymph-node metastases. Conclusions: Our results support a negative feedback loop between AR and MID1. Whereas the MID1/PP2A/a4 complex increases AR protein level the AR represses expression of MID1. This is a new concept of AR protein modulation in tumors that awaits further analysis with regard to impact on therapies targeting the AR function and the progression of prostate cancer to a castration-resistant tumor stage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2201. doi:1538-7445.AM2012-2201

935 A novel androgen receptor regulatory loop: Posttranscriptional androgen receptor regulation through the midline 1 ribonuclear protein complex

935 A novel androgen receptor regulatory loop: Posttranscriptional androgen receptor regulation through the midline 1 ribonuclear protein complex

The Ebola Virus VP24 Protein Prevents hnRNP C1/C2 Binding to Karyopherin α1 and Partially Alters its Nuclear Import

The Ebola virus (EBOV) protein VP24 inhibits type I and II interferon (IFN) signaling by binding to NPI-1 subfamily karyopherin α (KPNA) nuclear import proteins, preventing their interaction with tyrosine-phosphorylated STAT1 (phospho-STAT1). This inhibits phospho-STAT1 nuclear import. A biochemical screen now identifies heterogeneous nuclear ribonuclear protein complex C1/C2 (hnRNP C1/C2) nuclear import as an additional target of VP24. Co-immunoprecipitation studies demonstrate that hnRNP C1/C2 interacts with multiple KPNA family members, including KPNA1. Interaction with hnRNP C1/C2 occurs through the same KPNA1 C-terminal region (amino acids 424-457) that binds VP24 and phospho-STAT1. The ability of hnRNP C1/C2 to bind KPNA1 is diminished in the presence of VP24, and cells transiently expressing VP24 redistribute hnRNP C1/C2 from the nucleus to the cytoplasm. These data further define the mechanism of hnRNP C1/C2 nuclear import and demonstrate that the impact of EBOV VP24 on nuclear import extends beyond STAT1.

Phosphorylation of the NFAR proteins by the dsRNA-dependent protein kinase PKR constitutes a novel mechanism of translational regulation and cellular defense

Here, we describe a new mechanism of host defense that involves the nuclear factors associated with dsRNA (NFAR1 [90 kDa] and NFAR2 [110 kDa]), which constitute part of the shuttling ribonuclear protein (RNP) complex. Activation of the dsRNA-activated protein kinase PKR by viral RNA enabled phosphorylation of NFAR1 and NFAR2 on Thr 188 and Thr 315, an event found to be evolutionarily conserved in Xenopus. Phosphorylated NFAR1 and NFAR2 became dissociated from nuclear factor 45 (NF45), which was requisite for NFAR reshuttling, causing the NFARs to be retained on ribosomes, associate with viral transcripts, and impede viral replication. Cre-loxP animals with depletion of the NFARs in the thymus were exquisitely sensitive to the cytoplasmic replicating virus VSV (vesicular stomatitis virus). Thus, the NFARs constitute a novel, conserved mechanism of host defense used by the cell to detect and impede aberrant translation events.

Nuclear translocation of telomerase reverse transcriptase: a critical process in chemical induced hepatocellular carcinogenesis

Telomerase, a ribonuclearprotein complex, functions not only in cancer development but also in apoptosis, and senescence. As a catalytic subunit of telomerase, telomerase reverse transcriptase (TERT) has been confirmed to regulate telomerase activity in a rate-limiting manner. Although a lot of work has been done, the dynamic state of TERT protein and the relationship with telomerase have not been delineated systematically in cancer development. The purpose of this study was to do such an exploration. To investigate the role of TERT in the in vivo carcinogenesis, we performed immunofluorescence and Western blot analysis, respectively, to detect the alteration of TERT status as well as telomeric repeat amplification protocol (TRAP) assay to detect telomerase activity in diethyl nitrosoamine (DENA) induced rat hepatocellular carcinoma (HCC). The course of cancer development was divided into three main stages, which were inflammation ( <12 weeks), hepatocirrhosis (15 weeks), and hepatocarcinoma (18-21 weeks). In normal liver and its early inflammatory stage, concomitant with a weak positive TERT signal, which was detected exclusively in the cytoplasm, telomerase activity was very low at this stage. However, in late hepatocirrhosis and particularly cancer stage, high levels of TERT expression (P = 0.044 and P = 0.001, respectively) and telomerase activity (P = 0.02 and P = 0.01, respectively) were observed following TERT nuclear translocation. Our data suggest that TERT protein might regulate telomerase activity. TERT translocation from cytoplasm to the nucleus might be a turning point in cancer development. Therefore, TERT translocation might be more useful than TERT expression level and telomerase activity in predicting the progression of HCC.

HnRNP L binds to CA repeats in the 3′UTR of bcl-2 mRNA

We previously reported that the CA-repeat sequence in the 3′-untranslated region (3′UTR) of bcl-2 mRNA is involved in the decay of bcl-2 mRNA. However, the trans-acting factor for the CA element in bcl-2 mRNA remains unidentified. The heterogeneous nuclear ribonucleoprotein L (hnRNP L), an intron splicing factor, has been reported to bind to CA repeats and CA clusters in the 3′UTR of several genes. We reported herein that the CA repeats of bcl-2 mRNA have the potential to form a distinct ribonuclear protein complex in cytoplasmic extracts of MCF-7 cells, as evidenced by RNA electrophoretic mobility shift assays (REMSA). A super-shift assay using the hnRNP L antibody completely shifted the complex. Immunoprecipitation with the hnRNP L antibody and MCF-7 cells followed by RT-PCR revealed that hnRNP L interacts with endogenous bcl-2 mRNA in vivo. Furthermore, the suppression of hnRNP L in MCF-7 cells by the transfection of siRNA for hnRNP L resulted in a delay in the degradation of RNA transcripts including CA repeats of bcl-2 mRNA in vitro, suggesting that the interaction between hnRNPL and CA repeats of bcl-2 mRNA participates in destabilizing bcl-2 mRNA.