Mature mRNA Research Articles

Phosphorylation of pICln by the autophagy activating kinase ULK1 regulates snRNP biogenesis and splice activity of the cell

The spliceosome, responsible for all mature protein-coding transcripts of eukaryotic intron-containing genes, consists of small uridine-rich nuclear ribonucleoproteins (UsnRNPs). The assembly of UsnRNPs depends, on one hand, on the arginine methylation of Sm proteins catalyzed by the PRMT5 complex. On the other hand, it depends on the phosphorylation of the PRMT5 subunit pICln by the Uncoordinated Like Kinase 1 (ULK1). In consequence, phosphorylation of pICln affects the stability of the UsnRNP assembly intermediate, the so-called 6 S complex. The detailed mechanisms of phosphorylation-dependent integrity and subsequent UsnRNP assembly of the 6 S complex in vivo have not yet been analyzed.By using a phospho-specific antibody against ULK1-dependent phosphorylation sites of pICln, we visualize the intracellular distribution of phosphorylated pICln. Furthermore, we detect the colocaliphosphor-pICln1 with phospho-pICln by size-exclusion chromatography and immunofluorescence techniques. We also show that phosphorylated pICln is predominantly present in the 6 S complex. The addition of ULK1 to in vitro produced 6 S complex, as well as the reconstitution of ULK1 in ULK1-deficient cells, increases the efficiency of snRNP biogenesis. Accordingly, inhibition of ULK1 and the associated decreased pICln phosphorylation lead to accumulation of the 6 S complex and reduction in the spliceosomal activity of the cell.

Pseudouridine Identification and Functional Annotation with PIANO.

Pseudouridine (Ψ) is the first-discovered RNA modification abundantly present in many classes of RNAs, which plays a pivotal role in a series of biological processes. Accurately identifying the location of Ψ sites is helpful for relevant downstream researches. In this chapter, we introduce a website PIANO-for pseudouridine site (Ψ) identification and functional annotation, which enables researchers to predict human putative Ψ sites with a high-accuracy (average AUC of 0.955 under the full transcript model and 0.838 under the mature mRNA model when testing on six independent datasets). The posttranscriptional regulatory mechanisms of putative Ψ sites including miRNA-targets, RBP-binding regions, and splicing sites were also annotated. A comprehensive query database was also provided to deposit over 4300 human Ψ modifications, which is currently the most complete collection of experimental-derived Ψ sites. The PIANO website is freely accessible at: http://piano.rnamd.com or http://180.208.58.19/Ψ-WHISTLE .

Sequential Polyadenylation to Enable Alternative mRNA 3' End Formation.

In eukaryotic cells, a key RNA processing step to generate mature mRNA is the coupled reaction for cleavage and polyadenylation (CPA) at the 3' end of individual transcripts. Many transcripts are alternatively polyadenylated (APA) to produce mRNAs with different 3' ends that may either alter protein coding sequence (CDS-APA) or create different lengths of 3'UTR (tandem-APA). As the CPA reaction is intimately associated with transcriptional termination, it has been widely assumed that APA is regulated cotranscriptionally. Isoforms terminated at different regions may have distinct RNA stability under different conditions, thus altering the ratio of APA isoforms. Such differential impacts on different isoforms have been considered as post-transcriptional APA, but strictly speaking, this can only be considered "apparent" APA, as the choice is not made during the CPA reaction. Interestingly, a recent study reveals sequential APA as a new mechanism for post-transcriptional APA. This minireview will focus on this new mechanism to provide insights into various documented regulatory paradigms.

Regulation of age-dependent expression patterns of five transcription factors in Larix kaempferi.

To reveal the regulatory mechanisms underlying age-dependent expression patterns, we characterized seven age-related genes, LaDAL1, LaAGL2-2, LaAGL2-3, LaAGL11, LaSOC1-1, LaAP2-1, and LaAP2-2 in terms of transcription and intron splicing in Larix kaempferi. Based on the exon-intron structures, we quantified the pre-mRNA levels and mature mRNA levels of these seven genes using quantitative reverse transcription polymerase chain reaction experiments. We found that the pre-mRNA levels manifested age-related patterns, indicating that their transcription was primarily regulated by age. By comparing the increasing or decreasing rates of the pre-mRNA and spliced mRNA levels, we found that their splicing efficiencies also changed with age. These results clearly show that both pre-mRNA transcription and splicing of five age-related genes are regulated by age, indicating that age-dependent expression patterns are controlled at both transcriptional and post-transcriptional levels, and unveiling the underlying regulatory molecular mechanisms should focus on the transcription factors, epigenetic regulation, and RNA splicing. These data provide new insights into the age-mediated regulation of gene expression in woody perennials in terms of longevity.

Trypanosoma brucei RRP44: a versatile enzyme for processing structured and non-structured RNA substrates.

Rrp44/Dis3 is a conserved eukaryotic ribonuclease that acts on processing and degradation of nearly all types of RNA. It contains an endo- (PIN) and an exonucleolytic (RNB) domain and, its depletion in model organisms supports its essential function for cell viability. In Trypanosoma brucei, depletion of Rrp44 (TbRRP44) blocks maturation of ribosomal RNA, leading to disruption of ribosome synthesis and inhibition of cell proliferation. We have determined the crystal structure of the exoribonucleolytic module of TbRRP44 in an active conformation, revealing novel details of the catalytic mechanism of the RNB domain. For the first time, the position of the second magnesium involved in the two-metal-ion mechanism was determined for a member of the RNase II family. In vitro, TbRRP44 acts preferentially on non-structured uridine-rich RNA substrates. However, we demonstrated for the first time that both TbRRP44 and its homologue from Saccharomyces cerevisiae can also degrade structured substrates without 3'-end overhang, suggesting that Rrp44/Dis3 ribonucleases may be involved in degradation of a wider panel of RNA than has been assumed. Interestingly, deletion of TbRRP44 PIN domain impairs RNA binding to different extents, depending on the type of substrate.

SR Protein Kinase 1 Inhibition by TAF15.

Although SRPKs were discovered nearly 30 years ago, our understanding of their mode of regulation is still limited. Regarded as constitutively active enzymes known to participate in diverse biological processes, their prominent mode of regulation mainly depends on their intracellular localization. Molecular chaperones associate with a large internal spacer sequence that separates the bipartite kinase catalytic core and modulates the kinases' partitioning between the cytoplasm and nucleus. Besides molecular chaperones that function as anchoring proteins, a few other proteins were shown to interact directly with SRPK1, the most-studied member of SRPKs, and alter its activity. In this study, we identified TAF15, which has been involved in transcription initiation, splicing, DNA repair, and RNA maturation, as a novel SRPK1-interacting protein. The C-terminal RGG domain of TAF15 was able to associate with SRPK1 and downregulate its activity. Furthermore, overexpression of this domain partially relocalized SRPK1 to the nucleus and resulted in hypophosphorylation of SR proteins, inhibition of splicing of a reporter minigene, and inhibition of Lamin B receptor phosphorylation. We further demonstrated that peptides comprising the RGG repeats of nucleolin, HNRPU, and HNRNPA2B1, were also able to inhibit SRPK1 activity, suggesting that negative regulation of SRPK1 activity might be a key biochemical property of RGG motif-containing proteins.

The multi-faceted nature of 15 CFTR exonic variations: Impact on their functional classification and perspectives for therapy

BackgroundThe majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy. MethodsWe selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR-France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs. ResultsSix of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested. ConclusionThe 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments.

Clustered variants in the 5′ coding region of TRA2B cause a distinctive neurodevelopmental syndrome

PurposeTransformer2 proteins (Tra2α and Tra2β) control splicing patterns in human cells, and no human phenotypes have been associated with germline variants in these genes. The aim of this work was to associate germline variants in the TRA2B gene to a novel neurodevelopmental disorder. MethodsA total of 12 individuals from 11 unrelated families who harbored predicted loss-of-function monoallelic variants, mostly de novo, were recruited. RNA sequencing and western blot analyses of Tra2β-1 and Tra2β-3 isoforms from patient-derived cells were performed. Tra2β1-GFP, Tra2β3-GFP and CHEK1 exon 3 plasmids were transfected into HEK-293 cells. ResultsAll variants clustered in the 5′ part of TRA2B, upstream of an alternative translation start site responsible for the expression of the noncanonical Tra2β-3 isoform. All affected individuals presented intellectual disability and/or developmental delay, frequently associated with infantile spasms, microcephaly, brain anomalies, autism spectrum disorder, feeding difficulties, and short stature. Experimental studies showed that these variants decreased the expression of the canonical Tra2β-1 isoform, whereas they increased the expression of the Tra2β-3 isoform, which is shorter and lacks the N-terminal RS1 domain. Increased expression of Tra2β-3-GFP were shown to interfere with the incorporation of CHEK1 exon 3 into its mature transcript, normally incorporated by Tra2β-1. ConclusionPredicted loss-of-function variants clustered in the 5′ portion of TRA2B cause a new neurodevelopmental syndrome through an apparently dominant negative disease mechanism involving the use of an alternative translation start site and the overexpression of a shorter, repressive Tra2β protein.

The MITOCHONDRIAL TRANSCRIPT STABILITY FACTOR 4 (MTSF4) is essential for the accumulation of dicistronic rpl5-cob mRNAs in Arabidopsis thaliana.

The Arabidopsis thaliana genome harbors more than 450 nuclear genes encoding pentatricopeptide repeat (PPR) proteins that operate in the RNA metabolism of mitochondria and/or plastids. To date, the molecular function of many PPR proteins is still unknown. Here we analyzed the nucleus-encoded gene At4g19440 coding for a P-type PPR protein. Knockout of this gene interferes with normal embryo development and seed maturation. Two experimental approaches were applied to overcome lethality and to investigate the outcome of At4g19440 knockout in adult plants. These studies revealed changes in the abundance of several mitochondria-encoded transcripts. In particular, steady-state levels of dicistronic rpl5-cob RNAs were markedly reduced, whereas levels of mature ccmC and rpl2-mttB transcripts were clearly increased. Predictions according to the one repeat to one nucleotide code for PPR proteins indicate binding of the At4g19440 protein to a previously detected small RNA at the 3' termini of the dicistronic rpl5-cob transcripts. This potential interaction indicates a function of this protein in 3' end formation and stabilization of these RNA species, whereas the increase in the levels of the ccmC mRNA along with other mitochondria-encoded RNAs seems to be a secondary effect of At4g19440 knockout. Since the inactivation of At4g19440 influences the stability of several mitochondrial RNAs we call this gene MITOCHONDRIAL TRANSCRIPT STABILITY FACTOR 4 (MTSF4). This factor will be an interesting subject to study opposing effects of a single nucleus-encoded protein on mitochondrial transcript levels.

Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing

RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3′ end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3′ end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.

Inter-domain Flexibility of Human Ser/Arg-Rich Splicing Factor 1 Allows Variable Spacer Length in Cognate RNA's Bipartite Motifs.

Ser/Arg-rich splicing factor 1 (SRSF1 or ASF/SF2) is the prototypical member of SR proteins. SRSF1 binds to exonic splicing enhancers, which prompts inclusion of corresponding exons in the mature mRNA. The RNA-binding domain of SRSF1 consists of tandem RNA-recognition motifs (RRM1 and RRM2) separated by a 30 amino acid long linker. In this study, we investigate roles of RRM1, RRM2, and the linker in RNA binding. We find that although both RRMs are crucial to RNA binding, RRM2 plays the dominant role. The linker mildly contributes to RNA binding and remains flexible in the RNA-bound state. Flexibility of the linker allows the RRM1-cognate motif to be either upstream or downstream of the RRM2-cognate motif. In addition, we find that the spacer length between the bipartite motifs varies from 0 to 10 nucleotides. Our binding assays reveal that SRSF1 prefers RNA sequences with shorter spacers and the RRM1-cognate motif being placed upstream. Restrained by nuclear magnetic resonance data, we simulate RNA-bound complexes and demonstrate how tandem RRMs bind to RNA of different spacer lengths and swapped bipartite motifs. We find that when the RRM1-cognate motif is placed downstream, either the RRM1/RRM2 linker needs to be more extended or RNA needs to form a U turn, which may reduce conformational entropy. Our study suggests that the RNA-binding specificity of SRSF1 is broader than traditionally recapitulated by consensus sequences of 7 to 8 nucleotides. Instead, centered on the RRM2-cognate motif, an RNA fragment encompassing 10-nucleotide upstream and downstream should be scrutinized.

SUMO pathway is required for ribosome biogenesis

Ribosomes, acting as the cellular factories for protein production, are essential for all living organisms. Ribosomes are composed of both proteins and RNAs and are established through the coordination of several steps, including transcription, maturation of ribosomal RNA (rRNA), and assembly of ribosomal proteins. In particular, diverse factors required for ribosome biogenesis, such as transcription factors, small nucleolar RNA (snoRNA)-associated proteins, and assembly factors, are tightly regulated by various post-translational modifications. Among these modifications, small ubiquitin-related modifier (SUMO) targets lots of proteins required for gene expression of ribosomal proteins, rRNA, and snoRNAs, rRNA processing, and ribosome assembly. The tight control of SUMOylation affects functions and locations of substrates. This review summarizes current studies and recent progress of SUMOylation-mediated regulation of ribosome biogenesis. [BMB Reports 2022; 55(11): 535-540].

SlRIP1b is a global organellar RNA editing factor, required for normal fruit development in tomato plants.

RNA editing in plant organelles involves numerous C-U conversions, which often restore evolutionarily conserved codons and may generate new translation initiation and termination codons. These RNA maturation events rely on a subset of nuclear-encoded protein cofactors. Here, we provide evidence of the role of SlRIP1b on RNA editing of mitochondrial transcripts in tomato (Solanum lycopersicum) plants. SlRIP1b is a RIP/MORF protein that was originally identified as an interacting partner of the organellar editing factor SlORRM4. Mutants of SlRIP1b, obtained by CRISPR/Cas9 strategy, exhibited abnormal carpel development and grew into fruit with more locules. RNA-sequencing revealed that SlRIP1b affects the C-U editing of numerous mitochondrial pre-RNA transcripts and in particular altered RNA editing of various cytochrome c maturation (CCM)-related genes. The slrip1b mutants display increased H2 O2 and aberrant mitochondrial morphologies, which are associated with defects in cytochrome c biosynthesis and assembly of respiratory complex III. Taken together, our results indicate that SlRIP1b is a global editing factor that plays a key role in CCM and oxidative phosphorylation system biogenesis during fruit development in tomato plants. These data provide important insights into the molecular roles of organellar RNA editing factors during fruit development.

Human histone pre-mRNA assembles histone or canonical mRNA-processing complexes by overlapping 3'-end sequence elements.

Human pre-mRNA processing relies on multi-subunit macromolecularcomplexes, which recognize specific RNA sequence elements essential for assembly and activity. Canonical pre-mRNA processing proceeds via the recognition of a polyadenylation signal (PAS) and a downstream sequence element (DSE), and produces polyadenylated mature mRNAs, while replication-dependent (RD)histone pre-mRNA processingrequires association with a stem-loop (SL) motif and a histone downstream element (HDE), and produces cleaved but non-polyadenylated mature mRNAs. H2AC18 mRNA, a specific H2A RD histone pre-mRNA, can be processed to give either a non-polyadenylated mRNA, ending at the histone SL, or a polyadenylated mRNA. Here, we reveal how H2AC18 captures the two human pre-mRNA processing complexes in a mutually exclusive mode by overlapping a canonical PAS (AAUAAA) sequence element with a HDE. Disruption of the PAS sequence on H2AC18 pre-mRNA prevents recruitment of the canonical complex in vitro, without affecting the histone machinery. This shows how the relative position of cis-acting elements in histone pre-mRNAs allows the selective recruitment of distinct human pre-mRNA complexes, thereby expanding the capability to regulate 3' processing and polyadenylation.

TRFs and tRNA Halves: Novel Cellular Defenders in Multiple Biological Processes.

tRNA fragments derived from angiogenin or Dicer cleavage are referred to as tRNA-derived fragments (tRFs) and tRNA halves. tRFs and tRNA halves have been identified in both eukaryotes and prokaryotes and are precisely cleaved at specific sites on either precursor or mature tRNA transcripts rather than via random degradation. tRFs and tRNA halves are highly involved in regulating transcription and translation in a canonical or non-canonical manner in response to cellular stress. In this review, we summarize the biogenesis and types of tRFs and tRNA halves, clarify the biological functions and molecular mechanisms of tRNA fragments in both physiological and pathological processes with a particular focus on their cytoprotective roles in defending against oxidation and apoptosis, and highlight their potential application as biomarkers in determining cell fate.

Antisense oligonucleotide induced pseudoexon skipping and restoration of functional protein for Fukuyama muscular dystrophy caused by a deep-intronic variant.

Fukuyama congenital muscular dystrophy (FCMD) is an autosomal recessive disorder caused by fukutin (FKTN) gene mutations. FCMD is the second most common form of childhood muscular dystrophy in Japan, and the most patients possess a homozygous retrotransposal SINE-VNTR-Alu insertion in the 3'-untranslated region of FKTN. A deep-intronic variant (DIV) was previously identified as the second most prevalent loss-of-function mutation in Japanese patients with FCMD. The DIV creates a new splicing donor site in intron 5 that causes aberrant splicing and the formation of a 64-base pair pseudoexon in the mature mRNA, resulting in a truncated nonfunctional protein. Patients with FCMD carrying the DIV present a more severe symptoms, and currently, there is no radical therapy available for this disorder. In the present study, we describe in vitro evaluation of antisense oligonucleotide mediated skipping of pseudoexon inclusion and restoration of functional FKTN protein. A total of 16 19-26-mer antisense oligonucleotide sequences were designed with a 2'-O-methyl backbone and were screened in patient-derived fibroblasts, lymphoblast cells, and minigene splice assays. One antisense oligonucleotide targeting the exonic splice enhancer region significantly induced pseudoexon skipping and increased the expression of normal mRNA. It also rescued FKTN protein production in lymphoblast cells and restored functional O-mannosyl glycosylation of alpha-dystroglycan in patient-derived myotubes. Based on our results, ASO-based splicing correction should be investigated further as a potential treatment for patients with FCMD carrying the DIV.

Kinetics of mRNA nuclear export regulate innate immune response gene expression

The abundance and stimulus-responsiveness of mature mRNA is thought to be determined by nuclear synthesis, processing, and cytoplasmic decay. However, the rate and efficiency of moving mRNA to the cytoplasm almost certainly contributes, but has rarely been measured. Here, we investigated mRNA export rates for innate immune genes. We generated high spatio-temporal resolution RNA-seq data from endotoxin-stimulated macrophages and parameterized a mathematical model to infer kinetic parameters with confidence intervals. We find that the effective chromatin-to-cytoplasm export rate is gene-specific, varying 100-fold: for some genes, less than 5% of synthesized transcripts arrive in the cytoplasm as mature mRNAs, while others show high export efficiency. Interestingly, effective export rates do not determine temporal gene responsiveness, but complement the wide range of mRNA decay rates; this ensures similar abundances of short- and long-lived mRNAs, which form successive innate immune response expression waves.

Crystal Structure of the Metallo-Endoribonuclease YbeY from Staphylococcus aureus.

Endoribonuclease YbeY is specific to the single-stranded RNA of ribosomal RNAs and small RNAs. This enzyme is essential for the maturation and quality control of ribosomal RNA in a wide range of bacteria and for virulence in some pathogenic bacteria. In this study, we determined the crystal structure of YbeY from Staphylococcus aureus at a resolution of 1.9 Å in the presence of zinc chloride. The structure showed a zinc ion at the active site and two molecules of tricarboxylic acid citrate, which were also derived from the crystallization conditions. Our structure showed the zinc ion-bound local environment at the molecular level for the first time. Molecular comparisons were performed between the carboxylic moieties of citrate and the phosphate moiety of the RNA backbone, and a model of YbeY in complex with a single strand of RNA was subsequently constructed. Our findings provide molecular insights into how the YbeY enzyme recognizes single-stranded RNA in bacteria.

Developing Therapeutic Splice-Correcting Antisense Oligomers for Adult-Onset Pompe Disease with c.-32-13T>G Mutation.

The mutation c.-32-13T>G in the GAA gene impacts normal exon 2 splicing and is found in two-thirds of late-onset Pompe disease cases. We have explored a therapeutic strategy using splice modulating phosphorodiamidate morpholino oligomers to enhance GAA exon 2 inclusion in the mature mRNA of patients carrying this common mutation. We performed in silico analysis of the GAA gene transcript for potential splicing silencers and designed oligomers targeting motifs predicted to enhance exon 2 retention in the mature mRNA. Two patient-derived fibroblasts were obtained from Coriell Institute for Medical Research, and seven fibroblast strains from unrelated patients were supplied by Westmead Hospital in Sydney, Australia. Both fibroblasts and forced-myogenic cells were treated with optimized phosphorodiamidate morpholino oligomers supplied by Sarepta Therapeutics. Total RNA and protein were extracted from the cells after incubation with phosphorodiamidate morpholino oligomers, and RT-PCR and RT-qPCR were performed to confirmexon 2 inclusionis enhanced. Acid α-glucosidase activity and expression levels were also assessed to confirm therapeutic potential.

Exon-Skipping for a Pathogenic COL6A1 Variant in Ullrich Congenital Muscular Dystrophy.

Single nucleotide variants that alter splice sites or splicing regulatory elements can lead to the skipping of exons, retention of introns, or insertion of pseudo-exons (PE) into the mature mRNA transcripts. When translated, these changes can disrupt the function of the synthesized protein. Splice-switching antisense oligonucleotides (ASOs) are synthetic, modified nucleic acids that can correct these aberrant splicing events. They are currently in active clinical development for a number of conditions and have been approved by regulatory agencies for the treatment of neuromuscular disorders such as Duchenne muscular dystrophy and spinal muscular atrophy. We have previously reported that splice-switching ASOs effectively skip a pathogenic PE that causes Ullrich congenital muscular dystrophy (UCMD). This erroneous PE insertion is caused by a deep-intronic variant located within intron 11 of COL6A1 (c.930+189 C>T). Here, we describe the detailed protocols and workflow that our labs have used to assess the efficacy of ASOs to skip this PE in vitro. The protocols include designing ASOs; isolating, culturing, and transfecting fibroblasts; extracting RNA and protein; and validating splicing correction at the mRNA and protein levels using quantitative reverse transcription PCR (qRT-PCR) and western blot assays, respectively.