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In spliceosome assembly, the 5 splice site is initially recognized by U1 snRNA. U1 leaves the spliceosome during the assembly process, therefore other factors contribute to the maintenance of 5 splice site identity as it is loaded into the catalytic site. Recent structural data suggest that human tri-snRNP 27K (SNRP27) M141 and SNU66 H734 interact to stabilize the U4/U6 quasi-pseudo knot at the base of the U6 snRNA ACAGAGA box in pre-B complex. Previously, we found that mutations in Caenorhabditis elegans at SNRP-27 M141 promote changes in alternative 5ss usage. We tested whether the potential interaction between SNRP-27 M141 and SNU-66 H765 (the C. elegans equivalent position to human SNU66 H734) contributes to maintaining 5 splice site identity during spliceosome assembly. We find that SNU-66 H765 mutants promote alternative 5 splice site usage. Many of the alternative 5 splicing events affected by SNU-66(H765G) overlap with those affected SNRP-27(M141T). Double mutants of snrp-27(M141T) and snu-66(H765G) are homozygous lethal. We hypothesize that mutations at either SNRP-27 M141 or SNU-66 H765 allow the spliceosome to load alternative 5 splice sites into the active site. Tests with mutant U1 snRNA and swapped 5 splice sites indicate that the ability of SNRP-27 M141 and SNU-66 H765 mutants to affect a particular 5 splice alternative splicing event is dependent on both the presence of a weaker consensus 5ss nearby and potentially nearby splicing factor binding sites. Our findings confirm a new role for the C terminus of SNU-66 in maintenance of 5 splice site identity during spliceosome assembly.

RNA helicases drive necessary rearrangements and ensure fidelity during the pre-mRNA splicing cycle. DEAD-box helicase DDX41 has been linked to human disease and has recently been shown to interact with DEAH-box helicase PRP22 in the spliceosomal C* complex, yet its function in splicing remains unknown. Depletion of DDX41 homolog SACY-1 from somatic cells has been previously shown to lead to changes in alternative 3 splice site (3ss) usage. Here, we show by transcriptomic analysis of published and novel data sets that SACY-1 perturbation causes a previously unreported pattern in alternative 3 splicing in introns with pairs of 3 splice sites ≤18 nt away from each other. We find that both SACY-1 depletion and the allele sacy-1(G533R) lead to a striking unidirectional increase in the usage of the proximal (upstream) 3ss. We previously discovered a similar alternative splicing pattern between germline tissue and somatic tissue, in which there is a unidirectional increase in proximal 3ss usage in the germline for ∼200 events; many of the somatic SACY-1 alternative 3 splicing events overlap with these developmentally regulated events. We generated targeted mutant alleles of the Caenorhabditis elegans homolog of PRP22, mog-5, in the region of MOG-5 that is predicted to interact with SACY-1 based on the human C* structure. These viable alleles, and a mimic of the myelodysplastic syndrome-associated allele DDX41(R525H), all promote the usage of proximal alternative adjacent 3 splice sites. We show that PRP22/MOG-5 and DDX41/SACY-1 have overlapping roles in proofreading the 3ss.

Branching is a critical step in RNA splicing that is essential for 5 splice site selection. Recent spliceosome structures have led to competing models for the recognition of the invariant adenosine at the branch point. However, there are no structures of any splicing complex with the adenosine nucleophile docked in the active site and positioned to attack the 5 splice site. Thus we lack a mechanistic understanding of adenosine selection and splice site recognition during RNA splicing. Here we present a cryo-electron microscopy structure of a group II intron that reveals that active site dynamics are coupled to the formation of a base triple within the branch-site helix that positions the 2-OH of the adenosine for nucleophilic attack on the 5 scissile phosphate. This structure, complemented with biochemistry and comparative analyses to splicing complexes, supports a base triple model of adenosine recognition for branching within group II introns and the evolutionarily related spliceosome.

The DYNC2H1 gene has been associated with short‐rib polydactyly syndrome (SRPS), among other skeletal ciliopathies. Two cases are presented of distinctive phenotypes resulting from splicing variants in DYNC2H1. The first is a 14‐week‐old fetus with enlarged nuchal translucency, oral hamartoma, malformed uvula, bifid epiglottis, short ribs, micromelia, long bone agenesis, polysyndactyly, heart defect, pancreatic cysts, multicystic dysplastic kidney, megabladder and trident acetabulum. A ciliopathies NGS panel revealed two compound heterozygous variants in DYNC2H1: c.7840‐18T>G r.7841_7964del p.Gly2614Aspfs*5 and c.11070G>A r.11044_11116del p.Ile3682Aspfs*2. Both variants were initially classified as variants of uncertain significance but were reclassified as likely pathogenic after PCR‐based RNA testing. The second is an 11‐year‐old overweight male with multiple accessory oral frenula, median cleft lip and alveolar ridge, polysyndactyly, brachydactyly, normal rib length, and hypogonadism. Exome sequencing revealed two compound heterozygous variants in DYNC2H1: c.6315del p.(Thr2106Glnfs*7), classified as likely pathogenic, and c.3303‐16A>G p.(?), classified as a variant of uncertain significance. PCR‐based RNA testing suggested that c.3303‐16A>G induces an in‐frame deletion: r.3303_3458del p.Asp1102_Arg1153del, although the normal transcript is still produced. These results are consistent with both SRPS type I/III in the first case and orofaciodigital syndrome in the second, an unprecedented description. This work thus improves the clinical and molecular knowledge of the phenotypes associated with splicing variants in the DYNC2H1 gene. [ABSTRACT FROM AUTHOR]

Objectives: Methylmalonic acidemia (MMA) is a rare inborn genetic disorder that is characterized by increased levels of methylmalonic acid in blood plasma and urine. Isolated methylmalonic acidemia is one of the most common types of MMA and is caused by mutations in the gene encoding methyl-malonyl coenzyme A mutase (MMUT). In this study, we investigated the possible mechanisms underlying the symptoms of isolated MMA in a patient by molecular analysis. Methods: PCR amplification and Sanger sequencing analysis was performed to identify variants in the MMUT gene in the proband and his family. Furthermore, minigene constructs were generated to validate the splicing defects in the MMUT gene variant identified in the proband. Results: The 3-year-old patient was admitted to the hospital with symptoms of MMA, including fever, convulsions, and vomiting. He showed metabolic acidosis, high levels of methylmalonic acid in blood and urine, and normal blood homocysteine levels. Genetic analysis demonstrated that the patient was a compound heterozygous carrier of two variants in the MMUT gene: a missense c.278G > A variant that has already been reported in a patient with the severe mut⁰ phenotype; and a novel splice site variant c.2125-2A > G. RT-PCR analysis showed that, while the novel variant clearly alters splicing, a minor amount of a full-length transcript is generated, suggesting that a wild-type protein may be produced although at a lower quantitative level. The patient's condition improved after treatment with vitamin B12. Serious complications were not reported during follow-up at age 5. Conclusions: We identified a novel splice site variant that partially disrupts normal splicing of the MMUT pre-mRNA. Production of a reduced amount of full-length transcript is responsible for the mild clinical phenotype observed in this patient. Functional studies have proven useful in exploring the genotype‐phenotype association and in providing guidance for the genetic diagnosis of MMA.

De novo mutations affecting the pre-mRNA splicing factor U2AF2 are associated with developmental delays and intellectual disabilities, yet the molecular basis is unknown. Here, we demonstrated by fluorescence anisotropy RNA binding assays that recurrent missense mutants (Arg149Trp, Arg150His, or Arg150Cys) decreased the binding affinity of U2AF2 for a consensus splice site RNA. Crystal structures at 1.4 Å resolutions showed that Arg149Trp or Arg150His disrupted hydrogen bonds between U2AF2 and the terminal nucleotides of the RNA site. Reanalysis of publicly available RNaseq data confirmed that U2AF2 depletion altered splicing of transcripts encoding RNA binding proteins (RBPs). These results confirmed that the impaired RNA interactions of Arg149Trp and Arg150His U2AF2 variants could contribute to dysregulating an RBP-governed neurodevelopmental program of alternative splicing.

The spliceosome is the complex molecular machinery that sequentially assembles on eukaryotic messenger RNA precursors to remove introns (pre-mRNA splicing), a physiologically regulated process altered in numerous pathologies. We report transcriptome-wide analyses upon systematic knock down of 305 spliceosome components and regulators in human cancer cells and the reconstruction of functional splicing factor networks that govern different classes of alternative splicing decisions. The results disentangle intricate circuits of splicing factor cross-regulation, reveal that the precise architecture of late-assembling U4/U6.U5 tri-small nuclear ribonucleoprotein (snRNP) complexes regulates splice site pairing, and discover an unprecedented division of labor among protein components of U1 snRNP for regulating exon definition and alternative 5' splice site selection. Thus, we provide a resource to explore physiological and pathological mechanisms of splicing regulation.

The two transesterification reactions of pre-mRNA splicing require highly complex yet well-controlled rearrangements of small nuclear RNAs and proteins (snRNP) in the spliceosome. The efficiency and accuracy of these reactions are critical for gene expression, as almost all human genes pass through pre-mRNA splicing. Key parameters that determine the splicing outcome are the length of the intron, the strengths of its splicing signals and gaps between them, and the presence of splicing controlling elements. In particular, the gap between the branchpoint (BP) and the 3' splice site (ss) of introns is a major determinant of the splicing efficiency. This distance falls within a small range across the introns of an organism. The constraints exist possibly because BP and 3'ss are recognized by BP-binding proteins, U2 snRNP and U2 accessory factors (U2AF) in a coordinated manner. Furthermore, varying distances between the two signals may also affect the second transesterification reaction since the intervening RNA needs to be accurately positioned within the complex RNP machinery. Splicing such pre-mRNAs requires cis-acting elements in the RNA and many trans-acting splicing regulators. Regulated pre-mRNA splicing with BP-distant 3'ss adds another layer of control to gene expression and promotes alternative splicing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The mRNAs encoding the human papillomavirus type 16 (HPV16) E6 and E7 oncogene mRNAs are subjected to extensive alternative RNA splicing at multiple regulated splice sites. One of the most extensively used 5'-splice sites in the HPV16 genome is named SD880 and is located immediately downstream of the E7 open reading frame. Here, we show that a cluster of three GGG-motifs adjacent to HPV16 SD880 interacts with heterogeneous nuclear ribonucleoprotein (hnRNP) H that cooperates with SD880 to stimulate splicing to the upstream HPV16 3'-splice site SA742. This splice site is located in the E7 coding region and is required for the production of the HPV16 226^742 mRNA that encodes the E6^E7 fusion protein. Enhancement of HPV16 E6^E7 mRNA production by hnRNP H occurred at the expense of the intron-retained E6 mRNAs and the spliced E7 mRNAs, demonstrating that hnRNP H controls the relative levels of E6, E7, and E6^E7 proteins. Unexpectedly, overexpression of hnRNP H also promoted retention of the downstream E1 encoding intron and enhanced E1 protein production. We concluded that hnRNP H plays an important role in the HPV16 gene expression program.IMPORTANCEHere, we show that hnRNP H binds to multiple GGG-motifs downstream of human papillomavirus type 16 (HPV16) splice site SD880 and acts in concert with SD880 to promote expression of the HPV16 E6^E7 mRNA. The E6^E7 protein has been shown previously to stabilize the HPV16 E6 and E7 oncoproteins and may as such contribute to the carcinogenic properties of HPV16. In its capacity of major regulator of HPV16 oncogene expression, hnRNP H may be exploited as a target for antiviral drugs to HPV16., Competing Interests: The authors declare no conflict of interest.

The spliceosome is a eukaryotic multimegadalton RNA-protein complex that removes introns from transcripts. The spliceosome ensures the selection of each exon-intron boundary through multiple recognition events. Initially, the 5' splice site (5' SS) and branch site (BS) are bound by the U1 small nuclear ribonucleoprotein (snRNP) and the U2 snRNP, respectively, while the 3' SS is mostly determined by proximity to the branch site. A large number of splicing factors recognize the splice sites and recruit the snRNPs before the stable binding of the snRNPs occurs by base-pairing the snRNA to the transcript. Fidelity of this process is crucial, as mutations in splicing factors and U2 snRNP components are associated with many diseases. In recent years, major advances have been made in understanding how splice sites are selected in Saccharomyces cerevisiae and humans. Here, I review and discuss the current understanding of the recognition of splice sites by the spliceosome with a focus on recognition and binding of the branch site by the U2 snRNP in humans., (© 2024 Tholen; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Splice site recognition is essential for defining the transcriptome. Drugs like risdiplam and branaplam change how human U1 snRNP recognizes particular 5' splice sites (5'SS) and promote U1 snRNP binding and splicing at these locations. Despite the therapeutic potential of 5'SS modulators, the complexity of their interactions and snRNP substrates have precluded defining a mechanism for 5'SS modulation. We have determined a sequential binding mechanism for modulation of -1A bulged 5'SS by branaplam using a combination of ensemble kinetic measurements and colocalization single molecule spectroscopy (CoSMoS). Our mechanism establishes that U1-C protein binds reversibly to U1 snRNP, and branaplam binds to the U1 snRNP/U1-C complex only after it has engaged with a -1A bulged 5'SS. Obligate orders of binding and unbinding explain how reversible branaplam interactions cause formation of long-lived U1 snRNP/5'SS complexes. Branaplam targets a ribonucleoprotein, not only an RNA duplex, and its action depends on fundamental properties of 5'SS recognition., (© 2024. The Author(s).)

Loss of function of the RNA-binding protein TDP-43 (TDP-LOF) is a hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Here we describe TDP-REG, which exploits the specificity of cryptic splicing induced by TDP-LOF to drive protein expression when and where the disease process occurs. The SpliceNouveau algorithm combines deep learning with rational design to generate customizable cryptic splicing events within protein-coding sequences. We demonstrate that expression of TDP-REG reporters is tightly coupled to TDP-LOF in vitro and in vivo. TDP-REG enables genomic prime editing to ablate the UNC13A cryptic donor splice site specifically upon TDP-LOF. Finally, we design TDP-REG vectors encoding a TDP-43/Raver1 fusion protein that rescues key pathological cryptic splicing events, paving the way for the development of precision therapies for TDP43-related disorders.

Somatic mutations in genes encoding components of the RNA splicing machinery occur frequently in multiple forms of cancer. The most frequently mutated RNA splicing factors in cancer impact intronic branch site and 3' splice site recognition. These include mutations in the core RNA splicing factor SF3B1 as well as mutations in the U2AF1/2 heterodimeric complex, which recruits the SF3b complex to the 3' splice site. Additionally, mutations in splicing regulatory proteins SRSF2 and RBM10 are frequent in cancer, and there has been a recent suggestion that variant forms of small nuclear RNAs (snRNAs) may contribute to splicing dysregulation in cancer. Here, we describe molecular mechanisms by which mutations in these factors alter splice site recognition and how studies of this process have yielded new insights into cancer pathogenesis and the molecular regulation of splicing. We also discuss data linking mutant RNA splicing factors to RNA metabolism beyond splicing., Competing Interests: Declaration of interests O.A.-W. is a founder and scientific advisor of Codify Therapeutics, holds equity in this company, and receives research funding from this company. O.A.-W. has served as a consultant for Foundation Medicine Inc., Merck, Prelude Therapeutics, Amphista Therapeutics, MagnetBio, and Janssen, and is on the Scientific Advisory Board of Envisagenics Inc., Harmonic Discovery Inc., and Pfizer Boulder; O.A.-W. has received prior research funding from H3B Biomedicine, Nurix Therapeutics, Minovia Therapeutics, and LOXO Oncology unrelated to the current manuscript., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The process of splicing messenger RNA to remove introns plays a central role in creating genes and gene variants. We describe Splam, a novel method for predicting splice junctions in DNA using deep residual convolutional neural networks. Unlike previous models, Splam looks at a 400-base-pair window flanking each splice site, reflecting the biological splicing process that relies primarily on signals within this window. Splam also trains on donor and acceptor pairs together, mirroring how the splicing machinery recognizes both ends of each intron. Compared to SpliceAI, Splam is consistently more accurate, achieving 96% accuracy in predicting human splice junctions., (© 2024. The Author(s).)

Splicing is an important step of gene expression in all eukaryotes. Splice sites might be used with different efficiency, giving rise to alternative splicing products. At the same time, splice sites might be used at a variable rate. We used 5-ethynyl uridine labeling to sequence a nascent transcriptome of HeLa cells and deduced the rate of splicing for each donor and acceptor splice site. The following correlation analysis showed a correspondence of primary transcript features with the rate of splicing. Some dependencies we revealed were anticipated, such as a splicing rate decrease with a decreased complementarity of the donor splice site to U1 and acceptor sites to U2 snRNAs. Other dependencies were more surprising, like a negative influence of a distance to the 5' end on the rate of the acceptor splicing site utilization, or the differences in splicing rate between long, short, and RBM17-dependent introns. We also observed a deceleration of last intron splicing with an increase of the distance to the poly(A) site, which might be explained by the cooperativity of the splicing and polyadenylation. Additional analysis of splicing kinetics of SF3B4 knockdown cells suggested the impairment of a U2 snRNA recognition step. As a result, we deconvoluted the effects of several examined features on the splicing rate into a single regression model. The data obtained here are useful for further studies in the field, as they provide general splicing rate dependencies as well as help to justify the existence of slowly removed splice sites., (© 2024 Bolikhova et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Classification of introns, which is crucial to understanding their evolution and splicing, has historically been binary and has resulted in the naming of major and minor introns that are spliced by their namesake spliceosome. However, a broad range of intron consensus sequences exist, leading us to here reclassify introns as minor, minor-like, hybrid, major-like, major and non-canonical introns in 263 species across six eukaryotic supergroups. Through intron orthology analysis, we discovered that minor-like introns are a transitory node for intron conversion across evolution. Despite close resemblance of their consensus sequences to minor introns, these introns possess an AG dinucleotide at the -1 and -2 position of the 5' splice site, a salient feature of major introns. Through combined analysis of CoLa-seq, CLIP-seq for major and minor spliceosome components, and RNAseq from samples in which the minor spliceosome is inhibited we found that minor-like introns are also an intermediate class from a splicing mechanism perspective. Importantly, this analysis has provided insight into the sequence elements that have evolved to make minor-like introns amenable to recognition by both minor and major spliceosome components. We hope that this revised intron classification provides a new framework to study intron evolution and splicing., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

pre-mRNA splicing is a critical feature of eukaryotic gene expression. Both cis- and trans-splicing rely on accurately recognising splice site sequences by spliceosomal U snRNAs and associated proteins. Spliceosomal snRNAs carry multiple RNA modifications with the potential to affect different stages of pre-mRNA splicing. Here, we show that the conserved U6 snRNA m6A methyltransferase METT-10 is required for accurate and efficient cis- and trans-splicing of C. elegans pre-mRNAs. The absence of METT-10 in C. elegans and METTL16 in humans primarily leads to alternative splicing at 5' splice sites with an adenosine at +4 position. In addition, METT-10 is required for splicing of weak 3' cis- and trans-splice sites. We identified a significant overlap between METT-10 and the conserved splicing factor SNRNP27K in regulating 5' splice sites with +4A. Finally, we show that editing endogenous 5' splice site +4A positions to +4U restores splicing to wild-type positions in a mett-10 mutant background, supporting a direct role for U6 snRNA m6A modification in 5' splice site recognition. We conclude that the U6 snRNA m6A modification is important for accurate and efficient pre-mRNA splicing., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Alternative splicing plays a crucial role in protein diversity and gene expression regulation in higher eukaryotes, and mutations causing dysregulated splicing underlie a range of genetic diseases. Computational prediction of alternative splicing from genomic sequences not only provides insight into gene-regulatory mechanisms but also helps identify disease-causing mutations and drug targets. However, the current methods for the quantitative prediction of splice site usage still have limited accuracy. Here, we present DeltaSplice, a deep neural network model optimized to learn the impact of mutations on quantitative changes in alternative splicing from the comparative analysis of homologous genes. The model architecture enables DeltaSplice to perform "reference-informed prediction" by incorporating the known splice site usage of a reference gene sequence to improve its prediction on splicing-altering mutations. We benchmarked DeltaSplice and several other state-of-the-art methods on various prediction tasks, including evolutionary sequence divergence on lineage-specific splicing and splicing-altering mutations in human populations and neurodevelopmental disorders, and demonstrated that DeltaSplice outperformed consistently. DeltaSplice predicted ∼15% of splicing quantitative trait loci (sQTLs) in the human brain as causal splicing-altering variants. It also predicted splicing-altering de novo mutations outside the splice sites in a subset of patients affected by autism and other neurodevelopmental disorders (NDDs), including 19 genes with recurrent splicing-altering mutations. Integration of splicing-altering mutations with other types of de novo mutation burdens allowed the prediction of eight novel NDD-risk genes. Our work expanded the capacity of in silico splicing models with potential applications in genetic diagnosis and the development of splicing-based precision medicine., (© 2024 Xu et al.; Published by Cold Spring Harbor Laboratory Press.)

PARP inhibitor (PARPi) therapy has transformed outcomes for patients with homologous recombination DNA repair (HRR) deficient ovarian cancers, for example those with BRCA1 or BRCA2 gene defects. Unfortunately, PARPi resistance is common. Multiple resistance mechanisms have been described, including secondary mutations that restore the HR gene reading frame. BRCA1 splice isoforms △11 and △11q can contribute to PARPi resistance by splicing out the mutation-containing exon, producing truncated, partially functional proteins. However, the clinical impacts and underlying drivers of BRCA1 exon skipping are not fully understood.We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs) that drive exon skipping, confirmed using qRT-PCR, RNA sequencing, immunoblotting and minigene modelling. CRISPR/Cas9-mediated disruption of splicing functionally validated exon skipping as a mechanism of PARPi resistance. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials.Few PARPi resistance mechanisms have been confirmed in the clinical setting. While secondary/reversion mutations typically restore a gene's reading frame, we have identified secondary mutations in patient cohorts that hijack splice sites to enhance mutation-containing exon skipping, resulting in the overexpression of BRCA1 hypomorphs, which in turn promote PARPi resistance. Thus, BRCA1 SSMs can and should be clinically monitored, along with frame-restoring secondary mutations., (© 2024. The Author(s).)

Intronic deletions that critically shorten donor-to-branchpoint (D-BP) distance of a precursor mRNA impose biophysical space constraint on assembly of the U1/U2 spliceosomal complex, leading to canonical splicing failure. Here we use a series of β-globin (HBB) gene constructs with intron 1 deletions to define D-BP lengths that present low/no risk of mis-splicing and lengths which are critically short and likely elicit clinically relevant mis-splicing. We extend our previous observation in EMD intron 5 of 46 nt as the minimal productive D-BP length, demonstrating spliceosome assembly constraint persists at D-BP lengths of 47-56 nt. We exploit the common HBB exon 1 β-thalassemia variant that strengthens a cryptic donor (NM&#95;000518.5(HBB):c.79G > A) to provide a simple barometer for the earliest signs of space constraint, via cryptic donor activation. For clinical evaluation of intronic deletions, we assert D-BP lengths > 60 nt present low mis-splicing risk while space constraint increases exponentially with D-BP lengths < 55 nt, with critical risk and profound splicing abnormalities with D-BP lengths < 50 nt., (© 2024. The Author(s).)

Lowering expression of prion protein (PrP) is a well-validated therapeutic strategy in prion disease, but additional modalities are urgently needed. In other diseases, small molecules have proven capable of modulating pre-mRNA splicing, sometimes by forcing inclusion of cryptic exons that reduce gene expression. Here, we characterize a cryptic exon located in human PRNP's sole intron and evaluate its potential to reduce PrP expression through incorporation into the 5' untranslated region. This exon is homologous to exon 2 in nonprimate species but contains a start codon that would yield an upstream open reading frame with a stop codon prior to a splice site if included in PRNP mRNA, potentially downregulating PrP expression through translational repression or nonsense-mediated decay. We establish a minigene transfection system and test a panel of splice site alterations, identifying mutants that reduce PrP expression by as much as 78%. Our findings nominate a new therapeutic target for lowering PrP., Competing Interests: Conflict of interest S. M. V. acknowledges speaking fees from Ultragenyx, Illumina, Biogen, and Eli Lilly, consulting fees from Invitae and Alnylam, and research support from Ionis, Gate, and Sangamo. E.V.M. acknowledges speaking fees from Eli Lilly, consulting fees from Deerfield and Alnylam, and research support from Ionis, Gate, and Sangamo., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Congenital contractural arachnodactyly (CCA) is a rare connective tissue disorder characterized by arachnodactyly, multiple joint contractures, progressive kyphoscoliosis, pectus deformity and abnormal crumpled ears. FBN2 is the only gene currently known to be associated with CCA. In this study, we report on a prenatal case presented with skeletal, cardiac and spinal malformations. And his father had elongated limbs, contractures of the proximal interphalangeal joints, high myopia and scoliosis. We conducted whole exome sequencing (WES) on the fetus-parental trio and a heterozygous variant (hg19 chr5:127,673,685, c.3598 + 4A > G, NM&#95;001999.4) in intron 27 of the FBN2 gene was successfully identified, inherited from the father. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate the potential splicing effect of this variant, which confirmed that the variant caused a deletion of exon 27 (126 bp) by disrupting the splice-donor site and destroyed the 17th calcium-binding epidermal growth factor-like (cbEGF) domain. Our research not only finds the etiology of the disease in affected individuals and expands the mutation spectrum of FBN2 gene, but also provides genetic counseling and fertility guidance for this family., (© 2023. The Author(s).)

Identification of splice sites is a critical step in pre-messenger RNA (pre-mRNA) splicing because the definition of the exon/intron boundaries controls what nucleotides are incorporated into mature mRNAs. The intron boundary with the upstream exon is initially identified through interactions with the U1 small nuclear ribonucleoprotein (snRNP). This involves both base-pairing between the U1 snRNA and the pre-mRNA as well as snRNP proteins interacting with the 5' splice site (5'ss)/snRNA duplex. In yeast, this duplex is buttressed by two conserved protein factors, Yhc1 and Luc7. Luc7 has three human paralogs (LUC7L, LUC7L2, and LUC7L3), which play roles in alternative splicing. What domains of these paralogs promote splicing at particular sites is not yet clear. Here, we humanized the zinc finger (ZnF) domains of the yeast Luc7 protein in order to understand their roles in splice site selection using reporter assays, transcriptome analysis, and genetic interactions. Although we were unable to determine a function for the first ZnF domain, humanization of the second ZnF domain to mirror that found in LUC7L or LUC7L2 resulted in altered usage of nonconsensus 5'ss. In contrast, the corresponding ZnF domain of LUC7L3 could not support yeast viability. Further, humanization of Luc7 can suppress mutation of the ATPase Prp28, which is involved in U1 release and exchange for U6 at the 5'ss. Our work reveals a role for the second ZnF of Luc7 in splice site selection and suggests that different ZnF domains may have different ATPase requirements for release by Prp28., (© 2024 Carrocci et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The recognition of the 5' splice site (5' ss) is one of the earliest steps of pre-mRNA splicing. To better understand, the mechanism and regulation of 5' ss recognition, we selectively humanized components of the yeast U1 (yU1) snRNP to reveal the function of these components in 5' ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yU1 snRNA and the 5' ss RNA duplex. We replaced the zinc-finger (ZnF) domain of yeast U1C (yU1C) with its human counterpart, which resulted in a cold-sensitive growth phenotype and moderate splicing defects. We next added an auxin-inducible degron to yeast Luc7 (yLuc7) protein (to mimic the lack of Luc7Ls in human U1 snRNP). We found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of Prp40 and Snu71 (two other essential yU1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe 2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yU1 snRNP, and splicing plays a major role in the regulation of mitochondrial function in yeast., (© 2024 Chalivendra et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Bioinformatics is a contemporary interdisciplinary area focused on analyzing the growing number of genome sequences. Gene variants are differences in DNA sequences among individuals within a population. Splice site recognition is a crucial step in the process of gene expression, where the coding sequences of genes are joined together to form mature messenger RNA (mRNA). These genetic variants that disrupt genes are believed to be the primary reason for neuro-developmental disorders like ASD (Autism Spectrum Disorder) is a neuro-developmental disorder that is diagnosed in individuals, families, and society and occurs as the developmental delay in one among the hundred genes that are associated with these disorders. Missense variants, premature stop codons, or deletions alter both the quality and quantity of encoded proteins. Predicting genes within exons and introns presents main challenges, such as dealing with sequencing errors, short reads, incomplete genes, overlapping, and more. Although many traditional techniques have been utilized in creating an exon prediction system, the primary challenge lies in accurately identifying the length and spliced strand location classification of exons in conjunction with introns. From now on, the suggested approach utilizes a Deep Learning algorithm to analyze intricate and extensive genomic datasets. M-LSTM is utilized to categorize three binary combinations (EI as 1, IE as 2, and none as 3) using spliced DNA strands. The M-LSTM system is able to sequence extensive datasets, ensuring that long information can be stored without any impact on the current input or output. This enables it to recognize and address long-term connections and problems with rapidly increasing gradients. The proposed model is compared internally with Naïve Bayes and Random Forest to assess its efficacy. Additionally, the proposed model's performance is forecasted by utilizing probabilistic parameters like recall, F1-score, precision, and accuracy to assess the effectiveness of the proposed system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

CRISPR-Cas technology has transformed functional genomics, yet understanding of how individual exons differentially shape cellular phenotypes remains limited. Here, we optimized and conducted massively parallel exon deletion and splice-site mutation screens in human cell lines to identify exons that regulate cellular fitness. Fitness-promoting exons are prevalent in essential and highly expressed genes and commonly overlap with protein domains and interaction interfaces. Conversely, fitness-suppressing exons are enriched in nonessential genes, exhibiting lower inclusion levels, and overlap with intrinsically disordered regions and disease-associated mutations. In-depth mechanistic investigation of the screen-hit TAF5 alternative exon-8 revealed that its inclusion is required for assembly of the TFIID general transcription initiation complex, thereby regulating global gene expression output. Collectively, our orthogonal exon perturbation screens established a comprehensive repository of phenotypically important exons and uncovered regulatory mechanisms governing cellular fitness and gene expression., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Background: Nuclear introns in Euglenida have been understudied. This study aimed to investigate nuclear introns in Euglenida by identifying a large number of introns in Euglena gracilis (E. gracilis), including cis-spliced conventional and nonconventional introns, as well as trans-spliced outrons. We also examined the sequence characteristics of these introns., Results: A total of 28,337 introns and 11,921 outrons were identified. Conventional and nonconventional introns have distinct splice site features; the former harbour canonical GT/C-AG splice sites, whereas the latter are capable of forming structured motifs with their terminal sequences. We observed that short introns had a preference for canonical GT-AG introns. Notably, conventional introns and outrons in E. gracilis exhibited a distinct cytidine-rich polypyrimidine tract, in contrast to the thymidine-rich tracts observed in other organisms. Furthermore, the SL-RNAs in E. gracilis, as well as in other trans-splicing species, can form a recently discovered motif called the extended U6/5' ss duplex with the respective U6s. We also describe a novel type of alternative splicing pattern in E. gracilis. The tandem repeat sequences of introns in this protist were determined, and their contents were comparable to those in humans., Conclusions: Our findings highlight the unique features of E. gracilis introns and provide insights into the splicing mechanism of these introns, as well as the genomics and evolution of Euglenida., (© 2024. The Author(s).)

Tandem donor splice sites (5'ss) are unique regions with at least two GU dinucleotides serving as splicing cleavage sites. The Δ3 tandem 5'ss are a specific subclass of 5'ss separated by 3 nucleotides which can affect protein function by inserting/deleting a single amino acid. One 5'ss is typically preferred, yet factors governing particular 5'ss choice are not fully understood. A highly conserved exon 21 of the STAT3 gene was chosen as a model to study Δ3 tandem 5'ss splicing mechanisms. Based on multiple lines of experimental evidence, endogenous U1 snRNA most likely binds only to the upstream 5'ss. However, the downstream 5'ss is used preferentially, and the splice site choice is not dependent on the exact U1 snRNA binding position. Downstream 5'ss usage was sensitive to exact nucleotide composition and dependent on the presence of downstream regulatory region. The downstream 5'ss usage could be best explained by two novel interactions with endogenous U6 snRNA. U6 snRNA enables the downstream 5'ss usage in STAT3 exon 21 by two mechanisms: (i) binding in a novel non-canonical register and (ii) establishing extended Watson-Crick base pairing with the downstream regulatory region. This study suggests that U6:5'ss interaction is more flexible than previously thought., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNA splicing is pivotal in post-transcriptional gene regulation, yet the exponential expansion of intron length in humans poses a challenge for accurate splicing. Here, we identify hnRNPM as an essential RNA-binding protein that suppresses cryptic splicing through binding to deep introns, maintaining human transcriptome integrity. Long interspersed nuclear elements (LINEs) in introns harbor numerous pseudo splice sites. hnRNPM preferentially binds at intronic LINEs to repress pseudo splice site usage for cryptic splicing. Remarkably, cryptic exons can generate long dsRNAs through base-pairing of inverted ALU transposable elements interspersed among LINEs and consequently trigger an interferon response, a well-known antiviral defense mechanism. Significantly, hnRNPM-deficient tumors show upregulated interferon-associated pathways and elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity by repressing cryptic splicing and suggest that targeting hnRNPM in tumors may be used to trigger an inflammatory immune response, thereby boosting cancer surveillance., Competing Interests: Declaration of interests R.A.F. is a co-founder, board of directors member, and stockholder of GanNA Bio and a board of directors member and stockholder of Chronus Health. H.Y.C. is a co-founder of Accent Therapeutics, Boundless Bio, Cartography Biosciences, and Orbital Therapeutics, an advisor of 10× Genomics, Arsenal Biosciences, Chroma Medicine, and Spring Discovery, and a member of the Molecular Cell advisory board. E.L.V.N. is a co-founder, member of the board of directors, on the SAB, an equity holder, and a paid consultant for Eclipse BioInnovations, on the SAB of RNAConnect, and an inventor of intellectual property owned by University of California, San Diego. E.L.V.N.’s interests have been reviewed and approved by the Baylor College of Medicine in accordance with its conflict-of-interest policies., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Photoperiod insensitivity (auto-flowering) in drug-type Cannabis sativa circumvents the need for short day (SD) flowering requirements making outdoor cultivation in high latitudes possible. However, the benefits of photoperiod insensitivity are counterbalanced by low cannabinoid content and poor flower quality in auto-flowering genotypes. Despite recent studies in cannabis flowering, a mechanistic understanding of photoperiod insensitivity is still lacking. We used a combination of genome-wide association study and genetic fine-mapping to identify the genetic cause of auto-flowering in cannabis. We then used gene expression analyses and transient transformation assays to characterize flowering time control. Herein, we identify a splice site mutation within circadian clock gene PSEUDO-RESPONSE REGULATOR 37 (CsPRR37) in auto-flowering cannabis. We show that CsPRR37 represses FT expression and its circadian oscillations transition to a less repressive state during SD as compared to long days (LD). We identify several key circadian clock genes whose expression is altered in auto-flowering cannabis, particularly under non-inductive LD. Research into the pervasiveness of this mutation and others affecting flowering time will help elucidate cannabis domestication history and advance cannabis breeding toward a more sustainable outdoor cultivation system., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Mutations in ADNP result in Helsmoortel-Van der Aa syndrome. Here, we describe the first de novo intronic deletion, affecting the splice-acceptor site of the first coding ADNP exon in a five-year-old girl with developmental delay and autism. Whereas exome sequencing failed to detect the non-coding deletion, genome-wide CpG methylation analysis revealed an episignature suggestive of a Helsmoortel-Van der Aa syndrome diagnosis. This diagnosis was further supported by PhenoScore, a novel facial recognition software package. Subsequent whole-genome sequencing resolved the three-base pair ADNP deletion c.[-5-1&#95;-4del] with transcriptome sequencing showing this deletion leads to skipping of exon 4. An N-terminal truncated protein could not be detected in transfection experiments with a mutant expression vector in HEK293T cells, strongly suggesting this is a first confirmed diagnosis exclusively due to haploinsufficiency of the ADNP gene. Pathway analysis of the methylome indicated differentially methylated genes involved in brain development, the cytoskeleton, locomotion, behavior, and muscle development. Along the same line, transcriptome analysis identified most of the differentially expressed genes as upregulated, in line with the hypomethylated CpG episignature and confirmed the involvement of the cytoskeleton and muscle development pathways that are also affected in patient cell lines and animal models. In conclusion, this novel mutation for the first time demonstrates that Helsmoortel-Van der Aa syndrome can be caused by a loss-of-function mutation. Moreover, our study elegantly illustrates the use of EpiSignatures, WGS and Phenoscore as novel complementary diagnostic tools in case a of negative WES result., (© 2024. The Author(s), under exclusive licence to European Society of Human Genetics.)

The majority of genic transcription is intronic. Introns are removed by splicing as branched lariat RNAs which require rapid recycling. The branch site is recognized during splicing catalysis and later debranched by Dbr1 in the rate-limiting step of lariat turnover. Through generation of a viable DBR1 knockout cell line, we find the predominantly nuclear Dbr1 enzyme to encode the sole debranching activity in human cells. Dbr1 preferentially debranches substrates that contain canonical U2 binding motifs, suggesting that branchsites discovered through sequencing do not necessarily represent those favored by the spliceosome. We find that Dbr1 also exhibits specificity for particular 5' splice site sequences. We identify Dbr1 interactors through co-immunoprecipitation mass spectrometry. We present a mechanistic model for Dbr1 recruitment to the branchpoint through the intron-binding protein AQR. In addition to a 20-fold increase in lariats, Dbr1 depletion increases exon skipping. Using ADAR fusions to timestamp lariats, we demonstrate a defect in spliceosome recycling. In the absence of Dbr1, spliceosomal components remain associated with the lariat for a longer period of time. As splicing is co-transcriptional, slower recycling increases the likelihood that downstream exons will be available for exon skipping., (© 2024. The Author(s).)

Pre-mRNA splicing is a fundamental step in gene expression, conserved across eukaryotes, in which the spliceosome recognizes motifs at the 3' and 5' splice sites (SSs), excises introns, and ligates exons. SS recognition and pairing is often influenced by protein splicing factors (SFs) that bind to splicing regulatory elements (SREs). Here, we describe SMsplice, a fully interpretable model of pre-mRNA splicing that combines models of core SS motifs, SREs, and exonic and intronic length preferences. We learn models that predict SS locations with 83 to 86% accuracy in fish, insects, and plants and about 70% in mammals. Learned SRE motifs include both known SF binding motifs and unfamiliar motifs, and both motif classes are supported by genetic analyses. Our comparisons across species highlight similarities between non-mammals, increased reliance on intronic SREs in plant splicing, and a greater reliance on SREs in mammalian splicing.

It is now widely accepted that aberrant splicing of constitutive exons is often caused by mutations affecting cis-acting splicing regulatory elements (SREs), but there is a misconception that all exons have an equal dependency on SREs and thus a similar vulnerability to aberrant splicing. We demonstrate that some exons are more likely to be affected by exonic splicing mutations (ESMs) due to an inherent vulnerability, which is context dependent and influenced by the strength of exon definition. We have developed VulExMap, a tool which is based on empirical data that can designate whether a constitutive exon is vulnerable. Using VulExMap, we find that only 25% of all exons can be categorized as vulnerable, whereas two-thirds of 359 previously reported ESMs in 75 disease genes are located in vulnerable exons. Because VulExMap analysis is based on empirical data on splicing of exons in their endogenous context, it includes all features important in determining the vulnerability. We believe that VulExMap will be an important tool when assessing the effect of exonic mutations by pinpointing whether they are located in exons vulnerable to ESMs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Pharmacological modulation of RNA splicing by small molecules is an emerging facet of drug discovery. In this context, the SMN2 splicing modifier SMN-C5 was used as a prototype to understand the mode of action of small molecule splicing modifiers and propose the concept of 5'-splice site bulge repair. In this study, we combined in vitro binding assays and structure determination by NMR spectroscopy to identify the binding modes of four other small molecule splicing modifiers that switch the splicing of either the SMN2 or the HTT gene. Here, we determined the solution structures of risdiplam, branaplam, SMN-CX and SMN-CY bound to the intermolecular RNA helix epitope containing an unpaired adenine within the G-2A-1G+1U+2 motif of the 5'-splice site. Despite notable differences in their scaffolds, risdiplam, SMN-CX, SMN-CY and branaplam contact the RNA epitope similarly to SMN-C5, suggesting that the 5'-splice site bulge repair mechanism can be generalised. These findings not only deepen our understanding of the chemical diversity of splicing modifiers that target A-1 bulged 5'-splice sites, but also identify common pharmacophores required for modulating 5'-splice site selection with small molecules., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches., (© 2024. The Author(s).)

Introduction: A novel variant involving noncanonical splicing acceptor site (c.875-5 T > G) in propeptide coding region of von Willebrand factor (VWF) was identified in a patient with type 2A von Willebrand disease (VWD), who co-inherited with a null variant (p.Tyr271*) and presented characteristic discrepancy of plasma level of VWF antigen and activity, and a selective reduction of both intermediate-molecular-weight (IMWMs) and high-molecular-weight VWF multimers (HMWMs)., Materials and Methods: VWF mRNA transcripts obtained from peripheral leukocytes and platelets of the patients were investigated to analyze the consequence of c.875-5 T > G on splicing. The impact of the variant on expression and multimer assembly was further analyzed by in vitro expression studies in AtT-20 cells. The intracellular processing of VWF mutant and the Weibel-Palade bodies (WPBs) formation was evaluated by immunofluorescence staining and electron microscopy., Results: The mRNA transcript analysis revealed that c.875-5 T > G variant led to exon 8 skipping and an in-frame deletion of 41 amino acids in the D1 domain of VWF (p.Ser292&#95;Glu333delinsLys), yielding a truncated propeptide. Consistent with the patient's laboratory manifestations, the AtT-20 cells transfected with mutant secreted less VWF, with the VWF antigen level in conditioned medium 47 % of wild-type. A slight retention in the endoplasmic reticulum was observed for the mutant. Almost complete loss of IMWMs and HMWMs in the medium and impaired WPBs formation in the cell, indicating truncated VWF propeptide lost its chaperon-like function for VWF multimerization and tubular storage., Conclusions: The VWF splicing site variant (c.875-5 T > G) causes propeptide truncation, severely compromising VWF multimer assembly and tubular storage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Introduction: Molecular confirmation of pathogenic sequence variants in the CHM gene is required prior to enrolment in retinal gene therapy clinical trials for choroideremia. Individuals with mild choroideremia have been reported. The molecular basis of genotype-phenotype associations is of clinical relevance since it may impact on selection for retinal gene therapy., Methods and Materials: Genetic testing and RNA analysis were undertaken in a patient with mild choroideremia to confirm the pathogenicity of a novel intronic variant in CHM and to explore the mechanism underlying the mild clinical phenotype., Results: A 42-year-old male presented with visual field loss. Fundoscopy and autofluorescence imaging demonstrated mild choroideremia for his age. Genetic analysis revealed a variant at a splice acceptor site in the CHM gene (c.1350-3C > G). RNA analysis demonstrated two out-of-frame transcripts, suggesting pathogenicity, without any detectable wildtype transcripts. One of the two out-of-frame transcripts is present in very low levels in healthy controls., Discussion: Mild choroideremia may result from +3 or -3 splice site variants in CHM. It is presumed that the resulting mRNA transcripts may be partly functional, thereby preventing the development of the null phenotype. Choroideremia patients with such variants may present challenges for gene therapy since there may be residual transcript activity which could result in long-lasting visual function which is atypical for this disease.

Intellectual disability (ID) is associated with an increased risk of developing psychiatric disorders, suggesting a common underlying genetic factor. Importantly, altered signaling and/or expression of regulator of G protein signaling 6 (RGS6) is associated with ID and numerous psychiatric disorders. RGS6 is highly conserved and undergoes complex alternative mRNA splicing producing ~36 protein isoforms with high sequence similarity historically necessitating a global approach in functional studies. However, our recent analysis in mice revealed RGS6 is most highly expressed in CNS with RGS6L(+GGL) isoforms predominating. A previously reported genetic variant in intron 17 of RGS6 (c.1369-1G>C), associated with ID, may provide further clues into RGS6L(+GGL) isoform functional delineation. This variant was predicted to alter a highly conserved canonical 3' acceptor site creating an alternative branch point within exon 18 (included in a subset of RGS6L(+GGL) transcripts) and a frameshift forming an early stop codon. We previously identified this alternative splice site and demonstrated its use generates RGS6Lζ(+GGL) isoforms. Here, we show that the c.1369-1G>C variant disrupts the canonical, preferred (>90%) intron 17 splice site and leads to the exclusive use of the alternate exon 18 splice site, inducing disproportionate expression of a subset of isoforms, particularly RGS6Lζ(+GGL). Furthermore, RGS6 global knockout mice do not exhibit ID. Thus, ID caused by the c.1369-1G>C variant likely results from altered RGS6 isoform expression, rather than RGS6 isoform loss. In summary, these studies highlight the importance of proper RGS6 splicing and identify a previously unrecognized role of G protein signaling in ID., (© 2024. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)

Background: The disease-causing effects of genetic variations often depend on their location within a gene. Exonic changes generally lead to alterations in protein production, secretion, activity, or clearance. However, owing to the overlap between proteins and splicing codes, missense variants can also affect messenger RNA splicing, thus adding a layer of complexity and influencing disease phenotypes., Objectives: To extensively characterize a panel of 13 exonic variants in the F9 gene occurring at 6 different factor IX positions and associated with varying severities of hemophilia B (HB)., Methods: Computational predictions, splicing analysis, and recombinant factor IX assays were exploited to characterize F9 variants., Results: We demonstrated that 5 (38%) of 13 selected F9 exonic variants have pleiotropic effects. Although bioinformatic approaches accurately classified effects, extensive experimental assays were required to elucidate and deepen the molecular mechanisms underlying the pleiotropic effects. Importantly, their characterization was instrumental in developing tailored RNA therapeutics based on engineered U7 small nuclear RNA to mask cryptic splice sites and compensatory U1 small nuclear RNA to enhance exon definition., Conclusion: Overall, albeit a multitool bioinformatic approach suggested the molecular effects of multiple HB variants, the deep investigation of molecular mechanisms revealed insights into the HB phenotype-genotype relationship, enabling accurate classification of HB variants. Importantly, knowledge of molecular mechanisms allowed the development of tailored RNA therapeutics, which can also be translated to other genetic diseases., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

The minor spliceosome, which is responsible for the splicing of U12-type introns, comprises five small nuclear RNAs (snRNAs), of which only one is shared with the major spliceosome. In this work, we report the 3.3-angstrom cryo-electron microscopy structure of the fully assembled human minor spliceosome pre-B complex. The atomic model includes U11 small nuclear ribonucleoprotein (snRNP), U12 snRNP, and U4atac/U6atac.U5 tri-snRNP. U11 snRNA is recognized by five U11-specific proteins (20K, 25K, 35K, 48K, and 59K) and the heptameric Sm ring. The 3' half of the 5'-splice site forms a duplex with U11 snRNA; the 5' half is recognized by U11-35K, U11-48K, and U11 snRNA. Two proteins, CENATAC and DIM2/TXNL4B, specifically associate with the minor tri-snRNP. A structural analysis uncovered how two conformationally similar tri-snRNPs are differentiated by the minor and major prespliceosomes for assembly.

We report a case of Hb S/β 0 -thalassemia (Hb S/β 0 -thal) in a patient who is a compound heterozygote for the Hb Sickle mutation ( HBB :c.20A > T) and a mutation of the canonical splice acceptor sequence of IVS1 (AG > TG, HBB :c.93-2A > T). This is the fifth mutation involving the AG splice acceptor site of IVS1, all of which prevent normal splicing and cause β 0 -thal.

Cystinosis is a severe, monogenic systemic disease caused by variants in CTNS gene. Currently, there is growing evidence that exonic variants in many diseases can affect pre-mRNA splicing. The impact of CTNS gene exonic variants on splicing regulation may be underestimated due to the lack of routine studies at the RNA level. Here, we analyzed 59 exonic variants in the CTNS gene using bioinformatics tools and identified candidate variants that may induce splicing alterations by minigene assays. We identified six exonic variants that induce splicing alterations by disrupting the ratio of exonic splicing enhancers/exonic splicing silencers (ESEs/ESSs) or by interfering with the recognition of classical splice sites, or both. Our results help in the correct molecular characterization of variants in cystinosis and inform emerging therapies. Furthermore, our work suggests that the combination of in silico and in vitro assays facilitates to assess the effects of DNA variants driving rare genetic diseases on splicing regulation and will enhance the clinical utility of variant functional annotation., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Emerging evidence suggests that aberrant alternative splicing (AS) may play an important role in tuberculosis (TB). However, current knowledge regarding the value of AS in TB progression and prognosis remains unclear., Method: Public RNA-seq datasets related to TB progression and prognosis were searched and AS analyses were conducted based on SUPPA2. Percent spliced in (PSI) was used for quantifying AS events and multiple machine learning (ML) methods were employed to construct predictive models. Area under curve (AUC), sensitivity and specificity were calculated to evaluate the model performance., Results: A total of 1587 samples from 7 datasets were included. Among 923 TB-progression related differential AS events (DASEs), 3 events (GET1-skipping exon (SE), TPD52-alternative first exons (AF) and TIMM10-alternative 5' splice site (A5)) were selected as candidate biomarkers; however, their predictive performance was limited. For TB prognosis, 5 events (PHF23-AF, KIF1B-SE, MACROD2-alternative 3' splice site (A3), CD55-retained intron (RI) and GALNT11-AF) were selected as candidates from the 1282 DASEs. Six ML methods were used to integrate these 5 events and XGBoost outperformed than others. AUC, sensitivity and specificity of XGBoost model were 0.875, 81.1% and 83.5% in training set, while they were 0.805, 68.4% and 73.2% in test set., Conclusion: GET1-SE, TPD52-AF and TIMM10-A5 showed limited role in predicting TB progression, while PHF23-AF, KIF1B-SE, MACROD2-A3, CD55-RI and GALNT11-AF could well predict TB prognosis and work as candidate biomarkers. This work preliminarily explored the value of AS in predicting TB progression and prognosis and offered potential targets for further research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The B complex is a key intermediate stage of spliceosome assembly. To improve the structural resolution of monomeric, human spliceosomal B (hB) complexes and thereby generate a more comprehensive hB molecular model, we determined the cryo-EM structure of B complex dimers formed in the presence of ATP γ S. The enhanced resolution of these complexes allows a finer molecular dissection of how the 5' splice site (5'ss) is recognized in hB, and new insights into molecular interactions of FBP21, SNU23 and PRP38 with the U6/5'ss helix and with each other. It also reveals that SMU1 and RED are present as a heterotetrameric complex and are located at the interface of the B dimer protomers. We further show that MFAP1 and UBL5 form a 5' exon binding channel in hB, and elucidate the molecular contacts stabilizing the 5' exon at this stage. Our studies thus yield more accurate models of protein and RNA components of hB complexes. They further allow the localization of additional proteins and protein domains (such as SF3B6, BUD31 and TCERG1) whose position was not previously known, thereby uncovering new functions for B-specific and other hB proteins during pre-mRNA splicing., (© 2024. The Author(s).)

Drugs that target pre-mRNA splicing hold great therapeutic potential, but the quantitative understanding of how these drugs work is limited. Here we introduce mechanistically interpretable quantitative models for the sequence-specific and concentration-dependent behavior of splice-modifying drugs. Using massively parallel splicing assays, RNA-seq experiments, and precision dose-response curves, we obtain quantitative models for two small-molecule drugs, risdiplam and branaplam, developed for treating spinal muscular atrophy. The results quantitatively characterize the specificities of risdiplam and branaplam for 5' splice site sequences, suggest that branaplam recognizes 5' splice sites via two distinct interaction modes, and contradict the prevailing two-site hypothesis for risdiplam activity at SMN2 exon 7. The results also show that anomalous single-drug cooperativity, as well as multi-drug synergy, are widespread among small-molecule drugs and antisense-oligonucleotide drugs that promote exon inclusion. Our quantitative models thus clarify the mechanisms of existing treatments and provide a basis for the rational development of new therapies., (© 2024. The Author(s).)

Objectives: Methylmalonic acidemia (MMA) is a rare inborn genetic disorder that is characterized by increased levels of methylmalonic acid in blood plasma and urine. Isolated methylmalonic acidemia is one of the most common types of MMA and is caused by mutations in the gene encoding methyl-malonyl coenzyme A mutase ( MMUT ). In this study, we investigated the possible mechanisms underlying the symptoms of isolated MMA in a patient by molecular analysis., Methods: PCR amplification and Sanger sequencing analysis was performed to identify variants in the MMUT gene in the proband and his family. Furthermore, minigene constructs were generated to validate the splicing defects in the MMUT gene variant identified in the proband., Results: The 3-year-old patient was admitted to the hospital with symptoms of MMA, including fever, convulsions, and vomiting. He showed metabolic acidosis, high levels of methylmalonic acid in blood and urine, and normal blood homocysteine levels. Genetic analysis demonstrated that the patient was a compound heterozygous carrier of two variants in the MMUT gene: a missense c.278G > A variant that has already been reported in a patient with the severe mut⁰ phenotype; and a novel splice site variant c.2125-2A > G. RT-PCR analysis showed that, while the novel variant clearly alters splicing, a minor amount of a full-length transcript is generated, suggesting that a wild-type protein may be produced although at a lower quantitative level. The patient's condition improved after treatment with vitamin B12. Serious complications were not reported during follow-up at age 5., Conclusions: We identified a novel splice site variant that partially disrupts normal splicing of the MMUT pre-mRNA. Production of a reduced amount of full-length transcript is responsible for the mild clinical phenotype observed in this patient. Functional studies have proven useful in exploring the genotype-phenotype association and in providing guidance for the genetic diagnosis of MMA., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Exonic sequences contain both protein-coding and RNA splicing information but the interplay of the protein and splicing code is complex and poorly understood. Here, we have studied traditional and auxiliary splicing codes of human exons that encode residues coordinating two essential divalent metals at the opposite ends of the Irving-Williams series, a universal order of relative stabilities of metal-organic complexes. We show that exons encoding Zn2+-coordinating amino acids are supported much less by the auxiliary splicing motifs than exons coordinating Ca2+. The handicap of the former is compensated by stronger splice sites and uridine-richer polypyrimidine tracts, except for position -3 relative to 3' splice junctions. However, both Ca2+ and Zn2+ exons exhibit close-to-constitutive splicing in multiple tissues, consistent with their critical importance for metalloprotein function and a relatively small fraction of expendable, alternatively spliced exons. These results indicate that constraints imposed by metal coordination spheres on RNA splicing have been efficiently overcome by the plasticity of exon-intron architecture to ensure adequate metalloprotein expression., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Recurring mutations in genes encoding 3' splice-site recognition proteins, U2AF1 and ZRSR2 are associated with human cancers. Here, we determined binding sites of the proteins to reveal that U2-type and U12-type splice sites are recognized by U2AF1 and ZRSR2, respectively. However, some sites are spliced by both the U2-type and U12-type spliceosomes, indicating that well-conserved consensus motifs in some U12-type introns could be recognized by the U2-type spliceosome. Nucleotides flanking splice sites of U12-type introns are different from those flanking U2-type introns. Remarkably, the AG dinucleotide at the positions -1 and -2 of 5' splice sites of U12-type introns with GT-AG termini is not present. AG next to 5' splice site introduced by a single nucleotide substitution at the -2 position could convert a U12-type splice site to a U2-type site. The class switch of introns by a single mutation and the bias against G at the -1 position of U12-type 5' splice site support the notion that the identities of nucleotides in exonic regions adjacent to splice sites are fine-tuned to avoid recognition by the U2-type spliceosome. These findings may shed light on the mechanism of selectivity in U12-type intron splicing and the mutations that affect splicing., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

More than half of cases with suspected genetic disorders remain unsolved by genetic analysis using short-read sequencing such as exome sequencing (ES) and genome sequencing (GS). RNA sequencing (RNA-seq) and long-read sequencing (LRS) are useful for interpretation of candidate variants and detection of structural variants containing repeat sequences, respectively. Recently, adaptive sampling on nanopore sequencers enables target LRS more easily. Here, we present a Japanese girl with premature chromatid separation (PCS)/mosaic variegated aneuploidy (MVA) syndrome. ES detected a known pathogenic maternal heterozygous variant (c.1402-5A>G) in intron 10 of BUB1B (NM&#95;001211.6), a known responsive gene for PCS/MVA syndrome with autosomal recessive inheritance. Minigene splicing assay revealed that almost all transcripts from the c.1402-5G allele have mis-splicing with 4-bp insertion. GS could not detect another pathogenic variant, while RNA-seq revealed abnormal reads in intron 2. To extensively explore variants in intron 2, we performed adaptive sampling and identified a paternal 3.0 kb insertion. Consensus sequence of 16 reads spanning the insertion showed that the insertion consists of Alu and SVA elements. Realignment of RNA-seq reads to the new reference sequence containing the insertion revealed that 16 reads have 5' splice site within the insertion and 3' splice site at exon 3, demonstrating causal relationship between the insertion and aberrant splicing. In addition, immunoblotting showed severely diminished BUB1B protein level in patient derived cells. These data suggest that detection of transcriptomic abnormalities by RNA-seq can be a clue for identifying pathogenic variants, and determination of insert sequences is one of merits of LRS., (© 2023. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)