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Schematic of defective minor spliceosome induced SMA-associated phenotypes and competition model of minor intron recognition SMN is the upstream gene of SMA; a defective minor spliceosome results in inhibited splicing of minS-I- and minS-SS-containing neural genes, and directly induces SMA-associated phenotypes. Splicing recognition of U12-type or minor sensitive introns (minS-I) are proposed as: (i) only by the minor spliceosome; (ii & iii) 5′SS or 3′SS is competitively recognized by the minor and major spliceosomes; (iv) both SSs are competitively recognized by the two spliceosomes.
Source publication
The minor spliceosome is evolutionarily conserved in higher eukaryotes, but its biological significance remains poorly understood. Here, by precise CRISPR/Cas9-mediated disruption of the U12 and U6atac snRNAs, we report that a defective minor spliceosome is responsible for spinal muscular atrophy (SMA) associated phenotypes in Drosophila. Using a n...
Citations
... For additional comparisons to the Smn missense mutant proteomes, we used polyA + -RNA-seq datasets from two different Smn null mutant lines [27,43]; see Tables S6-S11. The Smn X7/D null mutant transcriptome identified an increase in BaraA2 and SPH93 (Serine protease homolog 93) transcripts in both T205I and V72G proteomes ( Fig. 1H and Tables S1-S3). ...
Background
Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by hypomorphic loss of function in the survival motor neuron (SMN) protein. SMA presents across a broad spectrum of disease severity. Unfortunately, genetic models of intermediate SMA have been difficult to generate in vertebrates and are thus unable to address key aspects of disease etiology. To address these issues, we developed a Drosophila model system that recapitulates the full range of SMA severity, allowing studies of pre-onset biology as well as late-stage disease processes.
Results
Here, we carried out transcriptomic and proteomic profiling of mild and intermediate Drosophila models of SMA to elucidate molecules and pathways that contribute to the disease. Using this approach, we elaborated a role for the SMN complex in the regulation of innate immune signaling. We find that mutation or tissue-specific depletion of SMN induces hyperactivation of the immune deficiency (IMD) and Toll pathways, leading to overexpression of antimicrobial peptides (AMPs) and ectopic formation of melanotic masses in the absence of an external challenge. Furthermore, the knockdown of downstream targets of these signaling pathways reduced melanotic mass formation caused by SMN loss. Importantly, we identify SMN as a negative regulator of a ubiquitylation complex that includes Traf6, Bendless, and Diap2 and plays a pivotal role in several signaling networks.
Conclusions
In alignment with recent research on other neurodegenerative diseases, these findings suggest that hyperactivation of innate immunity contributes to SMA pathology. This work not only provides compelling evidence that hyperactive innate immune signaling is a primary effect of SMN depletion, but it also suggests that the SMN complex plays a regulatory role in this process in vivo. In summary, immune dysfunction in SMA is a consequence of reduced SMN levels and is driven by cellular and molecular mechanisms that are conserved between insects and mammals.
... We then analyzed changes in pre-mRNA splicing in the deletion strains. To our surprise, fewer differential alternative splicing events (DASs) were identified in the singular U1-gene deletion strains (Fig 6A and S3 Table), compared to mutations or deletions of other splicing factor in Drosophila, such as the Sf3b1 mutants, U12 and U6atac deletion strains, which caused more than one thousand changed AS events but less than five hundred DEGs [42,61]. Consistent with the extent of defective phenotypes, the 82Eb Δ/Δ and 95Cb Δ/Δ strains also exhibited the most changed DASs (Fig 6A and S6A Fig); however, each only had 282 and 236 events respectively, unlike more than 1,000 DEGs in these two strains (Fig 5A). ...
... Briefly, for each strain, a transgenic pBID plasmid containing the upstream 1.5 kb of U1 gene, Kozac sequence, CDS of GFP, and the downstream 500 bp of U1 gene ( Fig 1B) , and incorporated in chromosome 2L. Five U1-gene deletion strains were constructed using CRISPR/Cas9-mediated gene editing [61]. Briefly, plasmids expressing guide RNAs (sgRNA) that specifically target the upstream and downstream sequences of each U1 snRNA locus were constructed (Fig 3A), and then micro-injected in embryos of nanos-Cas9 strain at the Core Facility of Drosophila Resource and Technology, CEMCS, CAS. ...
... Locomotion of the 3 rd instar larvae was detected with slight modification as described [61]. Eight of the larvae from each strain were randomly selected and placed in a 6-cm plate, and 0.5 ml of 1×PBS were added to keep the larval body wet. ...
Small nuclear RNAs (snRNAs) are structural and functional cores of the spliceosome. In metazoan genomes, each snRNA has multiple copies/variants, up to hundreds in mammals. However, the expressions and functions of each copy/variant in one organism have not been systematically studied. Focus on U1 snRNA genes, we investigated all five copies in Drosophila melanogaster using two series of constructed strains. Analyses of transgenic flies that each have a U1 promoter-driven gfp revealed that U1 : 21D is the major and ubiquitously expressed copy, and the other four copies have specificities in developmental stages and tissues. Mutant strains that each have a precisely deleted copy of U1-gene exhibited various extents of defects in fly morphology or mobility, especially deletion of U1 : 82Eb . Interestingly, splicing was changed at limited levels in the deletion strains, while large amounts of differentially-expressed genes and alternative polyadenylation events were identified, showing preferences in the down-regulation of genes with 1–2 introns and selection of proximal sites for 3’-end polyadenylation. In vitro assays suggested that Drosophila U1 variants pulled down fewer SmD2 proteins compared to the canonical U1. This study demonstrates that all five U1-genes in Drosophila have physiological functions in development and play regulatory roles in transcription and 3’-end formation.
... One possibility is that widespread splicing alterations cause the SMA phenotype. This scenario is founded on the identification of widespread splicing defects in tissues from SMA animal models [113,115,116,120]. Theories that widespread splicing alterations may cause selective motor neuron death in SMA are supported by mechanistic insights from recent work finding pervasive splicing defects, global DNA damage, and an activated stress response downstream of SMN-deficiency [116]. ...
In the cell, RNA exists and functions in a complex with RNA binding proteins (RBPs) that regulate each step of the RNA life cycle from transcription to degradation. Central to this regulation is the role of several molecular chaperones that ensure the correct interactions between RNA and proteins, while aiding the biogenesis of large RNA-protein complexes (ribonucleoproteins or RNPs). Accurate formation of RNPs is fundamentally important to cellular development and function, and its impairment often leads to disease. The survival motor neuron (SMN) protein exemplifies this biological paradigm. SMN is part of a multi-protein complex essential for the biogenesis of various RNPs that function in RNA metabolism. Mutations leading to SMN deficiency cause the neurodegenerative disease spinal muscular atrophy (SMA). A fundamental question in SMA biology is how selective motor system dysfunction results from reduced levels of the ubiquitously expressed SMN protein. Recent clarification of the central role of the SMN complex in RNA metabolism and a thorough characterization of animal models of SMA have significantly advanced our knowledge of the molecular basis of the disease. Here we review the expanding role of SMN in the regulation of gene expression through its multiple functions in RNP biogenesis. We discuss developments in our understanding of SMN activity as a molecular chaperone of RNPs and how disruption of SMN-dependent RNA pathways can contribute to the SMA phenotype.
... There is evidence that some accessory splicing factors regulating minor splicing are important for a normal function of the heart. For example, SMN1 has recently been identified as a splicing factor involved in minor splicing (Boulisfane et al., 2011;Li et al., 2020;Lotti et al., 2012). SMN1 is required for the survival of motor neurons, and mutations in this gene are responsible of spinal muscular atrophy. ...
Eukaryotic genomes contain a tiny subset of ‘minor class’ introns with unique sequence elements that require their own splicing machinery. These minor introns are present in certain gene families with specific functions, such as voltage-gated sodium and voltage-gated calcium channels. Removal of minor introns by the minor spliceosome has been proposed as a post-transcriptional regulatory layer, which remains unexplored in the heart. Here, we investigate whether the minor spliceosome regulates electrophysiological properties of cardiomyocytes by knocking-down the essential minor spliceosome component U6atac in neonatal rat ventricular myocytes. Loss of U6atac led to robust minor intron retention within Scn5a and Cacna1c, resulting in reduced protein levels of Nav1.5 and Cav1.2. Functional consequences were studied through path-clamp analysis, and revealed reduced sodium and L-type calcium currents after loss of U6atac. In conclusion, minor intron splicing modulates voltage-dependent ion channel expression and function in cardiomyocytes. This may be of particular relevance in situations in which minor splicing activity changes, such as in genetic diseases affecting minor spliceosome components, or in acquired diseases in which minor spliceosome components are dysregulated, such as heart failure.
... Alternative splicing is the consequence of competition/selection between multiple SSs. To address details of SS selection, we used our recently developed tool ΔUSS (Differential Usage of Splice Site) to evaluate all the individual SSs in the Drosophila transcriptome [74] (Fig 6A). In total, usages of 417 and 472 of 5 0 SSs, and 404 and 524 of 3 0 SSs were significantly changed in H698D and H698R, respectively (Fig 6B and S6 Table). ...
... The wild type (WT) Drosophila melanogaster used in this study is a w1118 isogenic strain (BDSC 5905). Point mutant strains were constructed using the CRISPR/Cas9 system [74]. In brief, the target sequence of each guide RNA (sgRNA) was selected, donor plasmids with point mutations and the adjacent 3 kb sequences as homologous arms were constructed using pMD18-T (Fig 1B), and the gRNA and donor plasmids were co-injected into embryos of the transgenic line nanos-Cas9 by UniHuaii Technology Company. ...
... Significant DS events were screened by conditions |ΔPSI| > 0.05 and FDR < 0.05. Differential splice site usage was analyzed by ΔUSS, which is modified from an Unused Index as described [74]. Significant ΔUSS were screened by conditions |ΔUSS| > 0.01, p value <0.05. ...
SF3B1 mutations occur in many cancers, and the highly conserved His662 residue is one of the hotspot mutation sites. To address effects on splicing and development, we constructed strains carrying point mutations at the corresponding residue His698 in Drosophila using the CRISPR-Cas9 technique. Two mutations, H698D and H698R , were selected due to their frequent presence in patients and notable opposite charges. Both the sf3b1-H698D and– H698R mutant flies exhibit developmental defects, including less egg-laying, decreased hatching rates, delayed morphogenesis and shorter lifespans. Interestingly, the H698D mutant has decreased resistance to fungal infection, while the H698R mutant shows impaired climbing ability. Consistent with these phenotypes, further analysis of RNA-seq data finds altered expression of immune response genes and changed alternative splicing of muscle and neural-related genes in the two mutants, respectively. Expression of Mef2-RB , an isoform of Mef2 gene that was downregulated due to splicing changes caused by H698R , partly rescues the climbing defects of the sf3b1-H698R mutant. Lariat sequencing reveals that the two sf3b1-H698 mutations cause aberrant selection of multiple intronic branch sites, with the H698R mutant using far upstream branch sites in the changed alternative splicing events. This study provides in vivo evidence from Drosophila that elucidates how these SF3B1 hotspot mutations alter splicing and their consequences in development and in the immune system.
... Ribonucleic acid samples were prepared as described (Li et al., 2020), and the construction of cDNA libraries and sequencing were performed using Illumina Hi-Seq 2000 (Stark et al., 2019). The head, body, and gonads of the female and male Drosophila were sequenced in the form of 150 bp fr-firststrand pair-end reads, and the embryo, 3L larvae, and adults were sequenced in the form of 100 bp fr-untstranded pair-end reads. ...
Interrupted exons in the pre-mRNA transcripts are ligated together through RNA splicing, which plays a critical role in the regulation of gene expression. Exons with a length ≤ 30 nt are defined as microexons that are unique in identification. However, microexons, especially those shorter than 8 nt, have not been well studied in many organisms due to difficulties in mapping short segments from sequencing reads. Here, we analyzed mRNA-seq data from a variety of Drosophila samples with a newly developed bioinformatic tool, ce-TopHat. In addition to the Flybase annotated, 465 new microexons were identified. Differentially alternatively spliced (AS) microexons were investigated between the Drosophila tissues (head, body, and gonad) and genders. Most of the AS microexons were found in the head and two AS microexons were identified in the sex-determination pathway gene fruitless.
... In addition, Siberian tiger Pcyt2 transcript (ENSPTIG00000010590) lacks the C-domain HXGH, gorilla variant (ENSGGOG00000067604.1) has the N-domain HXGH absent; zebrafish has several variants lacking either of the two HXGH catalytic sites. Excitingly, the most recent study in Drosophila established that dysfunctional minor spliceosome causes spinal muscular atrophy by direct contribution from three neural genes, Pcyt2, Zmynd10, and Fas3 as the main splicing targets [73]. This agreed with the existence of multiple Pcyt2 isoforms and implicates a strong splicing and self-inhibiting regulation of de novo PE synthesis by the Kennedy pathway. ...
Neurodegenerative diseases (NDs) are a diverse group of neuropathological diseases that are currently incurable due to the irreversible neuronal loss. At the present rate of the world population growth, it is projected that the number of ND cases will double by the year of 2050. With treatments only available for symptom management and relief, disease prevention may yield significant benefits. Recently, there had been association drawn between the disruption of phospholipid (PL) homeostasis and the progression of NDs. Pathological developments were observed in cellular processes including autophagy, maintenance of mitochondrial integrity, and management of tissue oxidative stress. As PLs actively participate in the regulation of these cellular pathways and in neuronal signal transduction for the maintenance of an optimally functioning nervous system, their homeostasis is tightly controlled via an intricate system of interconversion and metabolism. Therefore, in this review, the contribution of a homeostatic PL pool and the detrimental effects by the lack thereof, are discussed in detail as it relates to ND development.
RNA splicing is crucial in the multilayer regulatory networks for gene expression, making functional interactions with DNA‐ and other RNA‐processing machineries in the nucleus. However, these established couplings are all major spliceosome‐related; whether the minor spliceosome is involved remains unclear. Here, through affinity purification using Drosophila lysates, an interaction is identified between the minor spliceosomal 65K/RNPC3 and ANKRD11, a cofactor of histone deacetylase 3 (HDAC3). Using a CRISPR/Cas9 system, Deletion strains are constructed and found that both Dm65KΔ/Δ and Dmankrd11Δ/Δ mutants have reduced histone deacetylation at Lys9 of histone H3 (H3K9) and Lys5 of histone H4 (H4K5) in their heads, exhibiting various neural‐related defects. The 65K‐ANKRD11 interaction is also conserved in human cells, and the HsANKRD11 middle‐uncharacterized domain mediates Hs65K association with HDAC3. Cleavage under targets and tagmentation (CUT&Tag) assays revealed that HsANKRD11 is a bridging factor, which facilitates the synergistic common chromatin‐binding of HDAC3 and Hs65K. Knockdown (KD) of HsANKRD11 simultaneously decreased their common binding, resulting in reduced deacetylation of nearby H3K9. Ultimately, this study demonstrates that expression changes of many genes caused by HsANKRD11‐KD are due to the decreased common chromatin‐binding of HDAC3 and Hs65K and subsequently reduced deacetylation of H3K9, illustrating a novel and conserved coupling mechanism that links the histone deacetylation with minor spliceosome for the regulation of gene expression.
Catalyzed by spliceosomes in the nucleus, RNA splicing removes intronic sequences from precursor RNAs in eukaryotes to generate mature RNA, which also significantly increases proteome complexity and fine‐tunes gene expression. Most metazoans have two coexisting spliceosomes; the major spliceosome, which removes >99.5% of introns, and the minor spliceosome, which removes far fewer introns (only 770 at present have been predicted in the human genome). Both spliceosomes are large and dynamic machineries, each consisting of five small nuclear RNAs (snRNAs) and more than 100 proteins. However, the dynamic assembly, catalysis, and protein composition of the minor spliceosome are still poorly understood. With different splicing signals, minor introns are rare and usually distributed alone and flanked by major introns in genes, raising questions of how they are recognized by the minor spliceosome and how their processing deals with the splicing of neighboring major introns. Due to large numbers of introns and close similarities between the two machinery, cooperative, and competitive recognition by the two spliceosomes has been investigated. Functionally, many minor‐intron‐containing genes are evolutionarily conserved and essential. Mutations in the minor spliceosome exhibit a variety of developmental defects in plants and animals and are linked to numerous human diseases. Here, we review recent progress in the understanding of minor splicing, compare currently known components of the two spliceosomes, survey minor introns in a wide range of organisms, discuss cooperation and competition of the two spliceosomes in splicing of minor‐intron‐containing genes, and contributions of minor splicing mutations in development and diseases.
This article is categorized under: RNA Processing > Processing of Small RNAs
RNA Processing > Splicing Mechanisms
RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry
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 .
