# Spliceosome Complex: Structure, Mechanism, and Function in RNA Splicing ## Introduction to the Spliceosome Complex ### What is the Spliceosome? The spliceosome is a large, dynamic ribonucleoprotein (RNP) machine that catalyzes the removal of introns from precursor messenger RNA (pre-mRNA) and the ligation of exons to form mature mRNA. It is one of the most complex macromolecular assemblies in eukaryotic cells, comparable in size to the ribosome. A functional spliceosome is composed of five small nuclear RNAs (snRNAs)—U1, U2, U4, U5, and U6—and more than 100 associated proteins. These components assemble *de novo* on each intron in a highly ordered, stepwise manner, undergo extensive conformational rearrangements, and disassemble after a single round of splicing. The spliceosome is not a pre-formed, static enzyme. Rather, it is assembled anew on every intron that is spliced, a feature that distinguishes it from most other catalytic machines. This assembly–disassembly cycle is energy-intensive, requiring hydrolysis of ATP at multiple steps. The catalytic core of the spliceosome is formed by RNA—specifically U2 and U6 snRNAs—which positions the spliceosome within the class of ribozymes, albeit a ribozyme that requires a large protein scaffold for activity. For a detailed molecular map of the complex, see [Spliceosome Structure](/knowledge/molecular-biology/spliceosome-structure). ### Why Splicing Matters In eukaryotic genomes, protein-coding genes are interrupted by non-coding sequences called introns. The average human gene contains roughly 8 introns, and introns account for approximately 25% of the human genome. If introns were retained in mRNA, translation would produce aberrant, non-functional proteins. Splicing therefore is an essential step in gene expression. Beyond simple intron removal, splicing enables **[alternative splicing](/blog/guides/alternative-splicing)**, a process by which a single gene can produce multiple mRNA isoforms through differential exon inclusion or exclusion. It is estimated that over 95% of human multi-exon genes undergo alternative splicing, vastly expanding the coding capacity of the genome. Splicing also plays a role in mRNA export, nonsense-mediated decay, and translational regulation through the deposition of exon junction complexes. Defects in splicing are linked to numerous human diseases, including spinal muscular atrophy, retinitis pigmentosa, and many cancers. Understanding the spliceosome is therefore central to molecular biology, genetics, and medicine. ## Components of the Spliceosome ### snRNPs: U1, U2, U4/U6, U5 The core of the spliceosome is formed by five small nuclear ribonucleoprotein particles (snRNPs). Each snRNP consists of one (or two) snRNA molecules, a set of seven common Sm or LSm proteins, and particle-specific proteins. The snRNAs are 100–300 nucleotides long and are highly conserved across eukaryotes. **U1 snRNP.** U1 snRNA is approximately 164 nucleotides long in humans. Its 5′ end contains a sequence complementary to the 5′ splice site consensus sequence (AG/GURAGU, where R is a purine). The U1 snRNP recognizes and base-pairs with the 5′ splice site during the earliest steps of assembly. U1-specific proteins include U1-70K, U1-A, and U1-C. **U2 snRNP.** U2 snRNA (187 nucleotides in humans) base-pairs with the branch point sequence (BPS), which has the consensus YNYURAY (Y = pyrimidine, N = any nucleotide, R = purine) in humans. The bulged adenosine at the branch point is the nucleophile for the first transesterification reaction. U2-specific proteins include U2-A′, U2-B″, and the SF3a and SF3b complexes, which stabilize the U2–branch point interaction. **U4/U6 snRNP.** U4 and U6 snRNAs (144 and 106 nucleotides, respectively, in humans) exist as a single di-snRNP particle held together by extensive base-pairing between their complementary regions. U6 is the catalytically essential RNA; it base-pairs with U2 to form the catalytic core. U4 acts as a chaperone, sequestering U6 in an inactive conformation. During catalytic activation, U4 is displaced, allowing U6 to refold into its active structure. U4/U6-specific proteins include hPrp3, hPrp4, and hPrp31. **U5 snRNP.** U5 snRNA (116 nucleotides in humans) is highly conserved and participates in aligning the two exons for ligation. It base-pairs with exon sequences at both the 5′ and 3′ splice sites. U5-specific proteins include Prp8, Brr2, and Smu114 (in yeast) or hPrp8, hBrr2, and hSnu114 (in humans). Prp8 is the largest and most highly conserved spliceosomal protein, and it forms the structural heart of the catalytic core. For a comprehensive list of snRNP compositions and their protein components, see [Spliceosome Composed](/knowledge/molecular-biology/spliceosome-composed). ### Protein Factors and Accessory Proteins In addition to the core snRNPs, the spliceosome contains numerous non-snRNP proteins that are required for assembly, catalysis, and regulation. These include: - **DExD/H-box RNA helicases.** These enzymes use ATP hydrolysis to remodel RNA–RNA and RNA–protein interactions. Key examples include Prp5 (U2–branch point stabilization), Prp28 (U1–5′ splice site destabilization), Brr2 (U4/U6 unwinding), Prp2 (activation of the B* complex), Prp16 (proofreading of step 1), Prp22 (mRNA release), and Prp43 (disassembly). - **Prp19/CDC5 complex (NTC).** A large protein complex that associates with the spliceosome during activation and is required for stable U5/U6 association and catalytic activity. - **SR proteins and hnRNPs.** These are splicing regulators that bind to exonic and intronic splicing enhancers or silencers (discussed in Section 5). - **Cap-binding complex (CBC)** and **U2AF (U2 auxiliary factor).** These proteins facilitate early [spliceosome assembly](/knowledge/molecular-biology/spliceosome-assembly) by recognizing the 5′ cap and the polypyrimidine tract, respectively. The total protein content of the assembled spliceosome exceeds 150 distinct polypeptides, many of which are present in multiple copies. The [Spliceosome Proteins](/knowledge/molecular-biology/spliceosome-proteins) resource provides a detailed inventory of these factors and their functions. ## [Spliceosome Assembly](/knowledge/molecular-biology/spliceosome-assembly) and Disassembly Cycle ### Early Complexes: E, A, and B Spliceosome assembly proceeds through a series of defined complexes, each characterized by a distinct set of associated factors. The assembly is ordered and ATP-dependent at several steps. **Complex E (early/commitment complex).** Assembly begins with the recognition of the 5′ splice site by U1 snRNP via base-pairing. Concurrently, the splicing factor U2AF65 binds the polypyrimidine tract downstream of the branch point, and U2AF35 binds the AG dinucleotide at the 3′ splice site. The branch point is also recognized by the SF1/mBBP protein. This complex is ATP-independent and commits the pre-mRNA to the splicing pathway. **Complex A (pre-spliceosome).** In an ATP-dependent reaction catalyzed by the helicase Prp5, U2 snRNP stably associates with the branch point. U2 base-pairs with the BPS, bulging out the reactive adenosine. This interaction is stabilized by the SF3a and SF3b complexes. At this stage, the 5′ splice site is still held by U1, and the branch point is engaged by U2. **Complex B (pre-catalytic spliceosome).** The U4/U6·U5 tri-snRNP joins the complex. This large particle is pre-assembled in the nucleoplasm and recruited to complex A through protein–protein interactions. The resulting B complex contains all five snRNPs but is not yet catalytically active. U4 still base-pairs with U6, keeping U6 in an inactive conformation. ### Catalytic Activation and C Complex **B activation.** The transition from B to the activated B* complex requires the action of several RNA helicases. Prp28 destabilizes the U1–5′ splice site interaction, allowing U6 to base-pair with the 5′ splice site. Brr2 unwinds the U4/U6 duplex, releasing U4 and allowing U6 to refold. U6 then base-pairs with U2 to form the catalytic core, and U5 aligns the 5′ exon. The Prp19/CDC5 complex (NTC) associates and stabilizes these rearrangements. This step is ATP-dependent and irreversible. **B* to C complex.** The catalytically active B* complex performs the first transesterification reaction, producing the C complex. The C complex contains the lariat intron–3′ exon intermediate and the free 5′ exon. The helicase Prp16 then proofreads the first step and promotes conformational changes required for the second step. ### Disassembly and Recycling After the second transesterification reaction, the mature mRNA is released. The helicase Prp22 mediates mRNA release by unwinding the U5–exon interaction. The remaining intron–lariat complex is disassembled by Prp43, which unwinds the U2/U6 interaction and releases the snRNPs for recycling. The lariat intron is debranched by the enzyme Dbr1 and degraded. The entire assembly–disassembly cycle is summarized in the [Spliceosome Assembly](/knowledge/molecular-biology/spliceosome-assembly) pathway, which details the order of factor addition and removal. ## The Splicing Mechanism: Two Transesterification Steps ### Step 1: Branch Point Attack The chemistry of splicing is a two-step transesterification reaction, meaning that phosphodiester bonds are broken and formed in a coordinated manner without net hydrolysis. **Step 1: 5′ splice site cleavage and lariat formation.** The 2′-hydroxyl of the bulged adenosine at the branch point performs a nucleophilic attack on the phosphate at the 5′ splice site. This breaks the phosphodiester bond between the 5′ exon and the intron, producing two species: (1) the free 5′ exon with a 3′-hydroxyl group, and (2) the intron–3′ exon intermediate, in which the 5′ end of the intron is covalently linked to the branch point adenosine via a 2′–5′ phosphodiester bond. This branched structure is called a **lariat**. The catalytic core for this reaction is formed by U2 and U6 snRNAs. U6 positions a divalent metal ion (typically Mg²⁺) that coordinates the leaving group and stabilizes the transition state. The reaction is analogous to the first step of group II intron self-splicing, supporting the hypothesis that the spliceosome is an RNA-catalyzed machine. For a discussion of this evolutionary and mechanistic relationship, see [Spliceosome a Ribozyme](/knowledge/molecular-biology/spliceosome-a-ribozyme). ### Step 2: Exon Ligation **Step 2: 3′ splice site cleavage and exon ligation.** The 3′-hydroxyl of the free 5′ exon performs a nucleophilic attack on the phosphate at the 3′ splice site. This breaks the phosphodiester bond between the intron and the 3′ exon, and simultaneously forms a new phosphodiester bond between the 5′ and 3′ exons. The intron is released as a lariat and subsequently degraded. The second step requires the helicase Prp16, which promotes a conformational rearrangement after step 1. The U5 snRNA base-pairs with exon sequences at both the 5′ and 3′ splice sites, aligning the two exons for ligation. The protein Prp8, which contacts both the branch point and the 5′ splice site, also plays a critical role in positioning the substrates. The overall reaction can be written as: 1. 5′ exon–intron–3′ exon → 5′ exon-OH + intron-lariat–3′ exon 2. 5′ exon-OH + intron-lariat–3′ exon → 5′ exon–3′ exon + intron-lariat Both steps are reversible *in vitro*, but in the cell they are driven forward by the conformational changes and ATP hydrolysis that accompany each step. ## Alternative Splicing and Spliceosome Regulation ### Splice Site Selection The spliceosome must identify the correct 5′ and 3′ splice sites among a vast excess of decoy or cryptic sites. In humans, the average exon is only ~150 nucleotides long, while introns can be tens of thousands of nucleotides. The spliceosome achieves this specificity through a combination of weak, degenerate sequence motifs and a large network of regulatory proteins. The three core sequence elements are: 1. **5′ splice site:** consensus AG/GURAGU (the slash indicates the cleavage site) 2. **Branch point sequence:** YNYURAY (the underlined A is the branch point adenosine) 3. **3′ splice site:** a polypyrimidine tract followed by AG These sequences are recognized by U1 snRNP, U2 snRNP (with SF1 and U2AF), and U2AF35, respectively. However, these consensus sequences are degenerate, and many splice sites match the consensus poorly. This "weakness" is exploited by the cell: it allows regulation, but it also means that the spliceosome must be actively guided to the correct sites. ### Regulatory Elements: ESEs, ESSs, ISEs, ISSs Splicing regulation is mediated by *cis*-acting RNA elements and *trans*-acting protein factors. The cis-elements are classified by their location and effect: - **Exonic Splicing Enhancers (ESEs):** short sequences within exons that promote splicing. - **Exonic Splicing Silencers (ESSs):** sequences within exons that repress splicing. - **Intronic Splicing Enhancers (ISEs):** sequences within introns that promote splicing. - **Intronic Splicing Silencers (ISSs):** sequences within introns that repress splicing. These elements are typically 6–8 nucleotides long and are recognized by specific [RNA-binding proteins](/knowledge/molecular-biology/rna-binding-protein). ### SR Proteins and hnRNPs The two major families of *trans*-acting splicing regulators are: **SR proteins (Serine/Arginine-rich proteins).** These proteins contain one or two RNA recognition motifs (RRMs) and a C-terminal domain rich in arginine–serine dipeptides (the RS domain). SR proteins generally bind to ESEs and promote splicing by recruiting U1 snRNP to the 5′ splice site and U2AF to the 3′ splice site. They also promote exon definition, the process by which the spliceosome recognizes exons across short exon–long intron boundaries. Examples include SF2/ASF (SRSF1), SC35 (SRSF2), and SRp20 (SRSF3). **hnRNP proteins (heterogeneous nuclear ribonucleoproteins).** These proteins generally bind to ESSs and ISSs and repress splicing. They can antagonize SR proteins by competing for overlapping binding sites or by promoting the formation of inactive spliceosome complexes. Examples include hnRNP A1, hnRNP I (PTB), and hnRNP H. The balance between SR proteins and hnRNPs at any given splice site determines whether that site is used. This balance varies between cell types, developmental stages, and in response to cellular signals, giving rise to cell-type-specific alternative splicing patterns. ## Methods to Study the Spliceosome ### Genetic Screens in Yeast The budding yeast *Saccharomyces cerevisiae* has been instrumental in spliceosome research. Yeast is a powerful genetic system because it is haploid, has a short generation time, and its splicing machinery is highly conserved with humans. The first spliceosomal genes were identified through **temperature-sensitive (ts) mutants** that blocked pre-mRNA splicing at the non-permissive temperature (typically 37°C). These mutants were named *prp* (pre-mRNA processing) mutants. The *PRP* genes were later cloned and shown to encode RNA helicases, snRNP proteins, and other splicing factors. Genetic screens also identified *cis*-acting mutations in the 5′ splice site, branch point, and 3′ splice site that suppressed or enhanced splicing defects, defining the sequence requirements for splicing. Suppressor screens, in which second-site mutations restored splicing in a *prp* mutant background, identified RNA–RNA and RNA–protein interactions critical for spliceosome function. ### In Vitro Splicing Assays The development of **cell-free splicing extracts** in the 1980s was a landmark achievement. Nuclear extracts from HeLa cells (or whole-cell extracts from yeast) can splice exogenous pre-mRNA substrates *in vitro*. The standard assay uses a radiolabeled pre-mRNA substrate, typically derived from adenovirus major late (AdML) or β-globin genes, incubated in extract supplemented with ATP, MgCl₂, and creatine phosphate (an ATP-regenerating system). Reactions are incubated at 30°C for 60–90 minutes, and products are analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. This assay allowed researchers to: - Define the order of spliceosome assembly by native gel electrophoresis (complexes E, A, B, C). - Test the effects of mutations in the pre-mRNA or depletion of specific factors. - Perform kinetic analyses of the two catalytic steps. - Identify the ATP requirements at each step using non-hydrolyzable ATP analogs such as AMP-PNP. ### Cryo-Electron Microscopy and [X-ray Crystallography](/knowledge/molecular-biology/x-ray-crystallography) Structural biology has provided atomic-level views of the spliceosome. Because the spliceosome is large (~2–5 MDa) and conformationally dynamic, X-ray crystallography was initially limited to individual components, such as the U1 snRNP or the Prp8 protein. The breakthrough came with **cryo-electron microscopy (cryo-EM)**, which can determine structures of large, heterogeneous complexes without crystallization. Since 2015, cryo-EM structures have been determined for nearly every major spliceosomal complex from yeast and humans, including the B, B*, C, C*, and P complexes, at resolutions of 3–6 Å. These structures revealed: - The RNA catalytic core, with U2/U6 forming a triple-helix that coordinates two Mg²⁺ ions. - The role of Prp8 as a central scaffold that contacts the 5′ splice site, branch point, and 3′ splice site. - The conformational changes that occur between the first and second catalytic steps. - The mechanism of U4/U6 unwinding and U6 refolding during activation. These structures have transformed our understanding of splicing chemistry and are reviewed in detail in [Spliceosome Structure](/knowledge/molecular-biology/spliceosome-structure). ## Spliceosome Dysfunction and Human Disease ### Spliceosomopathies Mutations in core spliceosomal components cause a class of diseases known as **spliceosomopathies**. These are typically autosomal dominant disorders caused by haploinsufficiency or dominant-negative effects of mutant proteins. **Retinitis pigmentosa (RP).** Mutations in the genes encoding the U4/U6-associated proteins PRPF3, PRPF4, PRPF6, PRPF8, PRPF31, and SNRNP200 (Brr2) cause autosomal dominant retinitis pigmentosa, a degenerative disease of the retina. Why the retina is specifically affected is not fully understood, but it may reflect the high metabolic demand and splicing load of photoreceptor cells. **Spinal muscular atrophy (SMA).** SMA is caused by homozygous deletion or mutation of the *SMN1* gene. The SMN (survival of motor neuron) protein is required for the assembly of Sm core domains onto snRNAs. Loss of SMN leads to reduced snRNP levels, particularly affecting motor neurons. The related *SMN2* gene produces a partially functional protein but is inefficiently spliced due to an ESE mutation, resulting in exon 7 skipping. This makes SMA a disease of both snRNP biogenesis and splicing regulation. **Other spliceosomopathies.** Mutations in U2 snRNP components (SF3B1, U2AF1, ZRSR2) are recurrent in myelodysplastic syndromes (MDS) and chronic lymphocytic leukemia (CLL). These mutations alter splice site choice and are thought to contribute to pathogenesis through mis-splicing of genes involved in hematopoiesis. ### Cancer and Splicing Mutations Splicing dysregulation is a hallmark of cancer. This can arise through: 1. **Mutations in spliceosomal genes.** As noted, SF3B1, U2AF1, and SRSF2 are frequently mutated in hematologic malignancies. These mutations are often "hotspot" mutations that alter the RNA-binding specificity of the protein, leading to widespread changes in 3′ splice site choice. 2. **Mutations in cis-regulatory elements.** Mutations that create or destroy ESEs or ESSs can alter the splicing of [tumor suppressor genes](/knowledge/molecular-biology/tumor-suppressor-gene) or oncogenes. For example, mutations in the *TP53* gene can create cryptic splice sites that lead to loss of function. 3. **Altered expression of splicing factors.** Many tumors overexpress SR proteins such as SRSF1, which can drive the inclusion of alternative exons in genes that promote proliferation or invasion. 4. **Aberrant alternative splicing.** Tumors often express mRNA isoforms that are not present in normal tissues. These can encode truncated proteins with dominant-negative or oncogenic activity. A classic example is the CD44 gene, which undergoes complex alternative splicing to produce isoforms that promote metastasis. The development of splicing-modulating drugs, such as the SF3B1 inhibitor pladienolide B and the SMN2-splicing modifier risdiplam (approved for SMA), highlights the therapeutic potential of targeting the spliceosome. ## Common Pitfalls and Study Tips for Students ### Misconception: Spliceosome is a Static Enzyme A common error is to think of the spliceosome as a fixed complex that binds pre-mRNA, performs catalysis, and releases product, like a restriction enzyme. In reality, the spliceosome is **assembled de novo on every intron**. It is a dynamic machine that undergoes at least eight distinct conformational states, each requiring ATP hydrolysis. The U4/U6 snRNP, for example, is not present in the catalytically active complex—it is a delivery vehicle for U6. Students should trace the assembly and disassembly cycle step by step, noting which factors are present at each stage. ### Remembering the snRNP Names and Roles A reliable mnemonic for the snRNPs is their order of action: **U1, U2, U4/U6, U5**. Remember: - **U1** binds the **5′ splice site** (1 = first to bind). - **U2** binds the **branch point** (2 = second). - **U4/U6** is a **duplex** that must be **unwound** for activation. - **U5** holds the **exons** together for ligation. The catalytic core is **U2 + U6**, not U1 or U5. U6 is the catalytic RNA, and U2 provides the branch point helix. U4 is a chaperone that is released before catalysis. ### Visualizing the Splicing Cycle Students often struggle with the conformational rearrangements. It helps to draw the cycle as a circular diagram: 1. U1 binds 5′ SS → E complex 2. U2 binds BPS → A complex 3. U4/U6·U5 joins → B complex 4. U4 released, U6 refolds → B* complex 5. Step 1 catalysis → C complex 6. Step 2 catalysis → P complex 7. mRNA released, snRNPs recycled Label each step with the ATPase that drives it (Prp28, Brr2, Prp2, Prp16, Prp22, Prp43). This will help you connect the biochemistry to the structural transitions. ## Frequently Asked Questions ### What is the spliceosome complex? The spliceosome is a large, dynamic ribonucleoprotein machine that removes introns from pre-mRNA and ligates exons to produce mature mRNA. It is composed of five snRNPs (U1, U2, U4/U6, U5) and over 100 auxiliary proteins. It assembles anew on each intron and is catalytically driven by RNA (U2/U6), making it a ribozyme. See [Spliceosome Definition](/knowledge/molecular-biology/spliceosome-definition) for a concise overview. ### What are the main components of the spliceosome? The main components are the five snRNPs: U1 (recognizes the 5′ splice site), U2 (recognizes the branch point), U4/U6 (a duplex that delivers U6), and U5 (aligns exons). Each snRNP contains an snRNA and associated proteins. The catalytic core is formed by U2 and U6 snRNAs. Non-snRNP proteins include DExD/H-box helicases, the Prp19/CDC5 complex, and regulatory SR proteins and hnRNPs. For a full component list, see [Spliceosome Made](/knowledge/molecular-biology/spliceosome-made). ### How does the spliceosome recognize splice sites? Splice sites are recognized through base-pairing between snRNAs and the pre-mRNA, and through protein–RNA interactions. U1 snRNA base-pairs with the 5′ splice site; U2 snRNA base-pairs with the branch point; U2AF65 recognizes the polypyrimidine tract; and U2AF35 recognizes the 3′ splice site AG. These interactions are weak and degenerate, requiring auxiliary factors (SR proteins, hnRNPs) to ensure accurate recognition. ### What is the role of ATP in splicing? ATP is required for the conformational rearrangements that drive spliceosome assembly, activation, and disassembly. It is hydrolyzed by DExD/H-box RNA helicases, including Prp5 (U2 binding), Prp28 (U1 release), Brr2 (U4/U6 unwinding), Prp2 (B* activation), Prp16 (step 1 proofreading), Prp22 (mRNA release), and Prp43 (disassembly). ATP is not directly involved in the transesterification chemistry, which is driven by Mg²⁺ ions. ### What is alternative splicing? Alternative splicing is the process by which a single pre-mRNA can be spliced in multiple ways, producing different mRNA isoforms from the same gene. This is achieved through the differential use of 5′ splice sites, 3′ splice sites, or entire exons. It is regulated by cis-acting elements (ESEs, ESSs, ISEs, ISSs) and trans-acting factors (SR proteins and hnRNPs). Over 95% of human multi-exon genes undergo alternative splicing. ### Why is the spliceosome important in human disease? Mutations in spliceosomal components or in splicing regulatory elements cause or contribute to numerous diseases. These include retinitis pigmentosa (mutations in U4/U6-associated proteins), spinal muscular atrophy (SMN1 mutations), myelodysplastic syndromes (SF3B1, U2AF1 mutations), and many cancers. Splicing-modulating drugs are being developed to treat these conditions. ### How is the spliceosome studied experimentally? The spliceosome is studied using genetic screens in yeast (temperature-sensitive *prp* mutants), in vitro splicing assays with nuclear extracts, and structural biology techniques such as cryo-electron microscopy. These approaches have defined the assembly pathway, the catalytic mechanism, and the atomic structure of the spliceosome at multiple stages. ## Key Takeaways - The spliceosome is a dynamic ribonucleoprotein machine that assembles de novo on each intron and catalyzes intron removal via two transesterification reactions. - It is composed of five snRNPs (U1, U2, U4/U6, U5) and over 100 proteins; the catalytic core is formed by U2 and U6 snRNAs, making the [spliceosome a ribozyme](/knowledge/molecular-biology/spliceosome-a-ribozyme). - Assembly proceeds through defined complexes (E, A, B, B*, C, P) and requires ATP hydrolysis by DExD/H-box helicases at multiple steps. - The two catalytic steps are: (1) branch point adenosine attacks the 5′ splice site to form a lariat intermediate; (2) the free 5′ exon attacks the 3′ splice site to ligate exons and release the intron lariat. - Alternative splicing is regulated by cis-elements (ESEs, ESSs, ISEs, ISSs) and trans-factors (SR proteins, hnRNPs), enabling >95% of human genes to produce multiple mRNA isoforms. - Spliceosome dysfunction causes spliceosomopathies (retinitis pigmentosa, SMA) and contributes to cancer through mutations in splicing factors or regulatory elements. - Key experimental methods include yeast genetics, in vitro splicing assays, and cryo-EM structural biology, which together have revealed the molecular mechanism of splicing. ## Further Reading - Deutsch HM, Song Y, Li D. *Spliceosome complex and neurodevelopmental disorders*. Current opinion in genetics & development. 2025. [PubMed 40378521](https://doi.org/10.1016/j.gde.2025.102358) - Nilsen TW. *The spliceosome: the most complex macromolecular machine in the cell?*. BioEssays : news and reviews in molecular, cellular and [developmental biology](/blog/careers/developmental-biology). 2003. [PubMed 14635248](https://doi.org/10.1002/bies.10394) - Larsen NA. *The SF3b Complex is an Integral Component of the Spliceosome and Targeted by Natural Product-Based Inhibitors*. Sub-cellular biochemistry. 2021. [PubMed 33252738](https://doi.org/10.1007/978-3-030-58971-4_12) - Neubauer G. et al. *Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex*. Nature Genetics. 1998. [DOI 10.1038/1700](https://doi.org/10.1038/1700) - Michaud S. et al. *An ATP-independent complex commits pre-mRNA to the mammalian spliceosome assembly pathway*. Genes and Development. 1991. [DOI 10.1101/gad.5.12b.2534](https://doi.org/10.1101/gad.5.12b.2534)
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