Chimeric mRNA Research Articles

Reduced Expression of an RNA-binding Protein by Prolactin Leads to Translational Silencing of Programmed Cell Death Protein 4 and Apoptosis in Newt Spermatogonia

Recent studies indicate that the balance between cell survival and proapoptotic signals determines which cells commit to life or death. We have shown that the balance between follicle-stimulating hormone and prolactin determines differentiation or apoptosis in 7th generation spermatogonia during newt spermatogenesis; however, the molecular mechanisms specifying their fate are poorly understood. Here we show that the newt RNA-binding protein (nRBP) plays a critical role in determining their fate. nRBP was identified as a clone whose mRNA is decreased by prolactin, resulting in the reduction of the protein, which is otherwise expressed predominantly in the spermatogonia. nRBP protein associated with the mRNA for newt programmed cell death protein 4 (nPdcd4) at the 3'-untranslated region. nRBP reduction increased nPdcd4 mRNA but decreased its protein. In a cell-free system, cytoplasmic extracts containing reduced amounts of nRBP and nPdcd4 protein induced apoptosis, whereas adding nRBP protein to the extracts blocked apoptosis. Furthermore, overexpression of nRBP protected cells from apoptosis, stabilized the chimeric transcript containing the nPdcd4 3'-untranslated region, and accelerated its translation. These data suggest that, in the absence of nRBP, nPdcd4 mRNA is not stabilized and its translation is suppressed, leading to apoptosis in the spermatogonia.

Minor bcr/ablキメラmRNAを有し，好塩基球系への分化傾向を示した急性白血病

Minor bcr/ablキメラmRNAを有し，好塩基球系への分化傾向を示した急性白血病

再発時にp190 type bcr/ablキメラmRNAを認めた急性リンパ性白血病

再発時にp190 type bcr/ablキメラmRNAを認めた急性リンパ性白血病

T(8;15;21)(q22;p12;q22)を認め，経過中に新たな短縮型AML1-MTG8キメラmRNAが検出された幼児AML-M2

t(8;15;21)(q22;p12;q22)を認め，経過中に新たな短縮型AML1-MTG8キメラmRNAが検出された幼児AML-M2

Selective killing of human cancer cells by targeting a fusion mRNA between CYCLIN D1 and TROP2

e14569 Background: Trop-2 is a calcium signal transducer and a stem cell marker. Trop-2 is widely overexpressed by human cancers and stimulates their growth. A TROP2 mRNA was isolated as post-transcriptionally joined to CYCLIN D1 transcripts, suggesting this as one of the transforming mechanisms of TROP2. Methods: In vitro cell growth assays were utilized to assess the cell growth stimulatory capacity of the chimeric mRNA. Colony assays for growth in soft agarose and tumorigenicity assays in nude mice were utilized to assess for the transforming capacity of the fusion transcript. siRNA constructs were utilized for the stably shut-down of the expression of the CYCLIN D1-TROP2 mRNA. Results: The chimeric mRNA transforms primary cells in vitro and induces aggressive tumor growth in vivo in cooperation with activated RAS. The CYCLIN D1-TROP2 mRNA is expressed by a large fraction of human ovarian, endometrial and gastro-intestinal tumors. The chimera is coexpressed with activated RAS in a subset of tumors, consistent with a cooperative transforming activity. The chimeric mRNA is a bicistronic transcript that independently translates wild type Cyclin D1 and Trop-2 proteins, i.e. it does not generate chimeric, oncogenic proteins. On the other hand, joining to the stable TROP2 mRNA leads to a higher CYCLIN D1 mRNA stability, with inappropriate persistence during the cell cycle and acquisition of transforming capacity. As essentially no normal tissues express the chimeric mRNA, we targeted it for destruction in cancer cells with stably expressed siRNA constructs. Specific targeting led to essential annnihilation of the CYCLIN D1-TROP2 mRNA, in the absence of off-target effects. Silencing of the chimeric mRNA blocked the growth of expressing breast cancer cells. Conclusions: Our findings demonstrate a novel, widespread oncogenic mechanism in human cancers, and open novel avenues for mRNA-targeted anti-cancer therapies. Acknowledgments This work was supported by the the Fondazione of the Cassa di Risparmio della Provincia di Chieti, the Association for the Application of Biotechnology in Oncology (ABO and ABO Project S.p.A., grant no. VE01D0019) and the Marie Curie Transfer of Knowledge Fellowship, contract number 014541. No significant financial relationships to disclose.

A sequence-level map of chromosomal breakpoints in the MCF-7 breast cancer cell line yields insights into the evolution of a cancer genome

By applying a method that combines end-sequence profiling and massively parallel sequencing, we obtained a sequence-level map of chromosomal aberrations in the genome of the MCF-7 breast cancer cell line. A total of 157 distinct somatic breakpoints of two distinct types, dispersed and clustered, were identified. A total of 89 breakpoints are evenly dispersed across the genome. A majority of dispersed breakpoints are in regions of low copy repeats (LCRs), indicating a possible role for LCRs in chromosome breakage. The remaining 68 breakpoints form four distinct clusters of closely spaced breakpoints that coincide with the four highly amplified regions in MCF-7 detected by array CGH located in the 1p13.1-p21.1, 3p14.1-p14.2, 17q22-q24.3, and 20q12-q13.33 chromosomal cytobands. The clustered breakpoints are not significantly associated with LCRs. Sequences flanking most (95%) breakpoint junctions are consistent with double-stranded DNA break repair by nonhomologous end-joining or template switching. A total of 79 known or predicted genes are involved in rearrangement events, including 10 fusions of coding exons from different genes and 77 other rearrangements. Four fusions result in novel expressed chimeric mRNA transcripts. One of the four expressed fusion products (RAD51C-ATXN7) and one gene truncation (BRIP1 or BACH1) involve genes coding for members of protein complexes responsible for homology-driven repair of double-stranded DNA breaks. Another one of the four expressed fusion products (ARFGEF2-SULF2) involves SULF2, a regulator of cell growth and angiogenesis. We show that knock-down of SULF2 in cell lines causes tumorigenic phenotypes, including increased proliferation, enhanced survival, and increased anchorage-independent growth.

Alveolar Soft Part Sarcoma: A Bimarker Diagnostic Strategy Using TFE3 Immunoassay and ASPL-TFE3 Fusion Transcripts in Paraffin-Embedded Tumor Tissues

Alveolar soft part sarcoma (ASPS) is a malignancy with low incidence, but with poor prognosis if misdiagnosed. Immunohistochemical assay using TFE3 antibody has been shown to be a sensitive technique for ASPS diagnosis. A specific chromosomal translocation, t(X;17)(p11.2;q25), results in the ASPL-TFE3 fusion gene: it is detectable using reverse-transcription polymerase chain reaction (RT-PCR) in frozen tumor tissues of ASPS. However, the diagnostic usefulness of these markers has not been investigated in Chinese ASPS patients. Here, we report the first systematic study applying TFE3 immunoassay and ASPL-TFE3 fusion transcript detection to archival paraffin-embedded tissues in a large Chinese ASPS patient population. Sixteen patients had been diagnosed with ASPS (age, 3 to 58 y; 3 male patients and 13 female patients). Their tumors presented predominantly in the extremities (8/16), and were often located in the region of the orbit when affecting infants and children (3/16). Others had tumors in the chest wall, breast, and right pubis, respectively. One patient exhibited a tumor in the renal hilum, a location that had not been previously reported. Two patients had tumor metastases in the lung and the brain. ASPS tumors showed the best immunoreactivity to the TFE3 antibody (16/16). However, their immunoreactivity to other antibodies, including myoglobin (13/16), actin (10/16), desmin (2/16), and vimentin (2/16), was of various degrees. Positive staining was observed for the neural markers, NSE (9/16) and CgA (7/16), respectively. Using a strategy of RT-PCR, followed by a nested PCR with a different primer set, we were able to detect the expression of the chimeric ASPL-TFE3 mRNA in 11 of the 16 ASPS tumors. Of these 11, 7 were type 1 ASPL-TFE3 and 4 were type 2 ASPL-TFE3, including the tumor located in the renal hilum. No expression of ASPL-TFE3 fusion transcripts was detectable in all 38 control tumors. Our results demonstrate that the "bimarker strategy," a combination of TFE3 immunostaining and ASPL-TFE3 chimeric transcript detection, might have sufficient sensitivity and specificity in diagnosing most of the ASPSs. As both diagnostic techniques can be applied to widely available archival paraffin-embedded tissues, the usefulness of the strategy is largely implicated in routine pathology laboratories.

Chimeric Receptor mRNA Transfection as a Tool to Generate Antineoplastic Lymphocytes

Chimeric Receptor mRNA Transfection as a Tool to Generate Antineoplastic Lymphocytes

Chimeric receptor mRNA transfection as a tool to generate antineoplastic lymphocytes.

mRNA transfection is a useful approach for temporal cell reprogramming with minimal risk of transgene-mediated mutagenesis. We applied this to redirect lymphocyte cytotoxicity toward malignant cells. Using the chimeric immune receptor (CIR) constructs anti-CD19 CIR and 8H9 CIR, we achieved uniform expression of CIRs on virtually the entire population of lymphocytes. We reprogrammed CD3+ CD8+, CD3+ CD4+, and natural killer (NK ) cells toward autologous and allogeneic targets such as B cells, Daudi lymphoma, primary melanoma, breast ductal carcinoma, breast adenocarcinoma, and rhabdomyosarcoma. The reprogramming procedure is fast. Although most of the experiments were performed on lymphocytes obtained after 7-day activation, only 1-day activation of T cells with anti-CD3, anti-CD28 antibodies, and interleukin-2 is sufficient to develop both lymphocyte cytotoxicity and competence for mRNA transfer. The entire procedure, which includes lymphocyte activation and reprogramming, can be completed in 2 days. The efficiency of mRNA-modified human T cells was tested in a murine xenograft model. Human CD3+CD8+ lymphocytes expressing anti-CD19 CIR mRNA inhibited Daudi lymphoma growth in NOD=SCID mice. These results demonstrate that a mixed population of cytotoxic lymphocytes, including T cells together with NK cells, can be quickly and simultaneously reprogrammed by mRNA against autologous malignancies. With relatively minor modifications the described method of lymphocyte reprogramming can be scaled up for cancer therapy.

A Bicistronic CYCLIN D1-TROP2 mRNA Chimera Demonstrates a Novel Oncogenic Mechanism in Human Cancer

A chimeric CYCLIN D1-TROP2 mRNA was isolated from human ovarian and mammary cancer cells. The CYCLIN D1-TROP2 mRNA was shown to be a potent oncogene as it transforms naïve, primary cells in vitro and induces aggressive tumor growth in vivo in cooperation with activated RAS. Silencing of the chimeric mRNA inhibits the growth of breast cancer cells. The CYCLIN D1-TROP2 mRNA was expressed by a large fraction of the human gastrointestinal, ovarian, and endometrial tumors analyzed. It is most frequently detected in intestinal cell aneuploid cancers and it is coexpressed with activated RAS oncogenes, consistent with a cooperative transforming activity in human cancers. The chimeric mRNA is a bicistronic transcript of post transcriptional origin that independently translates the Cyclin D1 and Trop-2 proteins. This is a novel mechanism of CYCLIN D1 activation that achieves the truncation of the CYCLIN D1 mRNA in the absence of chromosomal rearrangements. This leads to a higher CYCLIN D1 mRNA stability, with inappropriate expression during the cell cycle. The stabilized CYCLIN D1 mRNA cooperates with TROP2 in stimulating the growth of the expressing cells. These findings show a novel epigenetic, oncogenic mechanism, which seems to be widespread in human cancers.

Half pint couples transcription and splicing of eIF4E-1,2 gene during fly development

There are seven genes encoding eight protein isoforms of the translation initiation factor 4E (eIF4E) in Drosophila. One of these genes, eIF4E-1,2, gives rise to three mRNAs with different 5′-untranslated regions (5′UTRs) through alternative splicing regulated by Haf pint (Hfp) in the ovary. eIF4E-1a and eIF4E-1b mRNAs encode the eIF4E-I protein, whereas eIF4E-2 mRNA encodes the eIF4E-II protein. We report here that Hfp regulates alternative splicing of eIF4E-1,2 pre-mRNA during Drosophila development in a sex-independent way. Hfp RNA interference-mediated knockdown in Drosophila S2 cells reproduces the eIF4E-1,2 gene expression found in development. Interestingly, 5′UTRs of eIF4E-1a and -1b isoforms, unlike the eIF4E-2 5′UTR, promote a significant increase in the translation efficiency of a luciferase-encoding chimeric mRNA. Thus, these results suggest that the eIF4E-1,2 gene is regulated by Hfp through a mechanism linked to transcription control and 3′ splice site selection, which determines the pattern and translation efficiency of eIF4E-1,2 mRNAs.

Design and kinetic analysis of hammerhead ribozyme and DNAzyme that specifically cleave TEL–AML1 chimeric mRNA

In order to develop the oligonucleotides to abolish an expression of TEL–AML1 chimeric RNA, which is a genetic aberration that causes the acute lymphoblastic leukemia (ALL), hammerhead ribozymes and deoxyoligoribozymes that can specifically cleave TEL–AML1 fusion RNA were designed. Constructs of the deoxyribozyme with an asymmetric substrate binding arm (Dz26) and the hammerhead ribozyme with a 4nt-bulged substrate binding arm in the stem III (buRz28) were able to cleave TEL–AML1 chimeric RNA specifically at sites close to the junction in vitro, without cleaving the normal TEL and AML1 RNA. Single-turnover kinetic analysis under enzyme-excess condition revealed that the buRz28 is superior to the Dz26 in terms of substrate binding and RNA-cleavage. In conjunction with current progress in a gene-delivery technology, the designed oligonucleotides that specifically cleave the TEL–AML1 chimeric mRNA are hoped to be applicable for the treatment of ALL in vivo.

Regulation of Plasminogen Activator Inhibitor-1 Expression by Tumor Suppressor Protein p53

H1299 lung carcinoma cells lacking p53 (p53-/-) express minimal amounts of plasminogen activator inhibitor-1 (PAI-1) protein as well as mRNA. p53(-/-) cells express highly unstable PAI-1 mRNA. Transfection of p53 in p53(-/-) cells enhanced PAI-1 expression and stabilized PAI-1 mRNA. On the contrary, inhibition of p53 expression by RNA silencing in non-malignant human lung epithelial (Beas2B) cells decreased basal as well as urokinase-type plasminogen activator-induced PAI-1 expression because of accelerated degradation of PAI-1 mRNA. Purified p53 protein specifically binds to the PAI-1 mRNA 3'-un-translated region (UTR), and endogenous PAI-1 mRNA forms an immune complex with p53. Treatment of purified p53 protein with anti-p53 antibody abolished p53 binding to the 3'-UTR of PAI-1 mRNA. The p53 binding region maps to a 70-nucleotide PAI-1 mRNA 3'-UTR sequence, and insertion of the p53-binding sequence into beta-globin mRNA destabilized the chimeric transcript. Deletion experiments indicate that the carboxyl-terminal region (amino acid residues 296-393) of p53 protein interacts with PAI-1 mRNA. These observations demonstrate a novel role for p53 as an mRNA-binding protein that regulates increased PAI-1 expression and stabilization of PAI-1 mRNA in human lung epithelial and carcinoma cells.

RNA secondary structures located in the interchromosomal region of human ACAT1 chimeric mRNA are required to produce the 56-kDa isoform

We have previously reported that the human ACAT1 gene produces a chimeric mRNA through the interchromosomal processing of two discontinuous RNAs transcribed from chromosomes 1 and 7. The chimeric mRNA uses AUG(1397-1399) and GGC(1274-1276) as translation initiation codons to produce normal 50-kDa ACAT1 and a novel enzymatically active 56-kDa isoform, respectively, with the latter being authentically present in human cells, including human monocyte-derived macrophages. In this work, we report that RNA secondary structures located in the vicinity of the GGC(1274-1276) codon are required for production of the 56-kDa isoform. The effects of the three predicted stem-loops (nt 1255-1268, 1286-1342 and 1355-1384) were tested individually by transfecting expression plasmids into cells that contained the wild-type, deleted or mutant stem-loop sequences linked to a partial ACAT1 AUG open reading frame (ORF) or to the ORFs of other genes. The expression patterns were monitored by western blot analyses. We found that the upstream stem-loop(1255-1268) from chromosome 7 and downstream stem-loop(1286-1342) from chromosome 1 were needed for production of the 56-kDa isoform, whereas the last stem-loop(1355-1384) from Chromosome 1 was dispensable. The results of experiments using both monocistronic and bicistronic vectors with a stable hairpin showed that translation initiation from the GGC(1274-1276) codon was mediated by an internal ribosome entry site (IRES). Further experiments revealed that translation initiation from the GGC(1274-1276) codon requires the upstream AU-constituted RNA secondary structure and the downstream GC-rich structure. This mechanistic work provides further support for the biological significance of the chimeric nature of the human ACAT1 transcript.

Functional T-cell responses generated by dendritic cells expressing the early HIV-1 proteins Tat, Rev and Nef

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutic strategies. One of the approaches that has gained prominence in recent years is therapeutic vaccination. We decided to assess the capacity of mature dendritic cells, derived from blood monocytes of HIV-1 infected patients, to generate functional T-cell responses. For this purpose, we constructed a chimeric mRNA encoding the proteins Tat, Rev and Nef. The TaReNef encoding information was linked to the HLA class II-targeting sequence of DC-LAMP. Broadly directed HIV-specific CD4 + and CD8 + cytotoxic T cells exhibiting a poly-functional cytokine secretion pattern were generated by co-culturing with autologous chimeric mRNA electroporated dendritic cells. Thus, administration of ex vivo generated dendritic cells expressing the early proteins Tat, Rev and Nef might offer a promising approach for therapeutic vaccination in HIV-1 infection.

Cis- andtrans-splicing of mRNAs mediated by tRNA sequences in eukaryotic cells

The formation of chimeric mRNAs is a strategy used by human cells to increase the complexity of their proteome, as revealed by the ENCODE project. Here, we use Saccharomyces cerevisiae to show a way by which trans-spliced mRNAs can be generated. We demonstrate that a pretRNA inserted into a premRNA context directs the splicing reaction precisely to the sites of the tRNA intron. A suppressor pretRNA gene was inserted, in cis, into the sequence encoding the third cytoplasmic loop of the Ste2 or Ste3 G protein-coupled receptor. The hybrid RNAs are spliced at the specific pretRNA splicing sites, releasing both functional tRNAs that suppress nonsense mutations and translatable mRNAs that activate the signal transduction pathway. The RNA molecules extracted from yeast cells were amplified by RT-PCR, and their sequences were determined, confirming the identity of the splice junctions. We then constructed two fusions between the premRNA sequence (STE2 or STE3) and the 5'- or 3'-pretRNA half, so that the two hybrid RNAs can associate with each other, in trans, through their tRNA halves. Splicing occurs at the predicted pretRNA sites, producing a chimeric STE3-STE2 receptor mRNA. RNA trans-splicing mediated by tRNA sequences, therefore, is a mechanism capable of producing new kinds of RNAs, which could code for novel proteins.

Novel intestinal splice variants of RNA-binding protein CUGBP2: isoform-specific effects on mitotic catastrophe

CUG triplet repeat-binding protein 2 (CUGBP2) is a RNA-binding protein that regulates mRNA translation and modulates apoptosis. Here, we report the identification of two splice variants (termed variants 2 and 3) in cultured human intestinal epithelial cells and in mouse gastrointestinal tract. The variants are generated from alternative upstream promoters resulting in the inclusion of additional NH(2)-terminal residues. Although variant 2 is the predominant isoform in normal intestine, its expression is reduced, whereas variant 1 is overexpressed following gamma-irradiation. All three variants bind cyclooxygenase-2 (COX-2) mRNA. However, only variant 1 inhibits the translation of the endogenous COX-2 mRNA and a chimeric luciferase mRNA containing the COX-2 3'untranslated region. Furthermore, whereas variant 1 is predominantly nuclear, variants 2 and 3 are predominantly cytoplasmic. These data imply that the additional amino acids affect CUGBP2 function. Previous studies have demonstrated that variant 1 induces intestinal epithelial cells to undergo apoptosis. However, in contrast to variant 1, the two novel variants do not affect proliferation or apoptosis of HCT116 cells. In addition, only variant 1 induced G(2)/M cell cycle arrest, which was overcome by prostaglandin E(2). Moreover, variant 1 increased cellular levels of phosphorylated p53 and Bax and decreased Bcl2. Caspase-3 and -9 were also activated, suggesting the initiation of the intrinsic apoptotic pathway. Furthermore, increased phosphorylation of checkpoint kinase (Chk)1 and Chk2 kinases and increased nuclear localization of Cdc2 and cyclin B1 suggested that cells were in mitotic transition. Taken together, these data demonstrate that cells expressing CUGBP2 variant 1 undergo apoptosis during mitosis, suggesting mitotic catastrophe.

Insertion of an intracisternal A particle retrotransposon element in plasma membrane calcium ATPase 2 gene attenuates its expression and produces an ataxic phenotype in joggle mutant mice

Using forward genetic analysis, we identified the insertion of an intracisternal A particle (IAP) retrotransposon element in the plasma membrane calcium ATPase 2 gene (Pmca2/Atp2b2) in the joggle mouse, a novel mutant that displays ataxic gait by postnatal day 12. Expression of Pmca2 mRNA in the joggle mouse is only 5% of that in the wild type mouse. The insertion is located 15 bp downstream of the donor splice site of the exon containing the initiation codon. Chimeric mRNA composed of the 5′-region of Pmca2 and the IAP element were detected, indicating that some of the primary transcripts are terminated by polyadenylation signals in long terminal repeats of the IAP element. We also identified cryptic splice sites in the IAP element that are likely involved in aberrant splicing of the Pmca2 primary transcripts that leads to rapid degradation of mRNA through nonsense mediated mRNA decay. Ataxia was observed in compound heterozygous mice carrying the joggle mutation and the wriggle mutation, a previously reported missense Pmca2 mutant. Thus, we attributed ataxia in joggle mice to reduced expression of Pmca2, resulting from insertion of the IAP element.

Atypical BCR-ABL mRNA transcripts in adult Acute lymphoblastic leukemia

RT-PCR detects chimeric BCR-ABL mRNA in approximately 25% of adult acute lymphoblastic leukemia (ALL) cases. Minor breakpoint transcripts (e1a2) are found in about 70% of positive cases and major breakpoint transcripts (e13a2, e14a2) in about 30% of cases. However, other atypical transcripts are sometimes observed. We report experience gained in the GMALL Study Group and identified 8 BCR-ABL-positive adult ALL cases with such atypical transcripts: 5 with e1a3, 2 with e13a3 (b2a3), and 1 with e6a2. This corresponds to a prevalence of 1-2% of all BCR-ABL-positive cases. The clinical courses are reported and diagnostic proposals are made.

Pitfalls of the CAT reporter gene for analyzing translational regulation in Leishmania

Heterologous reporter genes are widely used for the characterization of gene expression in many organisms. Particularly, constructs bearing reporter genes have greatly contributed to our understanding of gene regulation in kinetoplastids. In some specific circumstances, however, such heterologous reporter has a risk of resulting in irrelevant observations and conclusions, which are primarily due to the introduction of foreign sequence elements. This communication describes our recent experience using the chloramphenicol acetyltransferase (CAT) gene as a reporter for analysis of the translational regulation of HSP70 genes in Leishmania infantum. We show that chimeric mRNAs consisting of the CAT open reading frame (ORF) and the untranslated regions (UTRs) from HSP70-II genes behave differently as endogenous HSP70-II mRNAs and that this difference is due to the presence of CAT sequences. Thus, the main purpose of this communication is to alert researchers working in gene regulation to be cautious when interpreting results based on heterologous reporter genes.