Unspliced pre-mRNA Research Articles

Poly(A) Tail-Mediated Gene Regulation by Opposing Roles of Nab2 and Pab2 Nuclear Poly(A)-Binding Proteins in Pre-mRNA Decay

The 3' end of most eukaryotic transcripts is decorated by poly(A)-binding proteins (PABPs), which influence the fate of mRNAs throughout gene expression. However, despite the fact that multiple PABPs coexist in the nuclei of most eukaryotes, how functional interplay between these nuclear PABPs controls gene expression remains unclear. By characterizing the ortholog of the Nab2/ZC3H14 zinc finger PABP in Schizosaccharomyces pombe, we show here that the two major fission yeast nuclear PABPs, Pab2 and Nab2, have opposing roles in posttranscriptional gene regulation. Notably, we find that Nab2 functions in gene-specific regulation in a manner opposite to that of Pab2. By studying the ribosomal-protein-coding gene rpl30-2, which is negatively regulated by Pab2 via a nuclear pre-mRNA decay pathway that depends on the nuclear exosome subunit Rrp6, we show that Nab2 promotes rpl30-2 expression by acting at the level of the unspliced pre-mRNA. Our data support a model in which Nab2 impedes Pab2/Rrp6-mediated decay by competing with Pab2 for polyadenylated transcripts in the nucleus. The opposing roles of Pab2 and Nab2 reveal that interplay between nuclear PABPs can influence gene regulation.

Phase I Pharmacokinetic and Pharmacodynamic Study of the First-in-Class Spliceosome Inhibitor E7107 in Patients with Advanced Solid Tumors

To assess the safety, tolerability, pharmacokinetics, pharmacodynamics, and clinical activity of E7107 administered as 5-minute bolus infusions on days 1, 8, and 15 in a 28-day schedule. Patients with solid tumors refractory to standard therapies or with no standard treatment available were enrolled. Dose levels of 0.6 to 4.5 mg/m(2) were explored. Forty patients [24M/16F, median age 61 years (45-79)] were enrolled. At 4.5 mg/m(2), dose-limiting toxicity (DLT) consisted of grade 3 diarrhea, nausea, and vomiting and grade 4 diarrhea, respectively, in two patients. At 4.0 mg/m(2), DLT (grade 3 nausea, vomiting, and abdominal cramps) was observed in one patient. Frequently occurring side effects were mainly gastrointestinal. After drug discontinuation at 4.0 mg/m(2), one patient experienced reversible grade 4 blurred vision. The maximum tolerated dose (MTD) is 4.0 mg/m(2). No complete or partial responses during treatment were observed; one patient at 4.0 mg/m(2) had a confirmed partial response after drug discontinuation. Pharmacokinetic analysis revealed a large volume of distribution, high systemic clearance, and a plasma elimination half-life of 5.3 to 15.1 hours. Overall drug exposure increased in a dose-dependent manner. At the MTD, mRNA levels of selected target genes monitored in peripheral blood mononuclear cells showed a reversible 15- to 25-fold decrease, whereas unspliced pre-mRNA levels of DNAJB1 and EIF4A1 showed a reversible 10- to 25-fold increase. The MTD for E7107 using this schedule is 4.0 mg/m(2). Pharmacokinetics is dose-dependent and reproducible within patients. Pharmacodynamic analysis revealed dose-dependent reversible inhibition of pre-mRNA processing of target genes, confirming proof-of-principle activity of E7107.

Cotranscriptional recruitment of yeast TRAMP complex to intronic sequences promotes optimal pre-mRNA splicing

Most unwanted RNA transcripts in the nucleus of eukaryotic cells, such as splicing-defective pre-mRNAs and spliced-out introns, are rapidly degraded by the nuclear exosome. In budding yeast, a number of these unwanted RNA transcripts, including spliced-out introns, are first recognized by the nuclear exosome cofactor Trf4/5p-Air1/2p-Mtr4p polyadenylation (TRAMP) complex before subsequent nuclear-exosome-mediated degradation. However, it remains unclear when spliced-out introns are recognized by TRAMP, and whether TRAMP may have any potential roles in pre-mRNA splicing. Here, we demonstrated that TRAMP is cotranscriptionally recruited to nascent RNA transcripts, with particular enrichment at intronic sequences. Deletion of TRAMP components led to further accumulation of unspliced pre-mRNAs even in a yeast strain defective in nuclear exosome activity, suggesting a novel stimulatory role of TRAMP in splicing. We also uncovered new genetic and physical interactions between TRAMP and several splicing factors, and further showed that TRAMP is required for optimal recruitment of the splicing factor Msl5p. Our study provided the first evidence that TRAMP facilitates pre-mRNA splicing, and we interpreted this as a fail-safe mechanism to ensure the cotranscriptional recruitment of TRAMP before or during splicing to prepare for the subsequent targeting of spliced-out introns to rapid degradation by the nuclear exosome.

Regulation of Primary Response Genes in B Cells

Deregulated gene expression in B cells often results in various lymphoid malignancies and immune deficiencies. Therefore, understanding signal-induced gene regulatory pathways involved during B cell activation is important to tackle pathologies associated with altered B cell function. Primary response genes (PRGs) are rapidly induced upon signaling in B cells and other cell types and often encode oncogenic transcription factors, which are associated with various malignancies. However, an important issue that remains unclear is whether the fundamental mechanism of activation of these genes is essentially the same under such diverse conditions. c-fos is a PRG that is induced rapidly upon activation of B cells in response to a wide variety of stimuli. Using the c-fos gene as a candidate PRG, we addressed here how it is regulated in response to tumor-promoting and antigen-mimicking signals. Our results show that although the mRNA was induced and extinguished within minutes in response to both signals, surprisingly, apparently full-length unspliced pre-mRNA persisted for several hours in both cases. However, although the mitogenic signal resulted in a more sustained mRNA response that persisted for 4 h, antigenic signaling resulted in a more robust but very transient response that lasted for <1 h. Moreover, the pre-mRNA profile exhibited significant differences between the two signals. Additionally, the splicing regulation was also observed with egr-2, but not with c-myc. Together, these results suggest a previously underappreciated regulatory step in PRG expression in B cells.

The pre-mRNA retention and splicing complex controls tRNA maturation by promoting TAN1 expression

The conserved pre-mRNA retention and splicing (RES) complex, which in yeast consists of Bud13p, Snu17p and Pml1p, is thought to promote nuclear retention of unspliced pre-mRNAs and enhance splicing of a subset of transcripts. Here, we find that the absence of Bud13p or Snu17p causes greatly reduced levels of the modified nucleoside N4-acetylcytidine (ac4C) in tRNA and that a lack of Pml1p reduces ac4C levels at elevated temperatures. The ac4C nucleoside is normally found at position 12 in the tRNA species specific for serine and leucine. We show that the tRNA modification defect in RES-deficient cells is attributable to inefficient splicing of TAN1 pre-mRNA and the effects of reduced Tan1p levels on formation of ac4C. Analyses of cis-acting elements in TAN1 pre-mRNA showed that the intron sequence between the 5′ splice site and branchpoint is necessary and sufficient to mediate RES dependency. We also show that in RES-deficient cells, the TAN1 pre-mRNA is targeted for degradation by the cytoplasmic nonsense-mediated mRNA decay pathway, indicating that poor nuclear retention may contribute to the tRNA modification defect. Our results demonstrate that TAN1 pre-mRNA processing has an unprecedented requirement for RES factors and that the complex controls the formation of ac4C in tRNA.

Анализ интрон-содержащих пре-мРНК и сплайсированных мРНК в хлоропластах кукурузы посредством ОТ-ПЦР

The study of chloroplast RNA splicing is usually performed by such complicated methods as Northern blot hybridization, RNAse protection or primer extension. An application of simpler RT-PCR technique may lead to underestimation of unspliced pre-mRNA. We used five protein coding genes from maize plastome to analyze factors that can reduce a pool ofintron containing transcripts as compared to a mature RNA. We revealed that a mode of DNAse inactivation, a temperature of cDNA synthesis, and a type of DNA polymerase used are important for the proper detection of unspliced RNA level. The increase in a number of PCR cycles was accompanied with a concomitant decrease in the proportion of unspliced RNA for a one gene only. We demonstrated that after PCR intron containing and intron free strands of amplicons are able to form heteroduplexes. The information obtained let us to introduce the simple and effective method for the investigation of a chloroplast spliced mRNA and unspliced pre-mRNA.

Analysis of intron-containing pre-mRNA and spliced mRNA in maize chloroplasts by RT-PCR

Chloroplast RNA splicing is usually studied by complicated methods, such as Northern blot hybridization, RNAse protection, or primer extension assays. The use of a simpler RT-PCR technique frequently results in the underestimation of unspliced pre-mRNA levels. Five protein-coding genes from the maize plastome were studied to analyze which factors can lead to an apparent reduction in the pool of introncontaining transcripts compared to mature RNA. It was found that the accurate determination of the unspliced RNA level should take into account the DNase inactivation mode, the cDNA synthesis temperature, and the type of DNA polymerase. For one gene, the portion of unspliced RNAs decreased with increasing number of PCR cycles. It was found that intron-containing and intron-free strands of amplicons can form heteroduplexes after PCR. A simple and effective technique of investigating chloroplast spliced mRNA and unspliced pre-mRNA is suggested based on the obtained data.

IReckon: Simultaneous isoform discovery and abundance estimation from RNA-seq data

High-throughput RNA sequencing (RNA-seq) promises to revolutionize our understanding of genes and their role in human disease by characterizing the RNA content of tissues and cells. The realization of this promise, however, is conditional on the development of effective computational methods for the identification and quantification of transcripts from incomplete and noisy data. In this article, we introduce iReckon, a method for simultaneous determination of the isoforms and estimation of their abundances. Our probabilistic approach incorporates multiple biological and technical phenomena, including novel isoforms, intron retention, unspliced pre-mRNA, PCR amplification biases, and multimapped reads. iReckon utilizes regularized expectation-maximization to accurately estimate the abundances of known and novel isoforms. Our results on simulated and real data demonstrate a superior ability to discover novel isoforms with a significantly reduced number of false-positive predictions, and our abundance accuracy prediction outmatches that of other state-of-the-art tools. Furthermore, we have applied iReckon to two cancer transcriptome data sets, a triple-negative breast cancer patient sample and the MCF7 breast cancer cell line, and show that iReckon is able to reconstruct the complex splicing changes that were not previously identified. QT-PCR validations of the isoforms detected in the MCF7 cell line confirmed all of iReckon's predictions and also showed strong agreement (r2 = 0.94) with the predicted abundances.

Transcriptome-wide Analysis of Exosome Targets

SummaryThe exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. Here we apply in vivo RNA crosslinking (CRAC) to the nucleases (Rrp44, Rrp6), two structural subunits (Rrp41, Csl4) and a cofactor (Trf4) of the yeast exosome. Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of non-coding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome indicating that substrates follow multiple pathways to the nucleases.

Sequential RNA degradation pathways provide a fail-safe mechanism to limit the accumulation of unspliced transcripts in Saccharomyces cerevisiae

The nuclear exosome and the nonsense-mediated mRNA decay (NMD) pathways have been implicated in the degradation of distinct unspliced transcripts in Saccharomyces cerevisiae. In this study we show that these two systems can act sequentially on specific unspliced pre-mRNAs to limit their accumulation. Using steady-state and decay analyses, we show that while specific unspliced transcripts rely mostly on NMD or on the nuclear exosome for their degradation, some unspliced RNAs are stabilized only when both the nuclear exosome and NMD are inactivated. We found that the mechanism of degradation of these unspliced pre-mRNAs is not influenced by promoter identity. However, the specificity in the pre-mRNAs degradation pathways can be manipulated by changing the rate of export or retention of these mRNAs. For instance, reducing the nuclear export of pre-mRNAs mostly degraded by NMD results in a higher fraction of unspliced transcripts degraded by the nuclear exosome. Reciprocally, inactivating the Mlp retention factors results in a higher fraction of unspliced transcripts degraded by NMD for precursors normally targeted by the nuclear exosome. Overall, these results demonstrate that a functional redundancy exists between nuclear and cytoplasmic degradation pathways for unspliced pre-mRNAs, and suggest that the degradation routes of these species are mainly determined by the efficiency of their nuclear export rates. The presence of these two sequential degradation pathways for unspliced pre-mRNAs underscores the importance of limiting their accumulation and might serve as a fail-safe mechanism to prevent the expression of these nonfunctional RNAs.

Inactivation of a Single Copy of Crebbp Selectively Alters Pre-mRNA Processing in Mouse Hematopoietic Stem Cells

Global expression analysis of fetal liver hematopoietic stem cells (FL HSCs) revealed the presence of unspliced pre-mRNA for a number of genes in normal FL HSCs. In a subset of these genes, Crebbp+/− FL HSCs had less unprocessed pre-mRNA without a corresponding reduction in total mRNA levels. Among the genes thus identified were the key regulators of HSC function Itga4, Msi2 and Tcf4. A similar but much weaker effect was apparent in Ep300+/− FL HSCs, indicating that, in this context as in others, the two paralogs are not interchangeable. As a group, the down-regulated intronic probe sets could discriminate adult HSCs from more mature cell types, suggesting that the underlying mechanism is regulated with differentiation stage and is active in both fetal and adult hematopoiesis. Consistent with increased myelopoiesis in Crebbp hemizygous mice, targeted reduction of CREBBP abundance by shRNA in the multipotent EML cell line triggered spontaneous myeloid differentiation in the absence of the normally required inductive signals. In addition, differences in protein levels between phenotypically distinct EML subpopulations were better predicted by taking into account not only the total mRNA signal but also the amount of unspliced message present. CREBBP thus appears to selectively influence the timing and degree of pre-mRNA processing of genes essential for HSC regulation and thereby has the potential to alter subsequent cell fate decisions in HSCs.

The Translation Initiation Factor eIF4E Regulates the Sex-Specific Expression of the Master Switch Gene Sxl in Drosophila melanogaster

In female fruit flies, Sex-lethal (Sxl) turns off the X chromosome dosage compensation system by a mechanism involving a combination of alternative splicing and translational repression of the male specific lethal-2 (msl-2) mRNA. A genetic screen identified the translation initiation factor eif4e as a gene that acts together with Sxl to repress expression of the Msl-2 protein. However, eif4e is not required for Sxl mediated repression of msl-2 mRNA translation. Instead, eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs. Like other factors required for Sxl regulation of splicing, eif4e shows maternal-effect female-lethal interactions with Sxl. This female lethality can be enhanced by mutations in other co-factors that promote female-specific splicing and is caused by a failure to properly activate the Sxl-positive autoregulatory feedback loop in early embryos. In this feedback loop Sxl proteins promote their own synthesis by directing the female-specific alternative splicing of Sxl-Pm pre-mRNAs. Analysis of pre-mRNA splicing when eif4e activity is compromised demonstrates that Sxl-dependent female-specific splicing of both Sxl-Pm and msl-2 pre-mRNAs requires eif4e activity. Consistent with a direct involvement in Sxl-dependent alternative splicing, eIF4E is associated with unspliced Sxl-Pm pre-mRNAs and is found in complexes that contain early acting splicing factors—the U1/U2 snRNP protein Sans-fils (Snf), the U1 snRNP protein U1-70k, U2AF38, U2AF50, and the Wilms' Tumor 1 Associated Protein Fl(2)d—that have been directly implicated in Sxl splicing regulation.

Organization patterns of the AGFG genes: an evolutionary study

A number of proteins which are needed for the building of new immunodeficiency virus type 1 virions can only be translated from unspliced virus-derived pre-mRNAs. These unspliced mRNAs are shuttled through the nuclear pores reaching the cytosol when bound to the viral protein Rev. However, as a cellular co-factor Rev requires a Rev-binding protein of the AGFG family (nucleoporin-related Arf-GAP domain and FG repeats-containing proteins). In this article we address the evolution of the AGFGs by analyzing the first section of the coding mRNAs. This contains a “core module” which can be traced from Drosophilae to fish, amphibia, birds, and mammals, including man. In the subfamily of AGFG1 molecules the estimated conservation from Drosophilae to primates is 67% (with limited gaps). In some Drosophilae the core module is preceded by a long stretch of more than 300 coding nucleotides, but this additional module is absent in other Drosophilae and in all AGFG1s of other species. The AGFG2 molecules emerged later in evolution, possibly deriving from a duplication of AGFG1s. AGFG2s, present in mammals only, exhibit an additional module of about 50 coding nucleotides ahead of the core module, which is significantly less conserved (54%, with more remarkable gaps). This additional module does not seem to have homologies with the additional module of Drosophilae nor with the precoding section of AGFG1s. Interestingly, in birds a highly re-edited form of the AGFG1 core module (Gallus gallus, Galliformes) coexists with a typical form of the AGFG1 core module (Taeniopygia guttata, Passeriformes).

Proofreading and spellchecking: A two-tier strategy for pre-mRNA splicing quality control

Multi-tier strategies exist in many biochemical processes to ensure a maximal fidelity of the reactions. In this review, we focus on the two-tier quality control strategy that ensures the quality of the products of the pre-mRNA splicing reactions catalyzed by the spliceosome. The first step in the quality control process relies on kinetic proofreading mechanisms that are internal to the spliceosome and that are performed by ATP-dependent RNA helicases. The second quality control step, spellchecking, involves recognition of unspliced pre-mRNAs or aberrantly spliced mRNAs that have escaped the first proofreading mechanisms, and subsequent degradation of these molecules by degradative enzymes in the nucleus or in the cytoplasm. This two-tier quality control strategy highlights a need for high fidelity and a requirement for degradative activities that eliminate defective molecules. The presence of multiple quality control activities during splicing underscores the importance of this process in the expression of genetic information.

Nuclear Retention of Unspliced Pre-mRNAs by Mutant DHX16/hPRP2, a Spliceosomal DEAH-box Protein

Defective or imbalanced expression of spliceosomal factors has been linked to human disease; however, how a defective spliceosome affects intron-containing gene transcripts in human cells is largely unknown. DEAH-box protein DHX16 is a human orthologue of Saccharomyces cerevisiae spliceosomal protein Prp2, an RNA-dependent ATPase that activates the spliceosome before the first catalytic step of splicing. Yeast prp2 mutants accumulate unspliced RNAs from the vast majority of intron-containing genes. Here we used a genomic tiling microarray to screen transcripts from four chromosomes in human cells expressing a dominant negative DHX16 mutant and identified a number of gene transcripts that retained their introns. The mutant protein also affected gene transcripts that are sensitive to pladienolide, an SF3b inhibitor. The unspliced RNAs were retained in the nucleus, and block of nonsense-mediated decay did not affect their accumulation. Thus, a perturbation of human PRP2/DHX16 results in accumulation of unspliced transcripts, similar to the outcome in yeast prp2 mutants. The results further suggest that mutant DHX16/hPRP2 causes a defective spliceosome to retain unspliced gene transcripts in the nuclei of human cells.

U1 snRNP protects pre-mRNAs from premature cleavage and polyadenylation.

SummaryIn higher eukaryotes, U1 snRNP forms spliceosomes in equal stoichiometry with U2, U4, U5 and U6, however its abundance far exceeds that of the other snRNPs. Here, we used antisense morpholino oligonucleotide (AMO) to U1 snRNA for functional U1 snRNP knockdown in HeLa cells and identified accumulated unspliced pre-mRNAs by genomic tiling microarrays. Remarkably, in addition to inhibiting splicing, U1 snRNP knockdown caused premature cleavage and polyadenylation (PCPA) in numerous pre-mRNAs at cryptic polyadenylation signals (PASs), frequently in introns near (< 5 kb) the start of the transcript. This did not occur when splicing was inhibited with U2 snRNA AMO or the U2 snRNP inactivating drug, spliceostatin A, unless U1 AMO was also included. We further show that U1 snRNA-pre-mRNA base pairing was required to suppress PCPA from nearby cryptic PASs located in introns. These findings reveal a critical splicing-independent function for U1 snRNP in protecting the transcriptome, which we propose explains its overabundance.

The HPV-16 E7 oncoprotein is expressed mainly from the unspliced E6/E7 transcript in cervical carcinoma C33-A cells

The HPV-16 E6/E7 early transcripts are first produced as bicistronic or polycistronic mRNAs, and about 90% of the original pre-mRNA is spliced to produce three new alternative mRNAs. HPV-16 spliced transcripts are expressed heterogeneously in tumors and cell lines. Our results suggest that suboptimal splicing acceptor sites in E6/E7 intron 1 and the differential expression of splicing factors are involved in the production of the heterogeneous splicing profile in cell lines. The unspliced pre-mRNA and the alternative spliced transcripts contribute differentially to the production of E7 in stably transfected C33-A cells. The highest level of E7 was produced from the least prevalent transcript, the unspliced E6/E7(pre-mRNA). The order of relative expression of E7 was unspliced E6/E7(pre-mRNA)>E6*I/E7>E6*II/E7. Our findings suggest that E6/E7 alternative splicing may be a mechanism for differential expression of the E6 and E7 oncoproteins, which also affects the expression of their targets, the proteins p53 and pRb.

Cwc23, an Essential J Protein Critical for Pre-mRNA Splicing with a Dispensable J Domain

J proteins are structurally diverse, obligatory cochaperones of Hsp70s, each with a highly conserved J domain that plays a critical role in the stimulation of Hsp70's ATPase activity. The essential protein, Cwc23, is one of 13 J proteins found in the cytosol and/or nucleus of Saccharomyces cerevisiae. We report that a partial loss-of-function CWC23 mutant has severe, global defects in pre-mRNA splicing. This mutation leads to accumulation of the excised, lariat form of the intron, as well as unspliced pre-mRNA, suggesting a role for Cwc23 in spliceosome disassembly. Such a role is further supported by the observation that this mutation results in reduced interaction between Cwc23 and Ntr1 (SPP382), a known component of the disassembly pathway. However, Cwc23 is a very atypical J protein. Its J domain, although functional, is dispensable for both cell viability and pre-mRNA splicing. Nevertheless, strong genetic interactions were uncovered between point mutations encoding alterations in Cwc23's J domain and either Ntr1 or Prp43, a DExD/H-box helicase essential for spliceosome disassembly. These genetic interactions suggest that Hsp70-based chaperone machinery does play a role in the disassembly process. Cwc23 provides a unique example of a J protein; its partnership with Hsp70 plays an auxiliary, rather than a central, role in its essential cellular function.

Abstract C32: First-in-human clinical, pharmacokinetic (PK) and pharmacodynamic (PD) phase I study of the first-in-class spliceosome inhibitor E7107 administered IV (bolus) on days 1, 8, and 15 every 28 days to patients with solid tumors

Abstract Background: E7107 is a first-in-class inhibitor of the spliceosome, most likely by interacting with spliceosome-associated protein-130 (SAP 130). Splicing removes intron sequences from pre-mRNA and exons are fused resulting in the formation of mature mRNA. Alternative splicing frequently encodes oncoproteins. E7107 interferes the maturation process of pre-mRNA to mRNA, with consequent changes in protein expression profiles. Methods: Objectives of this study were to explore (1) tolerability and safety profile, (2) PK, (3) PD effects on pre-mRNA splicing, and (4) anti tumor activity of E7107 administered as bolus infusion on days 1, 8, 15 of a 28-day schedule Results: 36 patients (21M/15F, median age 61yrs (45–79)) received E7107 doses of 0.6 mg/m2 (n=4), 0.9 mg/m2 (n=3), 1.3 mg/m2 (n=3), 2.0 mg/m2 (n=3), 3.0 mg/m2 (n=4), 4.5 mg/m2 (n=3) and 4.0 mg/m2 (n=16). At 4.5 mg/m2 two episodes of DLT (grade 3 and 4 diarrhea) and at 4.0 mg/m2 one episode of DLT (a combination of grade 3 nausea, vomiting and abdominal cramps) were observed. Other frequently occurring side effects were mainly gastrointestinal. After drug discontinuation, one patient at 4.0 mg/m2 experienced reversible grade 4 blurred vision. The maximum tolerable dose (MTD) is 4.0 mg/m2. No complete or partial responses were observed. Pharmacokinetic analysis revealed large volume of distribution (Vss: 279 to 1369 L), high systemic clearance (CL: 111 to 253 L/hr), and moderate elimination half-life (t1/2: 5.3 to 15.1 hr). Systemic exposure on days 1 and 15 (Cmax, AUC0-∞) increased in a dose-dependent manner. At the MTD, mRNA levels of selected target genes (TRAPPC4, SLC25A19, GTF2H1), monitored in PBMC's, showed a 15–25-fold decrease, whereas unspliced pre-mRNA levels of DNAJB1 and EIF4A1 showed a 10–25-fold increase. Notably, at days 1 and 15, modulations generally peaked at 2–6 hr after end of the infusion and almost completely recovered to base-line levels at 24–48 hr. Conclusion: The MTD for E7107 using this schedule is 4.0 mg/m2. PK is dose-dependent and reproducible within subjects. PD analysis revealed dose-dependent reversible inhibition of pre-mRNA processing of target genes, confirming proof-of-principle activity of E7107. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C32.

The anti-inflammatory activity of the polyphenol resveratrol may be partially related to inhibition of tumour necrosis factor-α (TNF-α) pre-mRNA splicing

The present study shows for the first time that the polyphenol resveratrol (RESV) blocks processing of tumour necrosis factor-α (TNF-α) pre-mRNA in mature mRNA. This study was carried out in turbot (Psetta maxima (L.)), a fish species that we are using to evaluate the effects of RESV on the inflammatory response in vertebrates. Treatment of turbot head kidney leucocytes with polysaccharides from the seaweed Ulva rigida (ulvan) resulted in an increase in TNF-α expression. RESV did not inhibit transcription but almost completely inhibited the production of mRNA in ulvan-induced cells and caused a notable increase in the level of unspliced TNF-α pre-mRNA. RESV also induced accumulation of IL-1β pre-mRNA at the expense of mature mRNA, although the effects on IL-1β were less evident than those on TNF-α. However, the housekeeping gene was not affected by RESV. We also evaluated the effects of RESV in vivo under an inflammatory stimulus and found an inhibitory effect on TNF-α and IL-1β pre-mRNA splicing in turbot head kidney at 24 and 48h post-injection. In addition, RESV also reduced migration of cells to the peritoneal cavity under the same inflammatory stimulus. The results show that this fish species may be a useful model for analysing the effects of RESV on TNF-α and IL-1β expression, and suggest that RESV could be used to decrease the levels of pro-inflammatory cytokines in vivo and to reduce inflammatory reactions in certain inflammatory diseases.