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Spliceosome assembly. The interactions of the spliceosomal snRNPs and some selected non-snRNP protein complexes at various stages of spliceosome assembly (complexes E, A, B*, and C) are depicted schematically for both the U2- and U12-dependent spliceosomes. The Prp19/CDC5 complex is indicated by ‘19C’. Its association with the U12-dependent spliceosome is inferred from the major spliceosome and is therefore indicated with a question mark. (Adapted with permission from Ref 27. Copyright 2003 Macmillan Publishers Ltd)
Source publication
The removal of non-coding sequences, introns, from the mRNA precursors is an essential step in eukaryotic gene expression. U12-type introns are a minor subgroup of introns, distinct from the major or U2-type introns. U12-type introns are present in most eukaryotes but only account for less than 0.5% of all introns in any given genome. They are proc...
Similar publications
The molecular mechanisms of exon definition and back-splicing are fundamental unanswered questions in pre-mRNA splicing. Here we report cryo-electron microscopy structures of the yeast spliceosomal E complex assembled on introns, providing a view of the earliest event in the splicing cycle that commits pre-mRNAs to splicing. The E complex architect...
Citations
... The core component of spliceosome is small nuclear ribonucleoproteins (snRNPs) consisting of snRNA and Sm protein or like Sm (LSm) proteins. The U1, U2, U4, U5, and U6 snRNPs regulate >99% of splicing events, while their variant, so-called minor spliceosome formed by the U11, U12, U4atac and U6atac, and U5 regulate the rest of splicing events [18,19]. snRNPs difference of the two spliceosomes result in two types of introns. ...
The completion of the draft and complete human genome has revealed that there are only around 20,000 genes encoding proteins. Nonetheless, these genes can generate eight times more RNA transcript isoforms, while this number is still growing with the accumulation of high-throughput RNA sequencing (RNA-seq) data. In general, over 90% of genes generate various RNA isoforms emerging from variations at the 5′ and 3′ ends, as well as different exon combinations, known as alternative transcription start site (TSS), alternative polyadenylation (APA), and alternative splicing (AS). In this chapter, our focus will be on introducing the significance of these three types of isoform variations in gene regulation and their underlying molecular mechanisms. Additionally, we will highlight the historical, current, and prospective technological advancements in elucidating isoform regulations, from both the computational side such as deep-learning-based artificial intelligence, and the experimental aspect such as the long-read third-generation sequencing (TGS).
... Due to distinct splice sites and branchpoints, U12-type introns are removed by separate splicing machinery, called the minor spliceosome. U12-type introns are a small subset (< 0.5%) of all introns and are often found in genes that have been attributed a crucial role in RNA processing and cell cycle regulation (Turunen et al. 2013). Although several genes with U12-type introns have been implicated in cancerogenesis, the functional consequences of their aberrant expression due to minor intron retention require further investigation. ...
Alternative splicing (AS) is a strictly regulated process that generates multiple mRNA variants from a single gene, thus contributing to proteome diversity. Transcriptome-wide sequencing studies revealed networks of functionally coordinated splicing events, which produce isoforms with distinct or even opposing functions. To date, several mechanisms of AS are deregulated in leukemic cells, mainly due to mutations in splicing and/or epigenetic regulators and altered expression of splicing factors (SFs). In this review, we discuss aberrant splicing events induced by mutations affecting SFs ( SF3B1 , U2AF1 , SRSR2 , and ZRSR2 ), spliceosome components ( PRPF8 , LUC7L2 , DDX41, and HNRNPH1 ), and epigenetic modulators ( IDH1 and IDH2 ). Finally, we provide an extensive overview of the biological relevance of aberrant isoforms of genes involved in the regulation of apoptosis (e. g. BCL-X , MCL-1 , FAS , and c-FLIP ), activation of key cellular signaling pathways ( CASP8 , MAP3K7 , and NOTCH2 ), and cell metabolism ( PKM ).
... A novel ESS which is responsible for PDCD1 exon 3 skipping has been identified in this research. Generally, the splicing of mRNA requires the recognition of short wellconserved splice sites at the exon-intron boundaries by the spliceosome [29]. In addition, the cis-acting regulatory elements (enhancers and silencers), located in exons or introns, can regulate the splicing choice between the nearby splice sites [30]. ...
Objective
Immunotherapy targeting programmed cell death 1 (PDCD1 or PD-1) and its ligands has shown remarkable promise and the regulation mechanism of PD-1 expression has received arising attention in recent years. PDCD1 exon 3 encodes the transmembrane domain and the deletion of exon 3 produces a soluble protein isoform of PD-1 (sPD-1), which can enhance immune response by competing with full-length PD-1 protein (flPD-1 or surface PD-1) on T cell surface. However, the mechanism of PDCD1 exon 3 skipping is unclear.
Methods
The online SpliceAid program and minigene expression system were used to analyze potential splicing factors involved in the splicing event of PDCD1 exon 3. The potential binding motifs of heterogeneous nuclear ribonucleoprotein K (HNRNPK) on exon 3 predicted by SpliceAid were mutated by site-directed mutagenesis technology, which were further verified by pulldown assay. Antisense oligonucleotides (ASOs) targeting the exonic splicing silencer (ESS) on PDCD1 exon 3 were synthesized and screened to suppress the skipping of exon 3. The alternative splicing of PDCD1 exon 3 was analyzed by semiquantitative reverse transcription PCR. Western blot and flow cytometry were performed to detect the surface PD-1 expression in T cells.
Results
HNRNPK was screened as a key splicing factor that promoted PDCD1 exon 3 skipping, causing a decrease in flPD-1 expression on T cell membrane and an increase in sPD-1 expression. Mechanically, a key ESS has been identified on exon 3 and can be bound by HNRNPK protein to promote exon 3 skipping. Blocking the interaction between ESS and HNRNPK with an ASO significantly reduced exon 3 skipping. Importantly, HNRNPK can promote exon 3 skipping of mouse Pdcd1 gene as well.
Conclusions
Our study revealed a novel evolutionarily conserved regulatory mechanism of PD-1 expression. The splicing factor HNRNPK markedly promoted PDCD1 exon 3 skipping by binding to the ESS on PDCD1 exon 3, resulting in decreased expression of flPD-1 and increased expression of sPD-1 in T cells.
... So far, two different spliceosomal complexes, the major and the minor spliceosome, can be distinguished, whereby each of them splices its own introns (3,20). The major spliceosome contains the following five snRNAs: U1, U2, U4, U5, and U6, while the minor spliceosome has the five snRNAs U11, U12, U4atac, U6atac, and U5 (21). Depending on the organism, the major (also named U2dependent) spliceosome excises approximately 99.5% of introns, while the minor (or U12-dependent) spliceosome excises about 0.5% of introns. ...
Alternative splicing (AS) is an important molecular biological mechanism regulated by complex mechanisms involving a plethora of cis and trans-acting elements. Furthermore, AS is tissue specific and altered in various pathologies, including infectious, inflammatory, and neoplastic diseases. Recently developed immuno-oncological therapies include monoclonal antibodies (mAbs) and chimeric antigen receptor (CAR) T cells targeting, among others, immune checkpoint (ICP) molecules. Despite therapeutic successes have been demonstrated, only a limited number of patients showed long-term benefit from these therapies with tumor entity-related differential response rates were observed. Interestingly, splice variants of common immunotherapeutic targets generated by AS are able to completely escape and/or reduce the efficacy of mAb- and/or CAR-based tumor immunotherapies. Therefore, the analyses of splicing patterns of targeted molecules in tumor specimens prior to therapy might help correct stratification, thereby increasing therapy success by antibody panel selection and antibody dosages. In addition, the expression of certain splicing factors has been linked with the patients’ outcome, thereby highlighting their putative prognostic potential. Outstanding questions are addressed to translate the findings into clinical application. This review article provides an overview of the role of AS in (tumor) diseases, its molecular mechanisms, clinical relevance, and therapy response.
... Our results highlight the role of the 3 stem-loop of U12 snRNA in the minor spliceosome assembly-disassembly cycle. The significance of the 5 end of the U12 snRNA has long been recognized due to its function in the BPS recognition and the interactions with the U6atac snRNA in the catalytic core of the minor spliceosome ( 70 ). In contrast, the 3 -terminal stem-loop of the U12 snRNA has appeared as a static binding site for the U11 / U12-65K protein, necessary for the formation of the U11 / U12 di-snRNP. ...
Here, we identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of U11/U12-65K, a known unique component of the U11/U12 di-snRNP. Both proteins use their highly similar C-terminal RRMs to bind to 3′-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with U11/U12-65K, which uses its N-terminal region to interact with U11 snRNP during intron recognition. Finally, while RBM41 knockout cells are viable, they show alterations in U12-type 3′ splice site usage. Together, our results highlight the role of the 3′-terminal stem-loop of U12 snRNA as a dynamic binding platform for the U11/U12-65K and RBM41 proteins, which function at distinct stages of the assembly/disassembly cycle.
... This dynamic stepwise process involves concerted actions by ribonucleoproteins and splicing factors to ensure a precise selection of intron-exon sequences and their subsequent enzymatic processing [14]. Specifically, 98% of introns are processed by the major spliceosome, while the remaining are spliced by the minor spliceosome, which share most of their components but differ in a limited set of U RNAs and accompanying splicing factors [15]. Interestingly, there is now ample evidence that alternative splicing is commonly dysregulated in all tumors and cancers examined [16,17], including pancreatic NENs and SCLC [18][19][20][21]. ...
Background
Lung neuroendocrine neoplasms (LungNENs) comprise a heterogeneous group of tumors ranging from indolent lesions with good prognosis to highly aggressive cancers. Carcinoids are the rarest LungNENs, display low to intermediate malignancy and may be surgically managed, but show resistance to radiotherapy/chemotherapy in case of metastasis. Molecular profiling is providing new information to understand lung carcinoids, but its clinical value is still limited. Altered alternative splicing is emerging as a novel cancer hallmark unveiling a highly informative layer.
Methods
We primarily examined the status of the splicing machinery in lung carcinoids, by assessing the expression profile of the core spliceosome components and selected splicing factors in a cohort of 25 carcinoids using a microfluidic array. Results were validated in an external set of 51 samples. Dysregulation of splicing variants was further explored in silico in a separate set of 18 atypical carcinoids. Selected altered factors were tested by immunohistochemistry, their associations with clinical features were assessed and their putative functional roles were evaluated in vitro in two lung carcinoid-derived cell lines.
Results
The expression profile of the splicing machinery was profoundly dysregulated. Clustering and classification analyses highlighted five splicing factors: NOVA1 , SRSF1 , SRSF10 , SRSF9 and PRPF8 . Anatomopathological analysis showed protein differences in the presence of NOVA1, PRPF8 and SRSF10 in tumor versus non-tumor tissue. Expression levels of each of these factors were differentially related to distinct number and profiles of splicing events, and were associated to both common and disparate functional pathways. Accordingly, modulating the expression of NOVA1, PRPF8 and SRSF10 in vitro predictably influenced cell proliferation and colony formation, supporting their functional relevance and potential as actionable targets.
Conclusions
These results provide primary evidence for dysregulation of the splicing machinery in lung carcinoids and suggest a plausible functional role and therapeutic targetability of NOVA1, PRPF8 and SRSF10.
... The spliceosome is a macromolecular nucleic acid protein complex assembled from a variety of small nuclear RNAs (snRNAs) and a variety of proteins, including small nuclear ribonucleoprotein particles (snRNPs). With the participation of SF3B1, SRSF2, U2AF1, etc., the introns of the pre-mRNA are removed, and the exons are spliced through two transesterification reactions so that the eukaryotic pre-mRNA becomes mature mRNA [51,52]. Spliceosome and RNA metabolism genes are common in various myeloid neoplasms. ...
Chronic neutrophilic leukemia (CNL) is a rare type of myeloproliferative neoplasm (MPN). Due to its nonspecific clinical symptoms and lack of specific molecular markers, it was previously difficult to distinguish it from other diseases with increased neutrophils. However, the discovery of the CSF3R mutation in CNL 10 years ago and the update of the diagnostic criteria by the World Health Organization (WHO) in 2016 brought CNL into a new era of molecular diagnosis. Next-generation sequencing (NGS) technology has led to the identification of numerous mutant genes in CNL. While CSF3R is commonly recognized as the driver mutation of CNL, other mutations have also been detected in CNL using NGS, including mutations in other signaling pathway genes (CBL, JAK2, NARS, PTPN11) and chromatin modification genes (ASXL1, SETBP1, EZH2), DNA methylation genes (DNMT3A, TET2), myeloid-related transcription factor genes (RUNX1, GATA2), and splicing and RNA metabolism genes (SRSF2, U2AF1). The coexistence of these mutated genes and CSF3R mutations, as well as the different evolutionary sequences of clones, deepens the complexity of CNL molecular biology. The purpose of this review is to summarize the genetic research findings of CNL in the last decade, focusing on the common mutated genes in CNL and their clinical significance, as well as the clonal evolution pattern and sequence of mutation acquisition in CNL, to provide a basis for the appropriate management of CNL patients.
... The process of splicing and its delicate regulation is carried out by the spliceosome, a ribonucleoproteic complex that recognizes specific RNA sequences to precisely localize the introns and cut them, and subsequently bind the adjacent exons [19]. In mammals, there are two different spliceosomes that act separately: the major spliceosome that processes more than 99% of the introns, and the minor spliceosome that acts over a small and specific set of introns [20]. Accordingly, introns are classified as U2-type (or -dependent, GT-AT) and U12-type (or -dependent, AT-AC), depending on the spliceosome that processes them or the flanking sequences [21]. ...
... Both spliceosomes consist of a main core of small nuclear RNAs (snRNAs), known as RNU1, RNU2, RNU4, RNU5 and RNU6 for the major spliceosome; and RNU11, RNU12, RNU4ATAC and RNU6A-TAC (RNU5 is present in both), for the minor. These snRNAs are joined to proteins forming small nuclear ribonucleoproteins (snRNP; U1-U6) [19,20]. In addition, the spliceosomes closely interact with the splicing factors, a diverse set of more than 300 molecules that complete the splicing machinery, helping the snRNPs to select and process the precise sequences, and taking part dynamically in every step of the process, participating in both general tasks as well as very specific events [22,23]. ...
... A case as paradigmatic as intricate is the study of CD44, a multifunctional cell surface glycoprotein involved in structural and functional roles in cell-cell and cell-matrix interactions. The standard isoform of CD44 (CD44s or CD44h) only contains the five first exons [1][2][3][4][5] and last five exons [16][17][18][19][20][21], while the alternative variants CD44v have variable exons (v1-v10) that are alternatively spliced and incorporated between the exons 5-16, conditioning its final structure and thus its biological role [86]. The CD44 variants CD44v2 and CD44v6, can be detected in human PDAC tissue by immunohistochemistry, where their expression is connected to an increase in mortality rate [87][88][89]. ...
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers worldwide, mainly due to its late diagnosis and lack of effective therapies, translating into a low 5-year 12% survival rate, despite extensive clinical efforts to improve outcomes. International cooperative studies have provided informative multiomic landscapes of PDAC, but translation of these discoveries into clinical advances are lagging. Likewise, early diagnosis biomarkers and new therapeutic tools are sorely needed to tackle this cancer. The study of poorly explored molecular processes, such as splicing, can provide new tools in this regard. Alternative splicing of pre-RNA allows the generation of multiple RNA variants from a single gene and thereby contributes to fundamental biological processes by finely tuning gene expression. However, alterations in alternative splicing are linked to many diseases, and particularly to cancer, where it can contribute to tumor initiation, progression, metastasis and drug resistance. Splicing defects are increasingly being associated with PDAC, including both mutations or dysregulation of components of the splicing machinery and associated factors, and altered expression of specific relevant gene variants. Such disruptions can be a key element enhancing pancreatic tumor progression or metastasis, while they can also provide suitable tools to identify potential candidate biomarkers and discover new actionable targets. In this review, we aimed to summarize the current information about dysregulation of splicing-related elements and aberrant splicing isoforms in PDAC, and to describe their relationship with the development, progression and/or aggressiveness of this dismal cancer, as well as their potential as therapeutic tools and targets.
... Our results highlight the role of the 3' stem-loop of U12 snRNA in the minor spliceosome assembly-disassembly cycle. The significance of the 5' end of the U12 snRNA has long been recognized due to its function in the BPS recognition and the interactions with the U6atac snRNA in the catalytic core of the minor spliceosome (Turunen et al., 2013). In contrast, the 3'-terminal stem-loop of the U12 snRNA has appeared as a static binding site for the U11/U12-65K protein, necessary for the formation of the U11/U12 di-snRNP. ...
In this work, we identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of the U11/U12-65K protein, a known unique component of the minor spliceosome that functions during the early steps of minor intron recognition as a component of the U11/U12 di-snRNP. We show that both proteins use their highly similar C-terminal RRMs to bind to 3'-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with the U11/U12-65K protein, which uses the N-terminal region to interact with U11 snRNP during the intron recognition step. Finally, we show that while RBM41 knockout cells are viable, they show alterations in the splicing of U12-type introns, particularly differential U12-type 3' splice site usage. Together, our results highlight the role 3'-terminal stem-loop of U12 snRNA as a dynamic binding platform for the paralogous U11/U12-65K and RBM41 proteins, which function at distinct stages of minor spliceosome assembly/disassembly cycle.
... Punctuated and dramatic loss of minor introns is a hallmark feature of the minor splicing landscape, and it remains an outstanding question why certain lineages undergo either partial or complete loss of their ancestral minor introns while others do not ( 71 ). Previous work has delineated many groups that appear to lack either minor introns, minor splicing components or both ( 14 , 23 , 34 ), but the diversity and scope of more recently-available data motivated us to revisit this topic. ...
Spliceosomal introns are gene segments removed from RNA transcripts by ribonucleoprotein machineries called spliceosomes. In some eukaryotes a second ‘minor’ spliceosome is responsible for processing a tiny minority of introns. Despite its seemingly modest role, minor splicing has persisted for roughly 1.5 billion years of eukaryotic evolution. Identifying minor introns in over 3000 eukaryotic genomes, we report diverse evolutionary histories including surprisingly high numbers in some fungi and green algae, repeated loss, as well as general biases in their positional and genic distributions. We estimate that ancestral minor intron densities were comparable to those of vertebrates, suggesting a trend of long-term stasis. Finally, three findings suggest a major role for neutral processes in minor intron evolution. First, highly similar patterns of minor and major intron evolution contrast with both functionalist and deleterious model predictions. Second, observed functional biases among minor intron-containing genes are largely explained by these genes’ greater ages. Third, no association of intron splicing with cell proliferation in a minor intron-rich fungus suggests that regulatory roles are lineage-specific and thus cannot offer a general explanation for minor splicing’s persistence. These data constitute the most comprehensive view of minor introns and their evolutionary history to date, and provide a foundation for future studies of these remarkable genetic elements.
