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1. Reassessing the exon–foldon correspondence using frustration analysis

   https://doi.org/10.1073/pnas.2400151121  

   Galpern, Ezequiel A; Jaafari, Hana; Bueno, Carlos; Wolynes, Peter G; Ferreiro, Diego U (July 2024, Proceedings of the National Academy of Sciences)

   Muñoz, Victor (Ed.)

   Protein folding and evolution are intimately linked phenomena. Here, we revisit the concept of exons as potential protein folding modules across a set of 38 abundant and conserved protein families. Taking advantage of genomic exon–intron organization and extensive protein sequence data, we explore exon boundary conservation and assess the foldon-like behavior of exons using energy landscape theoretic measurements. We found deviations in the exon size distribution from exponential decay indicating selection in evolution. We show that when taken together there is a pronounced tendency to independent foldability for segments corresponding to the more conserved exons, supporting the idea of exon–foldon correspondence. While 45% of the families follow this general trend when analyzed individually, there are some families for which other stronger functional determinants, such as preserving frustrated active sites, may be acting. We further develop a systematic partitioning of protein domains using exon boundary hotspots, showing that minimal common exons correspond with uninterrupted alpha and/or beta elements for the majority of the families but not for all of them. 

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2. Expressed exome capture sequencing: A method for cost‐effective exome sequencing for all organisms

   https://doi.org/10.1111/1755-0998.12905  

   Puritz, Jonathan B.; Lotterhos, Katie E. (June 2018, Molecular Ecology Resources)

   Abstract Exome capture is an effective tool for surveying the genome for loci under selection. However, traditional methods require annotated genomic resources. Here, we present a method for creatingcDNAprobes from expressedmRNA, which are then used to enrich and capture genomicDNAfor exon regions. This approach, called “EecSeq,” eliminates the need for costly probe design and synthesis. We tested EecSeq in the eastern oyster,Crassostrea virginica, using a controlled exposure experiment. Four adult oysters were heat shocked at 36°C for 1 hr along with four control oysters kept at 14°C. StrandedmRNAlibraries were prepared for two individuals from each treatment and pooled. Half of the combined library was used for probe synthesis, and half was sequenced to evaluate capture efficiency. GenomicDNAwas extracted from all individuals, enriched via captured probes, and sequenced directly. We found that EecSeq had an average capture sensitivity of 86.8% across all known exons and had over 99.4% sensitivity for exons with detectable levels of expression in themRNAlibrary. For all mapped reads, over 47.9% mapped to exons and 37.0% mapped to expressed targets, which is similar to previously published exon capture studies. EecSeq displayed relatively even coverage within exons (i.e., minor “edge effects”) and even coverage across exonGCcontent. We discovered 5,951SNPs with a minimum average coverage of 80×, with 3,508SNPs appearing in exonic regions. We show that EecSeq provides comparable, if not superior, specificity and capture efficiency compared to costly, traditional methods. 

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3. An intronic RNA element modulates Factor VIII exon-16 splicing

   https://doi.org/10.1093/nar/gkad1034  

   Tse, Victor; Chacaltana, Guillermo; Gutierrez, Martin; Forino, Nicholas M; Jimenez, Arcelia G; Tao, Hanzhang; Do, Phong H; Oh, Catherine; Chary, Priyanka; Quesada, Isabel; et al (November 2023, Nucleic Acids Research)

   Abstract Pathogenic variants in the human Factor VIII (F8) gene cause Hemophilia A (HA). Here, we investigated the impact of 97 HA-causing single-nucleotide variants on the splicing of 11 exons from F8. For the majority of F8 exons, splicing was insensitive to the presence of HA-causing variants. However, splicing of several exons, including exon-16, was impacted by variants predicted to alter exonic splicing regulatory sequences. Using exon-16 as a model, we investigated the structure–function relationship of HA-causing variants on splicing. Intriguingly, RNA chemical probing analyses revealed a three-way junction structure at the 3′-end of intron-15 (TWJ-3–15) capable of sequestering the polypyrimidine tract. We discovered antisense oligonucleotides (ASOs) targeting TWJ-3–15 partially rescue splicing-deficient exon-16 variants by increasing accessibility of the polypyrimidine tract. The apical stem loop region of TWJ-3–15 also contains two hnRNPA1-dependent intronic splicing silencers (ISSs). ASOs blocking these ISSs also partially rescued splicing. When used in combination, ASOs targeting both the ISSs and the region sequestering the polypyrimidine tract, fully rescue pre-mRNA splicing of multiple HA-linked variants of exon-16. Together, our data reveal a putative RNA structure that sensitizes F8 exon-16 to aberrant splicing. 

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4. Targeted exon skipping with AAV-mediated split adenine base editors

   https://doi.org/10.1038/s41421-019-0109-7  

   Winter, Jackson; Luu, Alan; Gapinske, Michael; Manandhar, Sony; Shirguppe, Shraddha; Woods, Wendy S.; Song, Jun S.; Perez-Pinera, Pablo (August 2019, Cell Discovery)

   Abstract Techniques for exclusion of exons from mature transcripts have been applied as gene therapies for treating many different diseases. Since exon skipping has been traditionally accomplished using technologies that have a transient effect, it is particularly important to develop new techniques that enable permanent exon skipping. We have recently shown that this can be accomplished using cytidine base editors for permanently disabling the splice acceptor of target exons. We now demonstrate the application of CRISPR-Cas9 adenine deaminase base editors to disrupt the conserved adenine within splice acceptor sites for programmable exon skipping. We also demonstrate that by altering the amino acid sequence of the linker between the adenosine deaminase domain and the Cas9-nickase or by coupling the adenine base editor with a uracil glycosylase inhibitor, the DNA editing efficiency and exon-skipping rates improve significantly. Finally, we developed a split base editor architecture compatible with adeno-associated viral packaging. Collectively, these results represent significant progress toward permanent in vivo exon skipping through base editing and, ultimately, a new modality of gene therapy for the treatment of genetic diseases. 

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5. The Nopp140 gene in Drosophila melanogaster displays length polymorphisms in its large repetitive second exon

   https://doi.org/10.1007/s00438-019-01568-6  

   Baral, Sonu Shrestha; DiMario, Patrick J. (April 2019, Molecular Genetics and Genomics)

   Nopp140, often called the nucleolar and Cajal body phosphoprotein (NOLC1), is an evolutionarily conserved chaperone for the transcription and processing of rRNA during ribosome subunit assembly. Metazoan Nopp140 contains an amino terminal LisH dimerization domain and a highly conserved carboxyl domain. A large central domain consists of alternating basic and acidic motifs of low sequence complexity. Orthologous versions of Nopp140 contain variable numbers of repeating basic–acidic units. While vertebrate Nopp140 genes use multiple exons to encode the central domain, the Nopp140 gene in Drosophila uses exclusively exon 2 to encode the central domain. Here, we define three overlapping repeat sequence patterns (P, P′, and P″) within the central domain of D. melanogaster Nopp140. These repeat patterns are poorly conserved in other Drosophila species. We also describe a length polymorphism in exon 2 that pertains specifically to the P′ pattern in D. melanogaster. The pattern displays either two or three 96 base pair repeats, respectively, referred to as Nopp140-Short and Nopp140-Long. Fly lines homozygous for one or the other allele, or heterozygous for both alleles, show no discernible phenotypes. PCR characterization of the long and short alleles shows a poorly defined, artifactual bias toward amplifying the long allele over the short allele. The significance of this polymorphism will be in discerning the largely unknown properties of Nopp140’s large central domain in rDNA transcription and ribosome biogenesis. 

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