s, culminating in the two trans-esterification reactions that result in ligation of exons and excision of the lariat intron, followed by spliceosome disassembly and re-assembly for another round of splicing. Introns can be the precursors of other non-coding molecules such as microRNAs, which can also be transcribed from independent genes. Some exons are constitutively spliced; however, many exons, particularly in higher eukaryotes, are subject to alternative splicing (AS), resulting in multiple and different mature mRNAs generated from the same pre-mRNA, vastly expanding the proteome. The core splicing machinery is conserved from yeast to man; additional factors in the human system likely represent the greater demand for regulatory mechanisms and added functionality associated with alternative splicing. Small nuclear ribonucleoprotein particles (snRNPs) with uridine-rich (U-rich) U1, U2, U4, U5 and U6 snRNAs, in the case of the major spliceosome, and some ~100 to possibly 300 proteins, in yeast and human, respectively, are the key components. An additional minor spliceosome is present in some metazoans, including humans and in plants. A consensus major spliceosome pathway, alternative splicing and integration of splicing, transcription and chromatin machineries are presented.

Spliceosome pathway
A generic pre-mRNA sequence shows two exons separated by an intron with the 5' splice site (5'SS) and 3' splice site (3'SS) with GU and AG exon/intron border sequences, respectively, a branch point sequence (BPS) in between and, in metazoans, a polypyrimidine tract (PPT) between the BPS and 3'SS. In the two reactions catalyzed by the spliceosome first, the phosphodiester bond at the 5'SS is attacked by the 2'-hydroxyl of an adenosine in the BPS generating a free 5'end exon and an intron lariat-3' exon; then, the 3'-hydroxyl of the cleaved 5' exon attacks the phosphodiester bond at the 3'SS, leading to exon ligation and excision of the lariat intron. The small sequence size and in mammals, poor conservation and great intron size, render the correct identification and positioning of 5'SS and 3'SS a rather formidable challenge. Additional factors include the cis-elements represented by exonic and intronic splicing enhancers (ESE, ISE) and silencers (ESS, ISS). Trans-elements, which bind to enhancers and silencers, include the members of the Ser/Arg-rich (SR) and heterogeneous nuclear ribonucleoprotein (hnRNP) protein families, respectively. The U1, U2, U4 and U5 snRNAs have an Sm site, bound by a group of seven Sm proteins that form a heptametic ring through which the seven nucleotides (nts) of the Sm site are threaded. Shared and unique snRNP and non-snRNP proteins associate with as well as dissociate from the spliceosome at various stages of its assembly. Assembly begins with binding of U1 to the 5'SS. Metazoan U1 snRNP is composed of U1 snRNA, the seven Sm proteins and three U1-specific proteins. Additional factors Sf1 and U2af bind to BPS and PPT, respectively; Sf1 interacts with U2af2 (the 65kDa subunit of U2af) via its RNA recognition motif (RRM) while the 35 kDa subunit (U2uaf1) of the U2af heterodimer interacts with the 3'SS. Together, they represent the early complex, Complex E. U2 assembly leads to formation of complex A (pre-spliceosome); formation of this and subsequent complexes require the action of DexD/H-box RNA-dependent ATPases/helicases. Human U2 contains U2 snRNA, the Sm proteins and several U2-specific proteins, mostly components of Sf3a and b complexes, the latter important for BPS recognition. U4/U6, pre-assembled with U5 as a tri-snRNP, join the spliceosome to form the complex B (pre-catalytic spliceosome), still inactive. Nts within U6 snRNA are components of the active site but are initially in an inactive form through base pairing with U4. During activation U1 is displaced, U4/U6 interactions are disrupted and U4 is displaced, the conserved ACAGAG box of U6 snRNA engages the intron nts at the 5'SS, U6/U2 duplexes form. The substantial compositional and conformational rearrangements yield complex B* (activated spliceosome) and the first catalytic step. The resulting complex C (catalytic spliceosome) undergoes further rearrangements and the second catalytic step takes place followed by postcatalytic rearrangements (post-spliceosome complex) with subsequent release of spliced mRNA, excised lariat and disassembly of snRNPs. During transition to complex C, the core of the exon junction complex (EJC) is deposited; EJC is found in spliceosomes that have catalyzed the first splicing step, remains associated with mRNA after the second step and travels with it to the cytoplasm where it is removed from mRNA after the first round of translation. EJC is deposited upstream of the exon-exon splice junction thus marking the positions of former introns and is critical for distinguishing premature stop/termination codons (PTC) upstream of the last EJC (natural stop codons are downstream of the last EJC). Prpf8 (known as Prp8), a component of U5 which also shows footprints and crosslinking sites on U6, U2 and pre-mRNA, is thought to have an essential role in formation of the catalytic core/splicing reaction. It interacts with Aar2, a U5 assembly factor and with Snrnp200 (Brr2) and Eftud2 (Snu14), which in addition to being components of U5 are also a helicase and a GTPase, respectively (the only GTPase is required for splicing). Other U5 components may be lost upon activation of the spliceosome. The U6 snRNA associates with seven Sm-like (LSM) proteins at its 3' end. Prp24 (human homolog is SART3) is an essential component of U6, along with the core LSM proteins. Of the non-snRNP factors, the Prp19 complex and Prp19-associated proteins are involved in the activation of the spliceosome: they associate with the spliceosome during/after dissociation of U4, stabilize the B* complex and remain associated with C complex during the second catalytic step. Prpf18 and Slu7, along with helicases, promote the second catalytic step. Other proteins present in many stages of the spliceosome include members of SR and hnRNP protein families and other spliceosome-associated proteins (SAP). Many spliceosomal components undergo post-transcriptional modification (PTM), of which phosphorylation is the better documented type.

DExD/H-type ATPases/helicases, other enzymes and factors
The dynamics of RNA and proteins interactions and remodeling during spliceosome assembly require enzyme activity. At least eight DExD/H-type RNA-dependent ATPases/helicases are involved in the various steps of spliceosomal assembly. They act as platforms that assemble and reconfigure the snRNPs and also have RNA-related roles beyond splicing. Ddx39b (known as Uap56) and Ddx46 (Prp5) promote formation of complex A and the latter physically interacts with U2 snRNP. Ddx23 (Prp28) promotes complex B formation and the human protein, unlike the yeast, is found in the tri-snRNP U4/U6.U5. Snrnp200 (Brr2) - an integral component of U5, promotes unwinding of U4/U6, an activity stimulated by Prpf8. Eftud2 - the only GTPase required for splicing and also an U5 component, regulates U4/U6 unwinding. And Snrnp200 and Eftud2 activities promote complex B* formation. Dhx16 (Prp2) is thought to regulate the first catalytic step while Dhx38 (Prp16) and Dhx8 (Prp22) function in the second step (along with Prpf18 and Slu7); the molecular mechanisms of their function remain to be elucidated. Finally, Dhx15 (Prp43) plays a role in spliceosome disassembly; Eftud2 and possibly Snrnp200 also play a role (not shown).
Other enzymes include the peptidyl-prolyl-cis/trans insomerases (PPIases) of which several are associated with the human spliceosome. They are members of the cyclophilin family of the immunophilin superfamily and while their exact function is still to be established, a potential role in the structural transitions spliceosome assembly undergoes can be envisioned, as the isomerization of a peptide bond preceding a proline residue is often rate-limiting in protein folding and activity. As many have no yeast orthologs, a possible role in AS is speculated; others include aiding the assembly/disassembly of complexes or the coupling of transcription and splicing. Of note is PPIH which forms distinct complexes with either the Prpf4 component of U4 snRNP or Prpf18 which promotes the second catalytic step and neither complex obstructs/disrupts the active site of the enzyme.
Other factors include splicing regulators acting at various stages of spliceosome assembly or controlling splice site selection and those that are differentially expressed between various cells and tissue types. Examples include but are not limited to regulators such as Ptbp1 and 2, Mbnl, Srrm, Qki and Tial1.

Alternative splicing
Splice sites, depending on how close or divergent they are from the consensus sequence, are classified as strong or weak, respectively. While strong sites are associated with constitutive splicing, weak sites are subject to alternative splicing (AS) and the extent of their usage depends on both cis- and trans-regulatory elements. The cis-elements - exonic and intronic splicing enhancers (ESE, ISE) and silencers (ESS, ISS) mentioned above, important for correct splice recognition, play an essential role in AS. Alternative exons can be included or skipped (cassette exon) as single, multiple or partial; others are mutually exclusive, certain introns may be retained and alternative 5' and/or 3' splice sites can be used, in a tissue- and time-specific manner. The cis-elements are binding sites for the trans-factors - SR proteins for enhancers, hnRNP for silencers, which in turn recruit snRNP subunits or prevent their association. SR proteins bind both pre-mRNA and proteins with low affinity and specificity. Low binding affinity is a common feature not only of regulatory splicing factors but also of core components and is essential for the dynamic nature of the spliceosome. Both SR and hnRNP proteins are modular in nature, undergo nucleocytoplasmic shuttling and have functions beyond splicing. While promoters, sequences, relative abundance/presence of transcription factors and regulatory proteins, chromatin remodelers and other chromatin-affecting factors impact on transcription and the largely co-occurring splicing, a detailed picture of the molecular mechanisms of AS is still lacking. Two non-mutually exclusive mechanisms are proposed: recruitment coupling and kinetic coupling. In the first, splicing factors are recruited via the transcriptional machinery, particularly the carboxy-terminal domain (CTD) of RNA polymerase II and possibly the Mediator. The involvement of CTD in all aspects of pre-mRNA processing is relatively well documented; yet, the details of its role in AS are not well understood. In the second, the rate at which pol II elongates, the emergence of pre-mRNA, splice sites and regulatory sequences affects AS, with high rates leading to skipping and low rates to inclusion of alternative exons. AS not only augments the actual size of the proteome but also its functional versatility, as the protein domain/region encoded by an exon can have its underlying function 'visible/active' or 'hidden/silent', depending on whether that exon is included or skipped, respectively.

Integration of splicing, transcription and chromatin pathways
Splicing is largely co-transcriptional and whether the recruitment or the kinetic model is the preponderant one (likely, a combination of both), the unfolding of transcriptional events affects splicing and its outcomes. Chromatin dynamics and structure influence transcription, the coupling between transcription and splicing, and AS. Nucleosome positioning is preferential in exons and differential in included (higher) versus excluded exons. Histone marks/modifications are enriched in exons and can prevent/allow for the recruitment of specific factors that favor/disfavor exon inclusion. DNA methylation is also enriched in exonic compared to intronic regions; it may slow transcription elongation and thus favor inclusion. In turn, splicing can affect transcription and chromatin. Reports show that splicing enhances transcription in yeast and mouse models. Interactions between splicing factors/regulators and components of the transcriptional and/or chromatin modification machineries impact on all their outcomes. They can mediate recruitment of factors essential for pol II transcriptional elongation, and impact on the activity of histone modifying enzymes that affect chromatin structure and modulate the events associated with them. While the molecular mechanisms of differential gene expression, the detailed pictures of pol II transcription, chromatin modification/modulation and spliceosomal pathways are still to be unraveled, current evidence increasingly points to a bi-directional mode of interconnectivity. Mutations in the core splicing machinery and aberrant alternative splicing are associated with cancer.

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