Fabrice Lejeune

UV‐damaged DNA‐binding protein in the TFTC complex links DNA damage recognition to nucleosome acetylation

Initiation of transcription of protein‐encoding genes by RNA polymerase II (Pol II) was thought to require transcription factor TFIID, a complex comprised of the TATA box‐binding protein (TBP) and TBP‐associated factors (TAFIIs). In the presence of TBP‐free TAFII complex (TFTC), initiation of Pol II transcription can occur in the absence of TFIID. TFTC containing the GCN5 acetyltransferase acetylates histone H3 in a nucleosomal context. We have identified a 130 kDa subunit of TFTC (SAP130) that shares homology with the large subunit of UV‐damaged DNA‐binding factor. TFTC preferentially binds UV‐irradiated DNA, UV‐damaged DNA inhibits TFTC‐mediated Pol II transcription and TFTC is recruited in parallel with the nucleotide excision repair protein XP‐A to UV‐damaged DNA. TFTC preferentially acetylates histone H3 in nucleosomes assembled on UV‐damaged DNA. In agreement with this, strong histone H3 acetylation occurs in intact cells after UV irradiation. These results suggest that the access of DNA repair machinery to lesions within chromatin may be facilitated by TFTC via covalent modification of chromatin. Thus, our experiments reveal a molecular link between DNA damage recognition and chromatin modification.

Rescue of nonsense mutations by amlexanox in human cells

BackgroundNonsense mutations are at the origin of many cancers and inherited genetic diseases. The consequence of nonsense mutations is often the absence of mutant gene expression due to the activation of an mRNA surveillance mechanism called nonsense-mediated mRNA decay (NMD). Strategies to rescue the expression of nonsense-containing mRNAs have been developed such as NMD inhibition or nonsense mutation readthrough.MethodsUsing a dedicated screening system, we sought molecules capable to block NMD. Additionally, 3 cell lines derived from patient cells and harboring a nonsense mutation were used to study the effect of the selected molecule on the level of nonsense-containing mRNAs and the synthesis of proteins from these mutant mRNAs.ResultsWe demonstrate here that amlexanox, a drug used for decades, not only induces an increase in nonsense-containing mRNAs amount in treated cells, but also leads to the synthesis of the full-length protein in an efficient manner. We also demonstrated that these full length proteins are functional.ConclusionsAs a result of this dual activity, amlexanox may be useful as a therapeutic approach for diseases caused by nonsense mutations.

The CD44 Alternative v9 Exon Contains a Splicing Enhancer Responsive to the SR Proteins 9G8, ASF/SF2, and SRp20

The CD44 gene alternative exons v8, v9, and v10 are frequently spliced as a block by epithelial cells. By transfecting minigenes containing only one of these alternative exons, we show that splicing of each of them is under cell type-specific control. By using minigenes carrying short block mutations within exons v8 and v9, we detected a candidate exon splicing enhancer in each of these exons. These candidates activated splicing in vitro of a heterologous transcript and are thus true exon splicing enhancers. We analyzed further a v9 exon splicing enhancer covering ∼30 nucleotides. This enhancer can be UV cross-linked to SR proteins of 35 and 20 kDa in HeLa nuclear extract. By using individual recombinant SR proteins for UV cross-linking in S100 extract, these proteins were identified as 9G8, ASF/SF2, and SRp20. S100 complementation studies using recombinant 9G8, ASF/SF2, and SRp20 showed that all three proteins can activate splicing in vitro of a heterologous exon containing the v9 enhancer; the strongest activation was obtained with 9G8. Progressive truncation of the 30-nucleotide enhancer leads to a progressive decrease in splicing activation. We propose that 9G8, ASF/SF2, SRp20, and possibly other non-SR proteins cooperate in vivo to activate v9 exon splicing.

Alternative Splicing of Intron 3 of the Serine/Arginine-rich Protein 9G8 Gene IDENTIFICATION OF FLANKING EXONIC SPLICING ENHANCERS AND INVOLVEMENT OF 9G8 AS A TRANS-ACTING FACTOR

9G8 protein belongs to the conserved serine/arginine-rich (SR) protein family, whose members exhibit multiple functions in constitutive and alternative splicing. We have previously shown that 9G8 primary transcripts are subjected to alternative splicing by excision/retention of intron 3 and to a tissue specific modulation. Because both 5′- and 3′-splice sites of intron 3 appear to be suboptimal in vertebrates, we tested the 9G8 intron 3 as a novel model system of alternative splicing. By using an in vitro approach and a mutational analysis, we have identified two purine-rich exonic splicing enhancers (ESE) located in exon 4 and a (GAA)3 enhancer located in exon 3. These elements act in concert to promote efficient splicing activation both in vitro and in vivo. Titration experiments with an excess of exonic enhancers or SR-specific RNA targets strongly suggest that SR proteins are specifically involved in the activation process. Although ASF/SF2 was expected to interact the most efficiently with ESE according to the enhancer sequences, UV cross-linking coupled or not to immunopurification demonstrates that 9G8 is highly recruited by the three ESE, followed by SC35. In contrast, ASF/SF2 only binds significantly to the (GAA)3 motif. S100 complementation experiments with individual SR proteins demonstrate that only 9G8 is able to fully restore splicing of intron 3. These results, and the fact that the exon 3 and 4 ESE sequences are conserved in vertebrates, strongly suggest that the alternative splicing of intron 3 represents an important step in the regulation of the expression of 9G8.

Human RBMY regulates germline-specific splicing events by modulating the function of the serine/arginine-rich proteins 9G8 and Tra2-β

RBMY is a male germline RNA binding protein and potential alternative splicing regulator, but the lack of a convenient biological system has made its cellular functions elusive. We found that human RBMY fused to green fluorescent protein was strictly nuclear in transfected cells, but spatially enriched in areas around nuclear speckles with some components of the exon junction complex (EJC). Human RBMY (hRBMY) and the EJC components Magoh and Y14 also physically interacted but, unlike these two proteins, hRBMY protein did not shuttle to the cytoplasm. In addition, it relocalised into nucleolar caps after inhibition of RNA polymerase II transcription. Protein interactions were also detected between RBMY and splicing factors 9G8 and transformer-2 protein homolog β (Tra2-β), mediated by multiple regions of the RBMY protein that contain serine/arginine-rich dipeptides, but not by the single region lacking such dipeptides. These interactions modulated the splicing of several pre-mRNAs regulated by 9G8 and Tra2-β. Importantly, ectopic expression of hRBMY stimulated the inclusion of a testis-enriched exon from the Acinus gene, whereas 9G8 and Tra2-β repressed this exon. We propose that hRBMY associates with regions of the nucleus enriched in nascent RNA and participates in the regulation of specific splicing events in the germline by modulating the activity of constitutively expressed splicing factors.

Premature termination codon readthrough in human cells occurs in novel cytoplasmic foci and requires UPF proteins

ABSTRACT Nonsense-mutation-containing messenger ribonucleoprotein particles (mRNPs) transit through cytoplasmic foci called P-bodies before undergoing nonsense-mediated mRNA decay (NMD), a cytoplasmic mRNA surveillance mechanism. This study shows that the cytoskeleton modulates transport of nonsense-mutation-containing mRNPs to and from P-bodies. Impairing the integrity of cytoskeleton causes inhibition of NMD. The cytoskeleton thus plays a crucial role in NMD. Interestingly, disruption of actin filaments results in both inhibition of NMD and activation of premature termination codon (PTC) readthrough, while disruption of microtubules causes only NMD inhibition. Activation of PTC readthrough occurs concomitantly with the appearance of cytoplasmic foci containing UPF proteins and mRNAs with nonsense mutations but lacking the P-body marker DCP1a. These findings demonstrate that in human cells, PTC readthrough occurs in novel ‘readthrough bodies’ and requires the presence of UPF proteins. Summary: The cytoskeleton transports premature termination codon-containing mRNAs to be degraded by nonsense-mediated decay or to be read through in specific cytoplasmic foci called readthrough bodies.

Optimized approach for the identification of highly efficient correctors of nonsense mutations in human diseases

About 10% of patients with a genetic disease carry a nonsense mutation causing their pathology. A strategy for correcting nonsense mutations is premature termination codon (PTC) readthrough, i.e. incorporation of an amino acid at the PTC position during translation. PTC-readthrough-activating molecules appear as promising therapeutic tools for these patients. Unfortunately, the molecules shown to induce PTC readthrough show low efficacy, probably because the mRNAs carrying a nonsense mutation are scarce, as they are also substrates of the quality control mechanism called nonsense-mediated mRNA decay (NMD). The screening systems previously developed to identify readthrough-promoting molecules used cDNA constructs encoding mRNAs immune to NMD. As the molecules identified were not selected for the ability to correct nonsense mutations on NMD-prone PTC-mRNAs, they could be unsuitable for the context of nonsense-mutation-linked human pathologies. Here, a screening system based on an NMD-prone mRNA is described. It should be suitable for identifying molecules capable of efficiently rescuing the expression of human genes harboring a nonsense mutation. This system should favor the discovery of candidate drugs for treating genetic diseases caused by nonsense mutations. One hit selected with this screening system is presented and validated on cells from three cystic fibrosis patients.

Targeting nonsense-mediated mRNA decay in colorectal cancers with microsatellite instability

Nonsense-mediated mRNA decay (NMD) is responsible for the degradation of mRNAs with a premature termination codon (PTC). The role of this system in cancer is still quite poorly understood. In the present study, we evaluated the functional consequences of NMD activity in a subgroup of colorectal cancers (CRC) characterized by high levels of mRNAs with a PTC due to widespread instability in microsatellite sequences (MSI). In comparison to microsatellite stable (MSS) CRC, MSI CRC expressed increased levels of two critical activators of the NMD system, UPF1/2 and SMG1/6/7. Suppression of NMD activity led to the re-expression of dozens of PTC mRNAs. Amongst these, several encoded mutant proteins with putative deleterious activity against MSI tumorigenesis (e.g., HSP110DE9 chaperone mutant). Inhibition of NMD in vivo using amlexanox reduced MSI tumor growth, but not that of MSS tumors. These results suggest that inhibition of the oncogenic activity of NMD may be an effective strategy for the personalized treatment of MSI CRC.

Chapter 4 – Conclusions

These days, several choices are available to correct nonsense mutations with very encouraging results in cellulo, at least. Some of the molecules are reaching the clinical phase III, indicating that a treatment for nonsense mutation-associated diseases might be provided to patients in a near future. Nonsense mutation therapies represent a development of targeted therapy, rather than a development of personalized medicine. As new approaches, they are raising some ethical issues. One of them concerns the modification of the patient genome and his/her offspring for approaches targeting the gene, such as genome editing or gene therapy. Another ethical issue comes from therapeutic strategies that do not correct the nonsense mutation at the DNA level. Indeed, such approaches increase the possibility to stabilize nonsense mutations in the human DNA patrimony after several generations. These treatments might be available faster than the one modifying the patient genome, but will have to be replaced by those latter when their safety will be completely addressed and their in vivo efficiency demonstrated.

Pathologies Susceptible to be Targeted for Nonsense Mutation Therapies

Nonsense mutations are involved in about 10% of patients with genetic diseases. These genetic diseases can enter in the rare pathology category, or in the frequent disease class, making nonsense mutation therapies of interest for a significant number of patients. Among the rare pathology category, Duchenne muscular dystrophy (DMD), cystic fibrosis (CF), and spinal muscular atrophy (SMA) will be described. Cancer, metabolic diseases, and neurologic disorder represent three main classes of frequent diseases that will be discussed. The rate of nonsense mutations among all these pathologies is variable, from one gene to another, or from one tissue to another. However, the mechanism of gene silencing occurring as a consequence of a nonsense mutation is shared by all patients harboring a nonsense mutation in the gene responsible for their pathology. Consequently, common therapies can be applied to patients with various diseases, changing the way to see a pathology and an associated treatment.