Fig 3
Zebrafish Rnpc3 functions in the U12-type spliceosome. (A) Domain structure of zebrafish Rnpc3 and its human ortholog RNPC3, or U11/U12 di-snRNP 65-kDa protein (www.uniprot.org, accession no. Q96LT9). Red arrow in the C-terminal RRM denotes the approximate position of the premature stop codons in all clbn s846 isoforms. (B) Schematic of the human U11/U12 di-snRNP, with RNPC3 (65 K) bridging U12 snRNA and the 59 K protein (28). BPS, branch point sequence; "A" represents the branch point adenosine. (C) RT-PCR analysis of three different transcripts (sms, mapk3, mapk12) shows retention of U12-type introns (lane 4, Top three panels) in clbn s846 (5 dpf), but not of a U2-type intron (Bottom). Lanes 1 and 2 show-RT controls. (D and E) Glycerol gradient/Northern analysis revealing differential sedimentation of U11 (D) and U12 (E) snRNA-containing snRNPs in WT and clbn ZM extracts. Direction of sedimentation is left to right. Lane 1 contains total RNA. Full-length U11 or U12 signals were quantified using ImageQuant and expressed as a percentage of the fraction with the highest intensity. U12 snRNA showed persistent specific degradation (asterisks) without affecting its migration profile. (F) Northern analysis of WT and clbn s846 extracts resolved on 4% (80:1) native polyacrylamide gels and probed for U11 and U12 snRNAs. The predominant U11 snRNPs are disrupted in clbn compared with WT and the U12-containing particles are heavier and migrate more slowly (black arrowhead). (G) Northern analysis of WT and clbn s846 extracts resolved on native gels and probed for U5, U6atac, and U12 snRNAs shows retarded bands in clbn (red asterisks) compared with WT (blue asterisks). The same larval lysate was used in F and G. Data are representative of a total of 10 (D and E) and 5 (F and G) biological replicates.
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Significance
The accurate removal of introns by pre-mRNA splicing is a critical step in proper gene expression. Most eukaryotic genomes, from plant to human, contain a tiny subset of “minor class” introns with unique sequence elements that require their own splicing machinery. The significance of this second splicing pathway has intrigued RNA biolo...
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... Contains Incompletely Spliced Transcripts. The zebrafish rnpc3 gene encodes a 505-aa nuclear protein with two RNA-recognition motifs (RRM) (25) and the same domain structure as its human ortholog (Fig. 3A). Whereas the excision of U2-type introns is initiated by the binding of individual U1 and U2 monosnRNPs, U12-type introns in human cells are recognized and bound by a preformed U11/U12 di-snRNP (26). This di-snRNP comprises U11 and U12 snRNAs, RNPC3/65K and two other proteins unique to the U12-type spliceosome, the U11/U12 59-kDa and ...
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... introns in human cells are recognized and bound by a preformed U11/U12 di-snRNP (26). This di-snRNP comprises U11 and U12 snRNAs, RNPC3/65K and two other proteins unique to the U12-type spliceosome, the U11/U12 59-kDa and 48-kDa proteins. Together, these components form a "molecular bridge" between the 5′ ss and BPS of U12-type introns (27,28) (Fig. 3B). We used RT-PCR to assess the consequences of rnpc3 deficiency on the processing of mRNAs harboring U2-and U12-type introns in clbn and found that the U12-type introns in sms, mapk3, and mapk12 were retained in clbn larvae (Fig. 3C), whereas the excision of the U2-type intron in mapk12 was unaffected. Two-photon microscopy of WT and ...
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... Together, these components form a "molecular bridge" between the 5′ ss and BPS of U12-type introns (27,28) (Fig. 3B). We used RT-PCR to assess the consequences of rnpc3 deficiency on the processing of mRNAs harboring U2-and U12-type introns in clbn and found that the U12-type introns in sms, mapk3, and mapk12 were retained in clbn larvae (Fig. 3C), whereas the excision of the U2-type intron in mapk12 was unaffected. Two-photon microscopy of WT and clbn s846 larvae on the Tg(gutGFP) s854 background and (E) epifluorescence microscopy on the Tg(fabp10:RFP,ela3l: EGFP) gz12 background reveal smaller digestive organs in clbn s846 compared with WT. The two images in A were taken at ...
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... basis for the observed specific defect in U12-type splicing, we sought to compare the size and conformation of zebrafish U11-and U12-containing snRNPs in WT and clbn larvae using glycerol gradient sedimentation followed by Northern blot analysis for U11 and U12 snRNAs. Extracts of WT larvae gave rise to U11 peaks in fractions 15 and 16 (Fig. 3D, Upper), whereas the U11 peak in clbn extracts was found two fractions closer to the top of the gradient, corresponding to a lighter U11 species (Fig. 3D, Lower, and Fig. S3 A and B). In contrast, glycerol gradient analyses of U12 snRNA sedimentation in clbn showed a dramatic shift of the predominant U12 signal toward a heavier particle than ...
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... snRNPs in WT and clbn larvae using glycerol gradient sedimentation followed by Northern blot analysis for U11 and U12 snRNAs. Extracts of WT larvae gave rise to U11 peaks in fractions 15 and 16 (Fig. 3D, Upper), whereas the U11 peak in clbn extracts was found two fractions closer to the top of the gradient, corresponding to a lighter U11 species (Fig. 3D, Lower, and Fig. S3 A and B). In contrast, glycerol gradient analyses of U12 snRNA sedimentation in clbn showed a dramatic shift of the predominant U12 signal toward a heavier particle than in WT (Fig. 3E). We further investigated these findings using native gel electrophoresis followed by Northern blotting for U11 and U12 snRNAs (Fig. ...
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... larvae using glycerol gradient sedimentation followed by Northern blot analysis for U11 and U12 snRNAs. Extracts of WT larvae gave rise to U11 peaks in fractions 15 and 16 (Fig. 3D, Upper), whereas the U11 peak in clbn extracts was found two fractions closer to the top of the gradient, corresponding to a lighter U11 species (Fig. 3D, Lower, and Fig. S3 A and B). In contrast, glycerol gradient analyses of U12 snRNA sedimentation in clbn showed a dramatic shift of the predominant U12 signal toward a heavier particle than in WT (Fig. 3E). We further investigated these findings using native gel electrophoresis followed by Northern blotting for U11 and U12 snRNAs (Fig. 3F). Again, loss of ...
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... Upper), whereas the U11 peak in clbn extracts was found two fractions closer to the top of the gradient, corresponding to a lighter U11 species (Fig. 3D, Lower, and Fig. S3 A and B). In contrast, glycerol gradient analyses of U12 snRNA sedimentation in clbn showed a dramatic shift of the predominant U12 signal toward a heavier particle than in WT (Fig. 3E). We further investigated these findings using native gel electrophoresis followed by Northern blotting for U11 and U12 snRNAs (Fig. 3F). Again, loss of Rnpc3 was found to disrupt the predominant U11 snRNPs and cause the accumulation of a slower-migrating U12-containing particle. This unexpected finding suggests a function for Rnpc3 ...
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... species (Fig. 3D, Lower, and Fig. S3 A and B). In contrast, glycerol gradient analyses of U12 snRNA sedimentation in clbn showed a dramatic shift of the predominant U12 signal toward a heavier particle than in WT (Fig. 3E). We further investigated these findings using native gel electrophoresis followed by Northern blotting for U11 and U12 snRNAs (Fig. 3F). Again, loss of Rnpc3 was found to disrupt the predominant U11 snRNPs and cause the accumulation of a slower-migrating U12-containing particle. This unexpected finding suggests a function for Rnpc3 beyond its known role in the U11/U12 di-snRNP, potentially at later stages of the splicing cycle. U12-containing complexes that might be ...
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... known role in the U11/U12 di-snRNP, potentially at later stages of the splicing cycle. U12-containing complexes that might be stalled at any point after spliceosome activation would be expected to contain U6atac or U5 snRNAs (2). To test this theory we used native gel electrophoresis followed by Northern blotting for U12, U5, and U6atac snRNAs (Fig. 3G). To reduce potential nonspecific interactions and facilitate characterization of the stable complex accumulating in clbn embryos, heparin was added to the extracts before separation. Once again, we observed a strong signal corresponding to a heavier U12 snRNP complex ...
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... injected with WT rnpc3 RNA appear WT at 5 dpf (P < 0.0001, Fisher's exact test). There was no difference between uninjected and mutant rnpc3-injected groups (P = 0.9065). a, anus; e, eye; ib, intestinal bulb; sb, swim bladder; y, yolk. The brightfield images in D, E, and F were taken at the same magnification. in clbn larvae from both alleles (Fig. S3C), which appeared to comigrate with signals for both U5 and ...
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... RNPC3 is a component of one of the minor (U12-dependent) spliceosome complex, reported to act on 700-800 RNAs [60]. A zebrafish mutant, caliban (clbn), harboring a splicing mutation in the same RRM2 domain of rnpc3, leads to a severe and pleiotropic phenotype in developing zebrafish larvae with early lethality [74]. The authors showed that several genes involved in various steps of mRNA processing, including transcription, splicing, and nuclear export are disrupted in clbn mutants. ...
... The authors showed that several genes involved in various steps of mRNA processing, including transcription, splicing, and nuclear export are disrupted in clbn mutants. Interestingly, of the 38 downregulated genes, 4 are associated with human pathologies wherein hydrocephalus is a feature [74]. Though in human [75] and mice models [76,77] defects in minor spliceosome components are associated with several disorders characterized by microcephaly and dwarfism, a recent report describes a case of severe ventriculomegaly and mild growth retardation associated with compound heterozygote mutations in the non-coding region of RNA, U4atac Small Nuclear (RNU4ATAC) [78]. ...
Abstract Background Congenital hydrocephalus is characterized by ventriculomegaly, defined as a dilatation of cerebral ventricles, and thought to be due to impaired cerebrospinal fluid (CSF) homeostasis. Primary congenital hydrocephalus is a subset of cases with prenatal onset and absence of another primary cause, e.g., brain hemorrhage. Published series report a Mendelian cause in only a minority of cases. In this study, we analyzed exome data of PCH patients in search of novel causal genes and addressed the possibility of an underlying oligogenic mode of inheritance for PCH. Materials and methods We sequenced the exome in 28 unrelated probands with PCH, 12 of whom from families with at least two affected siblings and 9 of whom consanguineous, thereby increasing the contribution of genetic causes. Patient exome data were first analyzed for rare (MAF
... The Human Splicing Finder program predicted that c.6956+9C > G would result in a strong ectopic splicing site (HSF score of 80.6) [27]. In order to provide a better understanding of alternative tissue-specific splicing mechanism, in vivo minigene assay have been applied in the zebrafish and C. elegans [63,64]. It's not completely understood how some splice-site variants disrupt normal translation and produce unusual transcriptional products in the inner ear. ...
Pathogenic variants in MYO15A are known to cause autosomal recessive nonsyndromic hearing loss (ARNSHL), DFNB3. We have previously reported on one ARNSHL family including two affected siblings and identified MYO15A c.5964+3G > A and c.8375 T > C (p.Val2792Ala) as the possible deafness-causing variants. Eight year follow up identified one new affected individual in this family, who also showed congenital, severe to profound sensorineural hearing loss. By whole exome sequencing, we identified a new splice-site variant c.5531+1G > C (maternal allele), in a compound heterozygote with previously identified missense variant c.8375 T > C (p.Val2792Ala) (paternal allele) in MYO15A as the disease-causing variants. The new affected individual underwent unilateral cochlear implantation at the age of 1 year, and 5 year follow-up showed satisfactory speech and language outcomes. Our results further indicate that MYO15A-associated hearing loss is good candidates for cochlear implantation, which is in accordance with previous report. In light of our findings and review of the literatures, 58 splice-site variants in MYO15A are correlated with a severe deafness phenotype, composed of 46 canonical splice-site variants and 12 non-canonical splice-site variants.
... Rnpc3 is one of the seven integral proteins that are unique to the U12-dependent minor spliceosome, and its mutation has been associated with the human disease of isolated growth hormone deficiency [77]. Loss of Rnpc3 in zebrafish leads to pleiotropic phenotypes, mostly affecting highly proliferative tissues [78]. This suggests that Rnpc3 may regulate proliferation-related alternative splicing events during neuromast regeneration. ...
RNA-binding proteins (RBPs) regulate gene expression at the post-transcriptional level. They play major roles in the tissue- and stage-specific expression of protein isoforms as well as in the maintenance of protein homeostasis. The inner ear is a bi-functional organ, with the cochlea and the vestibular system required for hearing and for maintaining balance, respectively. It is relatively well documented that transcription factors and signaling pathways are critically involved in the formation of inner ear structures and in the development of hair cells. Accumulating evidence highlights emerging functions of RBPs in the post-transcriptional regulation of inner ear development and hair cell function. Importantly, mutations of splicing factors of the RBP family and defective alternative splicing, which result in inappropriate expression of protein isoforms, lead to deafness in both animal models and humans. Because RBPs are critical regulators of cell proliferation and differentiation, they present the potential to promote hair cell regeneration following noise- or ototoxin-induced damage through mitotic and non-mitotic mechanisms. Therefore, deciphering RBP-regulated events during inner ear development and hair cell regeneration can help define therapeutic strategies for treatment of hearing loss. In this review, we outline our evolving understanding of the implications of RBPs in hair cell formation and hearing disease with the aim of promoting future research in this field.
... RNA from two independent rnpc3 mutant alleles and their respective wildtype controls (n=3 for each genotype) were sequenced. Clbn s846 identified in an ethylynitrosourea mutagenesis screen encodes a T to A transition in intron 13, creating a novel 3'SS 10 nucleotides upstream of the canonical 3'SS (12). This results in aberrant transcripts all containing premature stop codons with no correctly spliced exon 13-14 junction transcript detected. ...
... This results in aberrant transcripts all containing premature stop codons with no correctly spliced exon 13-14 junction transcript detected. Clbn ZM harbors a retroviral insertion in intron 1 of rnpc3; both alleles are functionally null (12). Total RNA was extracted from pools of genotyped 72-hour post fertilization (hpf) larvae using TRIzol. ...
... Next, we studied intron retention and other de novo alternative splicing events around introns in twenty publicly available RNAseq datasets that covered various cell types in human, mouse, fruit fly and maize. Additionally, we performed RNAseq of zebrafish larvae with two different mutations in rnpc3, a critical minor spliceosomespecific protein (12). As expected, we found that bioinformatically classified minor introns were retained at higher levels upon minor spliceosome inhibition, reflected by a positive difference in mis-splicing index between the different experimental and their respective control conditions (Fig. S5-S9). ...
Despite the high conservation of minor introns across eukaryotic supergroups, specific lineages have completely lost minor intron splicing, which has raised questions about their evolution and purpose. Addressing these questions requires identification of the introns that are affected by minor spliceosome inhibition. To this end, we applied principles of Linnaean taxonomy combined with position-weight matrices to produce five intron classes: minor, minor-like, hybrid, major-like and major. We classified introns across the genomes of 263 species of six eukaryotic supergroups, which can be viewed at the Minor Intron Database (MIDB). Transcriptomic analysis revealed that ∼40% of the minor introns are responsive to minor spliceosome inhibition, while an additional 5% of the minor-like and hybrid introns are also affected. We propose that minor-like introns represent an intermediate in the conversion of minor to major introns and uncover the importance of a guanine at the −1 position of the 5’ splice site in facilitating this shift in spliceosome dependence. Finally, we find that minor introns are aberrantly spliced in fish and plants upon cold stress, thereby providing a potential explanation for their high degree of conservation in these lineages.
... Although many spliceosome components are essential for pre-implantation development in mouse, zebrafish lacking spliceosome components survive at least up to 2 days postfertilization (dpf ), when gastrulation has already completed ( Fig. 2B; Table S2). For example, even though knockout of the minor spliceosome component Rnpc3 in mice results in death before blastocyst formation around E3.5, mutation in rnpc3 in zebrafish results in lethality between 7 and 10 dpf, which is well into organogenesis (Doggett et al., 2018;Markmiller et al., 2014). This variability in the timing of lethality might be explained by differences in the scale and timing of the maternal-to-zygotic transition in these species. ...
... For example, it is known that early embryonic development is predominantly controlled by maternally deposited mRNAs and proteins, which in mice and humans are degraded by the 4-cell stage, but exist for longer in zebrafish (Vastenhouw et al., 2019). Accordingly, low levels of fully spliced, maternally deposited rnpc3 mRNAs are detected up to 24 h post fertilization (hpf ) in rnpc3-mutant zebrafish (Markmiller et al., 2014). Consequently, these larvae experience a delayed effect of rnpc3 loss-of-function on splicing until after the gastrulation stage. ...
... Interestingly, no signal is observed for these components in the heart during early organogenesis (Beauchamp et al., 2019;Ruiz-Lozano et al., 1997), suggesting that this tissue might be less affected by mutations in spliceosome components. Whole-mount in situ hybridization in zebrafish has revealed that most spliceosome components are widely expressed at early embryonic stages (An and Henion, 2012;Lei et al., 2017;Li et al., 2021;Liu et al., 2015;Markmiller et al., 2014;McElderry et al., 2019;Ríos et al., 2011;Wang et al., 2018;Yu et al., 2019). However, from 24-48 hpf, the expression of eftud2, dhx15, usp39, sf3b1, prpf31, bcas2 and prpf4 is enriched in the brain and eye, while rnpc3 expression is restricted to the eye and digestive organs. ...
Splicing is a crucial regulatory node of gene expression that has been leveraged to expand the proteome from a limited number of genes. Indeed, the vast increase in intron number that accompanied vertebrate emergence might have aided the evolution of developmental and organismal complexity. Here, we review how animal models for core spliceosome components have provided insights into the role of splicing in vertebrate development, with a specific focus on neuronal, neural crest and skeletal development. To this end, we also discuss relevant spliceosomopathies, which are developmental disorders linked to mutations in spliceosome subunits. Finally, we discuss potential mechanisms that could underlie the tissue-specific phenotypes often observed upon spliceosome inhibition and identify gaps in our knowledge that, we hope, will inspire further research.
... Our data on their differential expression during development suggests that the proteins encoded by each isoform perform specific functions which can be explored further using isoform-specific antibodies. zrsr2 is highly expressed in the anterior region of the embryos and intestinal bulb, as has also been reported for other components of the minor spliceosome, e.g., dhx15 and rnpc3 [38,39]. The Zrsr2deficient embryos developed normally for the first 3 days after fertilization, likely due to the presence of maternally-supplied WT zrsr2 mRNA. ...
... It is possible that these tissues are more dependent on the minor spliceosome to produce appropriate mRNA transcripts for their growth and differentiation. Similar to the embryonic lethality observed upon disruption of the other components of the minor spliceosome in Drosophila and zebrafish [34,38,39], zrsr2-knockout fish displayed lethality by 8 dpf. Thus, our study adds to the growing evidence that the proper functioning of the minor spliceosome is critical for early animal development. ...
... Similar to human MDS bone marrow samples from patients with ZRSR2 mutations, zrsr2-deficient zebrafish showed preferential retention of more than one-third of all U12-type introns (218/662). Specifically, the e2f and mapk gene families seem to be most affected by retained introns when the minor spliceosome function is disrupted [19,20,24,35,39]. Among the genes with retained introns in zrsr2 hg129/hg129 mutants, we also identified multiple genes from both the e2f and mapk gene families (e2f4, mapk3, mapk8a, mapk12a, mapk14a). ...
ZRSR2 (zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2) is an essential splicing factor involved in 3′ splice-site recognition as a component of both the major and minor spliceosomes that mediate the splicing of U2-type (major) and U12-type (minor) introns, respectively. Studies of ZRSR2-depleted cell lines and ZRSR2-mutated patient samples revealed its essential role in the U12-dependent minor spliceosome. However, the role of ZRSR2 during embryonic development is not clear, as its function is compensated for by Zrsr1 in mice. Here, we utilized the zebrafish model to investigate the role of zrsr2 during embryonic development. Using CRISPR/Cas9 technology, we generated a zrsr2-knockout zebrafish line, termed zrsr2hg129/hg129 (p.Trp167Argfs*9) and examined embryo development in the homozygous mutant embryos. zrsr2hg129/hg129 embryos displayed multiple developmental defects starting at 4 days post fertilization (dpf) and died after 8 dpf, suggesting that proper Zrsr2 function is required during embryonic development. The global transcriptome analysis of 3 dpf zrsr2hg129/hg129 embryos revealed that the loss of Zrsr2 results in the downregulation of essential metabolic pathways and the aberrant retention of minor introns in about one-third of all minor intron-containing genes in zebrafish. Overall, our study has demonstrated that the role of Zrsr2 as a component of the minor spliceosome is conserved and critical for proper embryonic development in zebrafish.
... Defects in pre-mRNA splicing by cis-or trans-acting mutations have been linked to approximately 60% of human diseases (Lim et al. 2011;Sterne-Weiler and Sanford 2014;Chabot and Shkreta 2016). While many mutations impact splicing of U2-type introns (Cooper et al. 2009), anomalous splicing of U12-type introns can cause developmental defects in both plants and animals (Kim et al. 2010;Edery et al. 2011;He et al. 2011;Jung and Kang 2014;Markmiller et al. 2014;Xu et al. 2016). Mutations in minor spliceosome factors impact normal splicing of a subset of MIGs in both humans and maize that result in cell differentiation defects (Fouquet et al. 2011;Madan et al. 2015;Gault et al. 2017). ...
... However, the mechanisms by which they impact cell development and differentiation are not well understood. Mutations in minor spliceosomal factors cause pleiotropic molecular defects affecting a large proportion of MIGs (Jafarifar et al. 2014;Jung and Kang 2014;Markmiller et al. 2014;Gault et al. 2017;Kwak et al. 2017;Verma et al. 2018). This has made it difficult to delineate the causative genes underlying the pleiotropic phenotypes of MIG spliceosomal factor mutants, even if they exhibit highly similar characteristics. ...
U12-type or minor introns are found in most multicellular eukaryotes and constitute ∼0.5% of all introns in species with a minor spliceosome. Although the biological significance for evolutionary conservation of U12-type introns is debated, mutations disrupting U12 splicing cause developmental defects in both plants and animals. In human hematopoietic stem cells, U12 splicing defects disrupt proper differentiation of myeloid lineages and are associated with myelodysplastic syndrome (MDS), predisposing individuals to acute myeloid leukemia. Mutants in the maize ortholog of RNA Binding Motif Protein 48 (RBM48) have aberrant U12-type intron splicing. Human RBM48 was recently purified biochemically as part of the minor spliceosome and shown to recognize the 5’ end of the U6atac snRNA. In this report, we use CRISPR/Cas9-mediated ablation of RBM48 in human K-562 cells to show the genetic function of RBM48. RNA-seq analysis comparing wild-type and mutant K-562 genotypes found that 48% of minor intron containing genes (MIGs) have significant U12-type intron retention in RBM48 mutants. Comparing these results to maize rbm48 mutants defined a subset of MIGs disrupted in both species. Mutations in the majority of these orthologous MIGs have been reported to cause developmental defects in both plants and animals. Our results provide genetic evidence that the primary defect of human RBM48 mutants is aberrant U12-type intron splicing, while a comparison of human and maize RNA-seq data identifies candidate genes likely to mediate mutant phenotypes of U12-type splicing defects.
... The copyright holder for this this version posted December 10, 2021. ; https://doi.org/10.1101/2021.12.09.471104 doi: bioRxiv preprint Primary tumor RNA-seq patient samples from The Cancer Genome Atlas (TCGA) were (http://github.com/Bioconductor/GenomicDataCommons). The minor intron retention pipeline developed by Olthof et. ...
Here we explored the role of minor spliceosome (MiS) function and minor intron-containing gene (MIG) expression in prostate cancer (PCa). We show MIGs are enriched as direct interactors of cancer-causing genes and their expression discriminates PCa progression. Increased expression of MiS U6atac snRNA, including others, and 6x more efficient minor intron splicing was observed in castration-resistant PCa (CRPC) versus primary PCa. Notably, androgen receptor signalling influenced MiS activity. Inhibition of MiS through siU6atac in PCa caused minor intron mis-splicing and aberrant expression of MIG transcripts and encoded proteins, which enriched for MAPK activity, DNA repair and cell cycle. Single cell-RNAseq confirmed cell cycle defects and lineage dependency on the MiS from primary to CRPC and neuroendocrine PCa. siU6atac was ∼50% more efficient in lowering tumor burden of CRPC cells and organoids versus current state-of-the-art combination therapy. In all, MiS is a strong therapeutic target for lethal PCa and potentially other cancers.
Graphical Abstract
U6atac expression, MiS activity, and minor intron splicing correlate with PCa therapy resistance and PCa progression to CRPC-adeno and transdifferentiation to CRPC-NE. One major MiS regulator during that process is the AR-axis, which is re-activated during CRPC-adeno and blocked in CRPC-NE. Molecularly, an increase in MiS dependent splicing promotes changes of transcriptome and proteome. This results in cell cycle activation, increased MAPK signalling and increased DNA repair. U6atac mediated MiS inhibition renders MiS splicing error-prone through increased intron retention and alternative splicing events, which results in cell cycle block and decreased MAPK signalling and DNA repair. MiS inhibition blocks all stages of PCa. Figure created with BioRender.com .
... This is the only rnpc3 loss of function in vivo model reported to date. Analysis of the zebrafish transcriptome revealed that efficient mRNA processing is critical for the growth and proliferation of cells during vertebrate development (54). Defective stimulation of the thyroid gland by TSH causes inadequate thyroid hormone biosynthesis and subsequent central, or secondary, hypothyroidism in affected patients, thus TSHD. ...
Human embryonic hypothalamo-pituitary (HP) development consists of complex molecular pathways reliant on an array of genes expressed at specific time points. The resulting intertwined signalling molecules work in synchronization to give rise to the five specialized anterior pituitary cell types that secrete the six vital hormones responsible for growth and homeostasis. Congenital hypopituitarism (CH) involves deficiencies in one or more of these hormones, and exists in many forms of severity ranging from an inability to sustain life, to mild hormone deficiencies that may go unnoticed. Accompanying phenotypic features affecting various target tissues, intellectual disability and dysmorphic features are often apparent in such patients, depending on the genes mutated. The rapid evolution in next generation sequencing (NGS) technology has revolutionized genotyping in such individuals, and stem cell research and CRISPR-Cas9 gene editing will doubtless allow a more detailed and physiologically relevant characterization of mutations identified. Future studies are likely to reveal an expanding list of new candidate genes associated with HP development. In this part we will focus on known genetic causes of CH and related disorders.
... Therefore, based on the exclusivity of minor introns to certain gene families and their incomplete functional loss in diseases, it is highly likely that they are implicated in diseases such as cancer, where alterations in these gene families contribute to the hallmarks of cancer. Indeed, mi-INTs are enriched in cancer-relevant oncogenes such as BRAF, ERK2, MAPK11/P38beta, and JNK1 [78,106]. Furthermore, p38MAPK, which has been implicated in mi-INTs splicing regulation [34], itself harbors an mi-INT. ...
Pre-mRNA splicing is an essential step in gene expression and is catalyzed by two machineries in eukaryotes: the major (U2 type) and minor (U12 type) spliceosomes. While the majority of introns in humans are U2 type, less than 0.4% are U12 type, also known as minor introns (mi-INTs), and require a specialized spliceosome composed of U11, U12, U4atac, U5, and U6atac snRNPs. The high evolutionary conservation and apparent splicing inefficiency of U12 introns have set them apart from their major counterparts and led to speculations on the purpose for their existence. However, recent studies challenged the simple concept of mi-INTs splicing inefficiency due to low abundance of their spliceosome and confirmed their regulatory role in alternative splicing, significantly impacting the expression of their host genes. Additionally, a growing list of minor spliceosome-associated diseases with tissue-specific pathologies affirmed the importance of minor splicing as a key regulatory pathway, which when deregulated could lead to tissue-specific pathologies due to specific alterations in the expression of some minor-intron-containing genes. Consequently, uncovering how mi-INTs splicing is regulated in a tissue-specific manner would allow for better understanding of disease pathogenesis and pave the way for novel therapies, which we highlight in this review.
