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Regulation of exon definition by intrinsic elements and by combination of tailored U1snRNA with antisense oligonucleotides
No full textMutazioni che coinvolgono l’RNA messaggero rappresentano una causa relativamente frequente di malattia; negli ultimi anni la ricerca ha potenziato l’interesse in questo ambito, promuovendo lo sviluppo di approcci terapeutici in grado di modulare questo processo molto complesso. Il lavoro di questa tesi riguarda lo sviluppo di strategie innovative, che vanno ad agire a livello dell’RNA, per tentare di correggere mutazioni presenti nel gene del fattore IX della coagulazione e nel gene ATR, e che causano rispettivamente patologie quali l’Emofilia B e la Sindrome di Seckel. Studi precedenti condotti nel nostro gruppo di ricerca hanno dimostrato che mediante l’utilizzo di varianti di U1 small nuclear RNA (U1snRNA), componente chiave della ribonucleoproteina U1 coinvolta nel processo di splicing, e in grado di legarsi a valle dell’esone contenente la mutazione (Exon-specific U1, ExSpeU1) si è in grado di ripristinare il corretto processamento del messaggero. Inoltre è stato visto che, in modelli cellulari, un’unica ExSpeU1fx9 è capace di ripristinare lo splicing aberrante causato da mutazioni che inducono il salto dell’esone e localizzate sia a livello del sito donatore (5’ss) o accettore (3’ss) nell’esone 5 del fattore IX della coagulazione. Attraverso studi di espressione di varianti mutate del fattore IX in modelli murini, è stato dimostrato per la prima volta che la ExSpeU1fix9 è in grado di ripristinare il corretto processamento del messaggero, risultante in livelli di proteina circolante e con attività coagulante nei due modelli di mutazioni al 5’ss (c.519A>G) e 3’ss (c.392-8T>G). Inoltre, attraverso l’espressione di minigeni in cellule eucariotiche, sono state caratterizzate numerose mutazioni (missenso e al 5’ss) nell’esone 2 del fattore IX della coagulazione, le quali producono uno splicing aberrante a causa dell’ utilizzo di un sito alternativo all’interno dell’esone. Esperimenti condotti utilizzando sia varianti naturali che non, hanno dimostrato che l’uso di questo sito alternativo è regolato dalla sinergia di elementi regolatori comprendenti un elemento silenziatore (Exonic splicing silencer ESS). Con lo scopo di ripristinare il corretto processamento del messaggero, sono stati testati degli oligonucleotidi antisenso (AON) per mascherare il sito alternativo. Tali AON sono risultati efficaci per le mutazioni esoniche ma hanno indotto il salto dell’esone 2 in presenza di mutazioni al sito donatore 5’ autentico. Parallelamente, è stata osservata una ridotta correzione dopo l’utilizzo di varianti di U1snRNA perfettamente complementari al sito donatore mutato. E’ interessante invece osservare che l’utilizzo combinato degli oligonucleotidi antisenso con le U1snRNA modificate ha indotto una correzione apprezzabile del processamento dell’RNA in presenza di mutazioni nel sito donatore.
Nel modello della Sindrome di Seckel (SS-1) è stata caratterizzata la mutazione sinonima c.A2101G presente nell’esone 9 del gene ATR, la quale provoca il salto dell’esone stesso introducendo un
elemento silenziatore (ESS) in un contesto già naturalmente poco definito. In maniera sinergica sono state esplorate due strategie di correzione basate su un AON, utilizzato per mascherare l’elemento silenziatore, e una U1snRNA modificata, con lo scopo di migliorare la definizione dell’esone 9. Utilizzando la strategia dei minigeni si è potuto osservare che entrambi gli approcci sono in grado di ripristinare l’inclusione dell’esone 9, che passa dal 6% al 100% e 63% a seconda dell’approccio utilizzato. La U1snRNA modificata è stata successivamente testata in fibroblasti murini umanizzati per la mutazione causativa di malattia (MEFSS-1). La veicolazione di questa U1snRNA mediante l’utilizzo di un lentivirus modificato ha portato a una parziale inclusione dell’esone 9 e, anche se in minor quantità, anche ad un aumento dell’espressione di ATR a livello proteico. Complessivamente, i dati ottenuti nei modelli di Emofilia B e Sindrome di Seckel dimostrano l’abilità di questi approcci nel correggere gli effetti delle mutazioni di splicing, e l’abilità nel ripristinare l’espressione genica. Questi dati incoraggiano lo sviluppo di terapie innovative basate sull’utilizzo di AON e U1snRNA modificate per la cura di malattie genetiche.The notion that a significant proportion of disease-causing mutations affects pre-mRNA boosted research towards the design of approaches modulating this very complex process for therapeutic purposes. This PhD project has been focused on the development of RNA-based correction approaches for Hemophilia B (HB) or Seckel syndrome, hemorrhagic or neurologic disorders due to mutations in the F9 or ATR genes, respectively. Previous studies by our research group on a panel of HB-causing mutations led to the development of variants of the U1 small nuclear RNA (U1snRNA), key component of the spliceosomal U1 small nuclear ribonucleoprotein, which can rescue exon inclusion by targeting intronic sequences downstream of a defective exon (Exon-specific U1snRNA, ExSpeU1). Most importantly, it has been proven in cellular models that a unique ExSpeU1fx9 can rescue aberrant splicing caused by multiple exon-skipping mutations occurring at the donor (5’ss) or acceptor (3’ss) splice sites of the F9 exon 5. Here, by expressing the human F9 splicing-defecting expression cassettes in mouse liver, we provided the first in vivo proof-of-concept that the selected ExSpeU1fix9 is able to restore F9 splicing and remarkably increase human factor IX (FIX) protein levels and coagulant activity in plasma in the presence of two model exon-skipping mutations at the 5’ss (c.519A>G) or 3’ss (c.392-8T>G) In the model of F9 exon 2, through the expression of F9 minigenes in mammalian cells, we characterized numerous HB-causing mutations, either missense or at the 5’ss , that promote aberrant splicing by inducing the usage of a strong exonic cryptic 5’ss. Splicing assays with natural and artificial F9 variants indicated that the cryptic 5’ss is regulated, among a network of regulatory elements, by an exonic splicing silencer (ESS). This finding supports a compensatory mechanism aimed at minimizing unproductive splicing. To recover splicing we tested antisense oligo-ribonucleotides (AON) masking the cryptic 5’ss, which were effective on exonic changes but promoted exon 2 skipping in the presence of mutations at the authentic 5’ss. On the other hand, we observed a very poor correction effect by U1snRNA variants with increased or perfect complementarity to the defective 5’ss. Noticeably, the combination of the mutant-specific U1snRNAs with antisense oligonucleotides produced appreciable amounts of correctly spliced transcripts from several mutants of the exon 2 5’ss. In the Seckel Syndrome (SS-1) model we characterized the synonymous c.A2101G change in exon 9 of the ATR gene that induces exon 9 skipping by creating an exonic splicing
silencer (ESS) in the poorly defined exon. Based on this mechanism, we explored two complementary correction strategies based on AON, designed to mask the ESS and modified U1snRNA, improving exon 9 definition. In ATR minigene assays demonstrated that both the AON and the U1snRNAATR induced a robust exon 9 inclusion raising from 6% to 100% and 63%, respectively. The U1snRNAATR was then challenged in the embryonic fibroblasts from the humanized SS-1 mouse model (MEFSS-1) harboring the SS1 splicing mutation. The lentiviral-mediated delivery of the U1snRNAATR in MEFSS-1 resulted in partial rescue of exon 9 inclusion and in the low but appreciable increase of ATR protein expression.
Taken together these data in the hemophilia B and Seckel syndrome models demonstrate the ability of appropriately designed RNA-based approaches to counteract splicing mutations and rescue gene expression, thus encouraging their exploitation for the development of innovative therapies for genetic disorders
A unique exon specific U1snRNA rescues different haemophilia B - causing splicing-defective factor IX variants in mice
No full textRegulation of a strong F9 cryptic 5'ss by intrinsic elements and by combination of tailored U1snRNAs with antisense oligonucleotides
No full textMutations affecting specific splicing regulatory elements offer suitable models to better understand their interplay and to devise therapeutic strategies. Here we characterize a meaningful splicing model in which numerous Hemophilia B-causing mutations, either missense or at the donor splice site (5'ss) of coagulation F9 exon 2, promote aberrant splicing by inducing the usage of a strong exonic cryptic 5'ss. Splicing assays with natural and artificial F9 variants indicated that the cryptic 5'ss is regulated, among a network of regulatory elements, by an exonic splicing silencer (ESS). This finding and the comparative analysis of the F9 sequence across species showing that the cryptic 5'ss is always paralleled by the conserved ESS support a compensatory mechanism aimed at minimizing unproductive splicing. To recover splicing we tested antisense oligoribonucleotides masking the cryptic 5'ss, which were effective on exonic changes but promoted exon 2 skipping in the presence of mutations at the authentic 5'ss. On the other hand, we observed a very poor correction effect by small nuclear RNA U1 (U1snRNA) variants with increased or perfect complementarity to the defective 5'ss, a strategy previously exploited to rescue splicing. Noticeably, the combination of the mutant-specific U1snRNAs with antisense oligonucleotides produced appreciable amounts of correctly spliced transcripts (from 0 to 20-40%) from several mutants of the exon 2 5'ss. Based on the evidence of an altered interplay among ESS, cryptic and the authentic 5'ss as a disease-causing mechanism, we provide novel experimental insights into the combinatorial correction activity of antisense molecules and compensatory U1snRNAs
Exploring Splicing-Switching Molecules For Seckel Syndrome Therapy
No full textThe c.2101 A > G synonymous change (p.G674G) in the gene for ATR, a key player in the DNA-damage response, has been the first identified genetic cause of Seckel Syndrome (SS), an orphan disease characterized by growth and mental retardation. This mutation mainly causes exon 9 skipping, through an ill-defined mechanism. Through ATR minigene expression studies, we demonstrated that the detrimental effect of this mutation (6 ± 1% of correct transcripts only) depends on the poor exon 9 definition (47 ± 4% in the ATRwt context), because the change was ineffective when the weak 5′ or the 3′ splice sites (ss) were strengthened (scores from 0.54 to 1) by mutagenesis. Interestingly, the exonic c.2101 A nucleotide is conserved across species, and the SS-causing mutation is predicted to concurrently strengthen a Splicing Silencer (ESS) and weaken a Splicing Enhancer (ESE). Consistently, the artificial c.2101 A > C change, predicted to weaken the ESE only, moderately impaired exon inclusion (28 ± 7% of correct transcripts). The observation that an antisense oligonucleotide (AONATR) targeting the c.2101 A position recovers exon inclusion in the mutated context supports a major role of the underlying ESS. A U1snRNA variant (U1ATR) designed to perfectly base-pair the weak 5'ss, rescued exon inclusion (63 ± 3%) in the ATRSS-allele. Most importantly, upon lentivirus-mediated delivery, the U1ATR partially rescued ATR mRNA splicing (from ~ 19% to ~ 54%) and protein (from negligible to ~ 6%) in embryonic fibroblasts derived from humanized ATRSS mice. Altogether these data elucidate the molecular mechanisms of the ATR c.2101 A > G mutation and identify two potential complementary RNA-based therapies for Seckel syndrome
Correction of aberrant splicing causing haemophilia B through the combination of compensatory U1snRNAs and antisense oligonucleotides
No full textRescue of missense and splicing mutations in Haemophilia A by a unique Exon Specific U1snRNA
No full textBackground:
In Hemophilia A (HA) patients, splicing mutations account for about 8-10% of all, a significant proportion if considered that they are largely underestimated. In fact, also the abundant missense mutations can trigger, besides potentially influencing protein biology, aberrant splicing by affecting splicing regulatory elements. The knowledge of molecular mechanisms leading to aberrant splicing led us to the development of new RNA-therapeutics, in particular the engineered spliceosomal U1snRNA targeting specific intronic sequences downstream of donor splice sites (Exon Specific U1, ExSpeU1). This approach enabled us to rescue mutations at donor (5’ss) or acceptor (3’ss) splice sites, as well as within exons. Here, the aim is to characterize the impact on splicing of different HA-causing mutations occurring in F8 exon 19 (at splice sites or within the exon) and develop ExSpeU1 able to correct them for therapeutic purposes.
Methods:
Expression of F8 minigenes in human hepatoma cells and dissection of splicing pattern by RT-PCR. Evaluation of ability of ExSpeU1, or the first generation U1, to rescue splicing.
Results:
The F8 variants c.6115+1G>A, c.6115+2T>C, c.6115+3G>T, c.6115+4A>G, c.6115+5G>A and c.6115+6T>A, expressed in HepG2 cells, led to complete skipping of exon 19. On the other hand, c.6115+9C>G, c.6113A>G (p.N2038S), c.6037G>A (p.G2013R), c.6053A>G (p.E2018G), c.6108C>T (p.Y2036Y) and c.5999G>C (p.G2000A) impaired to various extent exon 19 inclusion (exon skipping), with variable amounts of correct transcript. The impact of missense changes on protein function showed that the p.N2038S and p.G2013R impair both protein secretion and function (<10% of F8 wt). Notably, the p.E2018G has no effect on protein levels. By screening a panel of ExSpeU1s, we identified a unique ExSpeU1 (U1s1) able to rescue both different splicing-defective mutations, either at splice sites or exonic. In particular, the U1s1 promoted (from 0 to ~40%) exon inclusion in presence of c.6115+3G>T, c.6115+4A>G and c.6115+6T>A variants. Notably, the U1s1-mediated rescue, in presence of p.G2013R, p.N2038S and p.E2018G, was more robust (from ~30 to ~80%).
Conclusions:
We provide insights in the molecular mechanisms causing aberrant splicing in presence of splicing and missense changes in HA. We also provided the proof that a single ExSpeU1 can efficiently rescue multiple mutations and its effect, if translated into patients, could have therapeutic implication also for those missense changes with negligible impact on protein levels
Disease-causing variants of the conserved+2T of 5 ' splice sites can be rescued by engineered U1snRNAs
No full textThe ability of variants of the spliceosomal U1snRNA to rescue splicing has been proven in several human disease models, but not for nucleotide changes at the conserved GT nucleotide of 5 ' splice sites (5 ' ss), frequent and associated with severe phenotypes. Here, we focused on variants at the 5 ' ss of F9 intron 3, leading to factor IX (FIX) deficiency (hemophilia B). Through minigene expression, we demonstrated that all changes induce complete exon 3 skipping, which explains the associated hemophilia B phenotype. Interestingly, engineered U1snRNAs remarkably increased the proportion of correct transcripts in the presence of the c.277+4A>G (similar to 60%) and also c.277+2T>C mutation (similar to 20%). Expression of splicing-competent cDNA constructs indicated that the splicing rescue produces an appreciable increase of secreted FIX protein levels. These data provide the first experimental evidence that even part of variants at the conserved 5 ' ss +2T nucleotide can be rescued, thus expanding the applicability of this U1snRNA-based approach
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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