# [Spliceosome Composition](/knowledge/molecular-biology/spliceosome-composed): What the Spliceosome Is Made Of ## Introduction to the Spliceosome The spliceosome is a large, dynamic ribonucleoprotein (RNP) complex that catalyzes the removal of introns from precursor messenger RNA (pre-mRNA) and the ligation of exons to produce mature mRNA. This process, termed pre-mRNA splicing, is an essential step in eukaryotic gene expression. The spliceosome is not a single static entity but rather a collection of distinct complexes that assemble anew on each intron, perform the two transesterification reactions required for splicing, and then disassemble. In human cells, the spliceosome is composed of five small nuclear RNAs (snRNAs) and more than 150 distinct proteins, making it one of the most complex macromolecular machines in the cell. Its composition is remarkably conserved across eukaryotes, from yeast to humans, underscoring its fundamental importance. The spliceosome is often compared to the ribosome because both are large RNA-protein complexes that catalyze essential RNA processing reactions. However, the spliceosome differs in a critical way: while the ribosome is a stable structure that remains intact through many rounds of translation, the spliceosome is assembled *de novo* on each intron and undergoes extensive compositional rearrangements during its functional cycle. Understanding what the spliceosome is made of—its RNA components, its protein components, and how they are organized—is central to understanding how splicing is regulated and how mutations in splicing factors cause human disease. ## Core Components: snRNAs and snRNPs ### The Five snRNAs The spliceosome is built around five small nuclear RNAs, designated U1, U2, U4, U5, and U6 snRNA. These RNAs range in length from approximately 100 to 300 nucleotides in humans. Each snRNA performs a specific role in intron recognition and catalysis. U1 snRNA base-pairs with the 5′ splice site of the intron. U2 snRNA base-pairs with the branch point sequence, which contains the adenosine that initiates the first transesterification reaction. U4 and U6 snRNAs form an extensive base-paired complex that is disrupted during spliceosome activation; U6 then base-pairs with U2 and with the 5′ splice site to form the catalytic core. U5 snRNA aligns the two exons for ligation by interacting with exon sequences at both the 5′ and 3′ splice sites. The snRNAs are transcribed by RNA polymerase II (except U6, which is transcribed by RNA polymerase III) and undergo extensive post-transcriptional modification, including 2′-O-methylation, pseudouridylation, and the addition of a 5′ cap structure. U1, U2, U4, and U5 snRNAs carry a trimethylguanosine cap, while U6 snRNA has a γ-monomethyl phosphate cap. These modifications and cap structures are important for snRNA stability, nuclear import, and association with specific proteins. ### snRNP Structure Each snRNA associates with a set of proteins to form a small nuclear ribonucleoprotein particle (snRNP, pronounced "snurp"). The snRNPs are the fundamental building blocks of the spliceosome. Each snRNP contains a core of seven Sm proteins (B/B′, D1, D2, D3, E, F, and G) that assemble into a ring around a conserved sequence element called the Sm site, which is present in U1, U2, U4, and U5 snRNAs. U6 snRNA lacks an Sm site and instead associates with seven structurally related LSm (like-Sm) proteins (LSm2–LSm8) that form a similar ring. In addition to the Sm core, each snRNP contains specific proteins unique to that particle. The U1 snRNP contains three U1-specific proteins: U1-70K, U1A, and U1C. The U2 snRNP contains U2A′, U2B″, and the SF3a and SF3b complexes, which together comprise more than ten proteins. The U4/U6.U5 tri-snRNP is a pre-assembled complex containing the U4, U6, and U5 snRNAs along with more than twenty proteins, including Prp31, Prp3, Prp4, and the U5-specific proteins Prp8, Brr2, and Prp6. Prp8 is the largest and most highly conserved protein in the spliceosome, with a mass of approximately 280 kDa in humans. The assembly of snRNPs is a highly ordered process. The Sm proteins are assembled onto the Sm site of the snRNA by the survival of motor neuron (SMN) complex in the cytoplasm, a process that requires the methyltransferase Tgs1 to hypermethylate the 5′ cap. The assembled snRNP is then imported into the nucleus, where it undergoes further maturation. The biogenesis of snRNPs is a quality-controlled process; improperly assembled particles are degraded. ## Protein Composition of the Spliceosome ### Sm and LSm Proteins The Sm proteins are a family of seven evolutionarily conserved proteins (B/B′, D1, D2, D3, E, F, G) that form a heteroheptameric ring around the Sm site of U1, U2, U4, and U5 snRNAs. Each Sm protein contains a conserved Sm domain, a fold consisting of an N-terminal helix followed by a five-stranded antiparallel β-sheet. The Sm ring binds the single-stranded Sm site RNA in a sequence-specific manner, with the RNA passing through the central hole of the ring. The Sm proteins are essential for snRNP stability, nuclear localization, and interactions with other splicing factors. The LSm proteins are structurally related to the Sm proteins but assemble around U6 snRNA. The LSm2–LSm8 ring binds the 3′ end of U6 snRNA, protecting it from exonuclease degradation and stabilizing the U6 snRNA. LSm proteins also play roles in other RNA processing pathways, including mRNA decapping and degradation, reflecting the broad functional versatility of the Sm/LSm protein fold. ### Non-snRNP Splicing Factors Beyond the snRNPs, the spliceosome contains numerous non-snRNP proteins that are not stably associated with snRNAs but are recruited to the spliceosome at specific stages of assembly. These proteins include the serine/arginine-rich (SR) proteins, which are essential for exon definition and splice site recognition; the heterogeneous nuclear ribonucleoproteins (hnRNPs), which generally antagonize SR protein function; and a large number of ATP-dependent RNA helicases that remodel the spliceosome. The SR proteins, such as SRSF1 (SF2/ASF) and SRSF2 (SC35), contain one or two RNA recognition motifs (RRMs) and a C-terminal arginine/serine-rich (RS) domain. They bind to exonic splicing enhancers (ESEs) and promote [spliceosome assembly](/knowledge/molecular-biology/spliceosome-assembly) by recruiting U1 snRNP to the 5′ splice site and U2 snRNP to the branch point. The RS domain is extensively phosphorylated by SR protein kinases, such as SRPK1 and Clk/Sty, and this phosphorylation regulates SR protein localization and activity. The RNA helicases are particularly important for the dynamic rearrangements that occur during [spliceosome assembly](/knowledge/molecular-biology/spliceosome-assembly) and catalysis. These enzymes use the energy of ATP hydrolysis to unwind RNA duplexes, disrupt RNA-protein interactions, and remodel the spliceosome. Key helicases include Prp5 (which promotes U2 snRNP recruitment), Prp28 (which destabilizes U1 snRNP binding to the 5′ splice site), Brr2 (which unwinds the U4/U6 duplex), Prp2 (which remodels the B* complex before catalysis), Prp16 (which promotes the first transesterification reaction), Prp22 (which releases the mRNA after the second reaction), and Prp43 (which disassembles the spliceosome). In human cells, these helicases are often part of larger complexes, such as the nineteen complex (NTC) and the NTC-related complex, which contain additional proteins required for catalytic activation. The total number of proteins in the human spliceosome is estimated at approximately 150, though this number varies depending on the purification method and the stage of the splicing reaction. The protein composition is not static; it changes dramatically as the spliceosome progresses through its assembly, catalytic, and disassembly phases. ## Assembly and Disassembly of the Spliceosome ### Assembly Pathway The spliceosome assembles on pre-mRNA in a stepwise manner, passing through several biochemically distinct complexes. The assembly pathway is highly ordered and requires ATP hydrolysis at multiple steps. 1. **E complex (early complex or commitment complex):** U1 snRNP base-pairs with the 5′ splice site through complementarity between the 5′ end of U1 snRNA and the intron sequence. Concurrently, the branch point binding protein (SF1 in humans, BBP in yeast) binds the branch point, and the U2 auxiliary factor (U2AF) binds the polypyrimidine tract and the 3′ splice site. This complex is ATP-independent and commits the pre-mRNA to the splicing pathway. 2. **A complex (pre-spliceosome):** U2 snRNP is recruited to the branch point in an ATP-dependent manner, requiring the helicase Prp5. U2 snRNA base-pairs with the branch point sequence, bulging out the branch point adenosine. This base-pairing is stabilized by the SF3a and SF3b proteins, which protect the U2-branch point interaction. 3. **B complex (pre-catalytic spliceosome):** The pre-assembled U4/U6.U5 tri-snRNP joins the A complex. This step requires the helicase Prp28, which destabilizes U1 snRNP binding to the 5′ splice site, allowing U6 snRNA to take over this interaction. The B complex contains all five snRNPs and is catalytically inactive. 4. **B* complex (activated spliceosome):** The helicase Brr2 unwinds the U4/U6 duplex, releasing U4 snRNP from the complex. This triggers a major conformational rearrangement in which U6 snRNA refolds to form the catalytic core with U2 snRNA. The NTC and NTC-related complexes are recruited, and the helicase Prp2 remodels the complex to form the catalytically active B* complex. 5. **B* to C complex (first catalytic step):** The B* complex catalyzes the first transesterification reaction, in which the 2′-hydroxyl of the branch point adenosine attacks the 5′ splice site phosphate, producing a free 5′ exon and a lariat intermediate. This converts the B* complex to the C complex. 6. **C complex (second catalytic step):** The helicase Prp16 promotes a conformational rearrangement required for the second transesterification reaction, in which the 3′-hydroxyl of the 5′ exon attacks the 3′ splice site phosphate. This ligates the two exons and releases the lariat intron. The C complex is then converted to the P complex (post-splicing complex). ### Catalytic Core and Disassembly The catalytic core of the spliceosome is formed by a specific three-dimensional arrangement of U2 and U6 snRNAs and the pre-mRNA substrate. U6 snRNA forms an intramolecular stem-loop and base-pairs with U2 snRNA through two intermolecular helices (helix Ia and helix Ib). The U6 snRNA also base-pairs with the 5′ splice site, while U2 snRNA base-pairs with the branch point. This network of RNA-RNA interactions positions the reactive groups of the two transesterification reactions in close proximity. The U5 snRNA loops interact with exon sequences at both splice sites, helping to align the exons for the second catalytic step. After the second transesterification reaction, the mRNA is released from the spliceosome by the helicase Prp22, which translocates along the mRNA and disrupts the mRNA-spliceosome interaction. The lariat intron remains associated with the spliceosome and is released by the helicase Prp43, which also promotes the disassembly of the remaining snRNPs. The snRNPs are then recycled for additional rounds of splicing. The disassembly process is ATP-dependent and is essential for maintaining the pool of free snRNPs available for new splicing reactions. ## The Catalytic Core: RNA or Protein? A central question in the study of the spliceosome is whether catalysis is performed by RNA or by protein. The answer is now clear: the spliceosome is a ribozyme, and the catalytic chemistry is performed by U6 snRNA. This conclusion is supported by several lines of evidence. First, the chemistry of the splicing reaction is analogous to that catalyzed by group II introns, which are self-splicing RNAs found in bacteria and organelles. Both reactions proceed through two transesterification steps with the same stereochemistry, and both generate a lariat intermediate. The structural similarity between the U2/U6 snRNA network and the catalytic domain of group II introns is striking, strongly suggesting an evolutionary relationship. Second, mutational analyses have identified specific nucleotides in U6 snRNA that are essential for catalysis. The invariant ACAGAGA sequence at the 5′ end of U6 snRNA base-pairs with the 5′ splice site and is required for the first transesterification reaction. A metal ion binding site in U6 snRNA, formed by a specific arrangement of phosphate groups, coordinates catalytic magnesium ions. These metal ions are directly involved in the chemistry of both transesterification reactions, stabilizing the developing negative charge on the leaving group and the attacking nucleophile. Third, high-resolution cryo-electron microscopy (cryo-EM) structures of the spliceosome have revealed that the active site is devoid of protein side chains. The catalytic magnesium ions are coordinated exclusively by RNA phosphates from U6 snRNA and the pre-mRNA substrate. Proteins surround the catalytic core but do not directly participate in the chemistry; instead, they position the RNA elements, stabilize the active conformation, and regulate the conformational changes required for catalysis. This is not to say that proteins are unimportant. The spliceosome contains many proteins that are essential for splicing, but their roles are structural and regulatory rather than catalytic. Proteins such as Prp8, which is positioned at the heart of the spliceosome, stabilize the RNA network and ensure the correct geometry of the active site. The helicases provide the directional remodeling required for assembly and disassembly. The SR proteins and hnRNPs regulate splice site selection. However, the bond-breaking and bond-forming chemistry is performed by RNA. The classification of the [spliceosome as a ribozyme](/knowledge/molecular-biology/spliceosome-a-ribozyme) has important implications for our understanding of the evolution of catalysis. The spliceosome is the most complex ribozyme known, and its existence supports the [RNA world hypothesis](/blog/guides/rna-world-hypothesis), which posits that early life was based on RNA catalysis before the evolution of proteins. ## Methods to Study Spliceosome Composition ### Biochemical Purification The study of spliceosome composition has relied heavily on biochemical purification. The classic approach is to assemble spliceosomes on radiolabeled pre-mRNA substrates in nuclear extracts, then fractionate the complexes by glycerol gradient sedimentation or native gel electrophoresis. This approach allowed the identification of the E, A, B, and C complexes and the determination of their protein compositions. A major advance was the development of affinity purification methods. By incorporating affinity tags into the pre-mRNA substrate or into specific splicing factors, researchers can purify spliceosomal complexes at specific stages of assembly. For example, the use of a pre-mRNA substrate containing a 3′ terminal hairpin that binds the MS2 coat protein allows the purification of spliceosomes by binding to an MS2-agarose column. This approach, combined with mass spectrometry, has been used to comprehensively identify the protein composition of each spliceosomal complex. ### Mass Spectrometry and Cryo-EM Mass spectrometry has been instrumental in defining the protein composition of the spliceosome. By digesting purified spliceosomal complexes with proteases and analyzing the resulting peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS), researchers have identified more than 150 proteins in the human spliceosome. Quantitative mass spectrometry approaches, such as stable isotope labeling by amino acids in cell culture (SILAC), have been used to compare the composition of different spliceosomal complexes and to identify proteins that are differentially associated with specific stages of the splicing reaction. Cryo-EM has revolutionized the study of [spliceosome structure](/knowledge/molecular-biology/spliceosome-structure) and composition. The spliceosome is a large, dynamic complex that is difficult to crystallize, but cryo-EM can be used to determine the structures of complexes in solution. In recent years, cryo-EM structures have been determined for the yeast and human spliceosome at various stages of the splicing cycle, at resolutions of 3–6 Å. These structures have revealed the precise positions of the snRNAs and proteins within the complex and have provided unprecedented insight into the architecture of the catalytic core. For example, the cryo-EM structure of the human C complex showed that the catalytic core is formed by U2 and U6 snRNAs, with the pre-mRNA substrate positioned in the active site, and that Prp8 forms a scaffold that surrounds the catalytic RNA network. The combination of biochemical purification, mass spectrometry, and cryo-EM has provided a near-complete picture of spliceosome composition. However, it is important to note that the spliceosome is a dynamic machine, and its composition varies not only between species but also between different cell types and in response to cellular signals. The composition described here represents the consensus view derived from studies in yeast and human cells. ## Common Misconceptions and Pitfalls Several misconceptions about spliceosome composition are common among students and even some researchers. Understanding these pitfalls is essential for a correct grasp of the material. **Misconception 1: The spliceosome is made only of proteins.** This is incorrect. The spliceosome is a ribonucleoprotein complex, meaning it contains both RNA and protein. The five snRNAs are essential components, and U6 snRNA is directly responsible for catalysis. Removing the snRNAs abolishes splicing. **Misconception 2: The spliceosome is a static complex.** The spliceosome is highly dynamic. It assembles on each intron, undergoes extensive conformational rearrangements, and disassembles after splicing. Its protein composition changes dramatically during the splicing cycle. It is not a pre-formed machine that binds pre-mRNA as a unit. **Misconception 3: The spliceosome is the same as the ribosome.** While both are large RNP complexes, they are fundamentally different. The ribosome is a stable structure that remains intact through many rounds of translation. The spliceosome assembles *de novo* on each intron. Furthermore, the ribosome is composed of rRNA and ribosomal proteins, whereas the spliceosome is composed of snRNAs and splicing factors. The two complexes share no common components. **Misconception 4: All splicing is performed by the same spliceosome.** In eukaryotes, there are two types of spliceosomes: the major (U2-dependent) spliceosome, which processes the vast majority of introns, and the minor (U12-dependent) spliceosome, which processes a small subset of introns. The minor spliceosome contains U11, U12, U4atac, U6atac, and U5 snRNAs, which are functional analogs of the major spliceosome snRNAs but are distinct molecules. The minor spliceosome also contains a different set of associated proteins. **Misconception 5: The spliceosome is made of enzymes.** While the spliceosome contains proteins that are enzymes (such as the RNA helicases), the splicing reaction itself is catalyzed by RNA. The spliceosome is a ribozyme, not a protein enzyme. The proteins in the spliceosome are structural and regulatory factors, not the catalytic agents. **Misconception 6: The spliceosome recognizes the intron as a whole.** The spliceosome recognizes short, degenerate sequence elements at the 5′ splice site, the branch point, and the 3′ splice site. It does not recognize the entire intron sequence. The consensus sequences at these sites are short (approximately 6–10 nucleotides) and are recognized by base-pairing with snRNAs and by protein-RNA interactions. ## Frequently Asked Questions ### What is the spliceosome made of? The spliceosome is made of five small nuclear RNAs (U1, U2, U4, U5, and U6 snRNA) and more than 150 proteins. The snRNAs associate with proteins to form snRNPs, which are the core building blocks of the spliceosome. In addition to the snRNP proteins, the spliceosome contains many non-snRNP proteins, including SR proteins, hnRNPs, RNA helicases, and the NTC complex. ### Is the spliceosome made of RNA or protein? The spliceosome is made of both RNA and protein. It is a ribonucleoprotein complex. The RNA components are the five snRNAs, and the protein components include the Sm and LSm proteins, snRNP-specific proteins, and numerous non-snRNP splicing factors. The catalytic activity of the spliceosome is performed by U6 snRNA, making it a ribozyme. ### What are spliceosomes made of? Spliceosomes are made of snRNPs and non-snRNP proteins. The snRNPs are U1, U2, U4/U6, and U5, each containing a specific snRNA and associated proteins. The non-snRNP proteins include splicing factors that regulate splice site recognition, helicases that remodel the complex, and the NTC complex that is required for catalytic activation. ### Are spliceosomes made of ribosomes? No. Spliceosomes and ribosomes are both large ribonucleoprotein complexes, but they are entirely distinct. Spliceosomes are made of snRNPs and splicing factors, while ribosomes are made of ribosomal RNA (rRNA) and ribosomal proteins. The two complexes share no common components and perform different functions: spliceosomes remove introns from pre-mRNA, while ribosomes synthesize proteins. ### What is the function of the spliceosome? The spliceosome removes introns from pre-mRNA and ligates exons to produce mature mRNA. This process, called pre-mRNA splicing, is essential for gene expression in eukaryotes. Splicing also contributes to proteome diversity through [alternative splicing](/blog/guides/alternative-splicing), in which different combinations of exons are joined to produce multiple mRNA isoforms from a single gene. ### How many proteins are in the spliceosome? The human spliceosome contains approximately 150 distinct proteins. This number varies depending on the stage of the splicing reaction and the method of analysis. The core snRNPs contain about 40 proteins, and the remaining proteins are non-snRNP factors that associate with the spliceosome at specific stages of assembly and catalysis. ### Is the spliceosome made of enzymes? The spliceosome contains enzymes, but it is not itself a protein enzyme. The splicing reaction is catalyzed by U6 snRNA, making the [spliceosome a ribozyme](/knowledge/molecular-biology/spliceosome-a-ribozyme). The protein components include ATP-dependent RNA helicases (which are enzymes) that remodel the complex, but these proteins do not catalyze the transesterification reactions. ## Key Takeaways - The spliceosome is a large ribonucleoprotein complex composed of five snRNAs (U1, U2, U4, U5, U6) and more than 150 proteins. - The snRNAs associate with Sm and LSm proteins to form snRNPs, which are the core building blocks of the spliceosome. - The spliceosome assembles stepwise on each intron through the E, A, B, B*, and C complexes, and disassembles after splicing. - The catalytic core of the spliceosome is formed by U2 and U6 snRNAs, and the splicing chemistry is performed by U6 snRNA, making the spliceosome a ribozyme. - Proteins in the spliceosome play structural, regulatory, and remodeling roles, but do not catalyze the splicing reaction. - The spliceosome is a dynamic machine whose composition changes during the splicing cycle, not a static complex. - The major (U2-dependent) and minor (U12-dependent) spliceosomes are distinct complexes that process different classes of introns. ## Related Topics - [Spliceosome Structure](/knowledge/molecular-biology/spliceosome-structure) - [Spliceosome Proteins](/knowledge/molecular-biology/spliceosome-proteins) - [Spliceosome Assembly](/knowledge/molecular-biology/spliceosome-assembly) - [Spliceosome Complex](/knowledge/molecular-biology/spliceosome-complex) - [Spliceosome Definition](/knowledge/molecular-biology/spliceosome-definition) ## Related Clinical & Scientific Guides * [MAPK Pathway: Mechanism, Function, and Clinical Relevance](/knowledge/molecular-biology/mapk-pathway) * [Mammalian Cell Culture Bioreactors: A Practical Guide](/knowledge/molecular-biology/mammalian-cell-culture-bioreactor) * [Nucleotide Formation: Biosynthesis and Assembly of DNA/RNA Building Blocks](/knowledge/molecular-biology/nucleotide-formation)