The spliceosome is a complex molecular machine in eukaryotic cells that plays a role in gene expression. This machinery is responsible for RNA splicing, a process where non-coding regions, called introns, are removed from precursor messenger RNA (pre-mRNA) molecules. Concurrently, coding regions, known as exons, are precisely joined to form a mature messenger RNA (mRNA). This mature mRNA then carries the genetic instructions for building proteins.
Building Blocks of the Spliceosome
The spliceosome is not a fixed structure but a dynamic assembly of multiple components. These include five main small nuclear ribonucleoproteins (snRNPs) and a wide array of non-snRNP proteins. Each snRNP is a complex of a small nuclear RNA (snRNA) molecule and associated proteins.
The five major snRNPs are U1, U2, U4, U5, and U6. For instance, U1 snRNP recognizes the 5′ splice site of an intron, while U2 snRNP binds to a specific branch point sequence within the intron. These snRNAs provide specificity by recognizing critical splicing signals.
Beyond the snRNPs, the spliceosome incorporates over 300 non-snRNP proteins, often called splicing factors. These proteins contribute to the spliceosome’s assembly, regulation, and catalytic activity. The coordination of all these components is necessary for the spliceosome to function.
The Splicing Process
The splicing process involves a series of coordinated steps that transform pre-mRNA into mature mRNA. It begins with the spliceosome recognizing specific sequences at the boundaries of introns and exons, known as splice sites. These include a 5′ splice site, a 3′ splice site, and a branch point sequence within the intron.
The assembly of the spliceosome on the pre-mRNA is a stepwise process. Initially, U1 snRNP binds to the 5′ splice site, forming an early complex. U2 snRNP is then recruited to the branch point sequence, involving interactions with other non-snRNP proteins.
Following these initial recognition events, the U4/U6-U5 tri-snRNP complex joins the assembly. Structural rearrangements occur within the spliceosome, which destabilize U1 and U4 snRNPs, with U1 being displaced by U6. This prepares the spliceosome for two sequential transesterification reactions.
In the first transesterification reaction, a bulged adenosine at the branch point attacks the phosphodiester bond at the 5′ splice site. This cleaves the 5′ end of the intron and forms a lariat intermediate. The second transesterification reaction then joins the 3′ end of the upstream exon to the 5′ end of the downstream exon, releasing the intron as a lariat and creating a continuous coding sequence. The snRNAs within the snRNPs directly participate in these catalytic reactions.
Alternative Splicing
Alternative splicing allows a single gene to produce multiple different mRNA transcripts and, consequently, different proteins. This occurs by selectively including or excluding specific exons or by using alternative splice sites within a pre-mRNA molecule. This process expands the protein diversity encoded by a limited number of genes.
The biological significance of alternative splicing is extensive. It generates tissue-specific protein forms, contributing to the specialized functions of different cell types. For example, the muscle protein titin has various forms produced through alternative splicing, which differ in fetal and adult hearts.
Alternative splicing is also involved in various developmental processes and cellular adaptations. Its regulation involves specific sequences on the pre-mRNA, known as splicing enhancers and silencers, which are recognized by regulatory proteins. The secondary structure of the pre-mRNA transcript can also influence splicing outcomes by masking or revealing binding sites for splicing factors.
Spliceosome Dysfunction and Disease
Malfunctions of the spliceosome or disruptions in splicing can impact cellular health. Even minor errors can lead to the production of non-functional or harmful proteins. Such mis-splicing can arise from mutations within spliceosome components or in the DNA sequences that define splice sites.
Spliceosome dysfunction is linked to a range of human diseases. Certain cancers involve mutations in splicing factors. For instance, some breast cancer subtypes show altered snRNA expression patterns.
Neurodegenerative disorders are another category of diseases impacted by splicing errors. Examples include spinal muscular atrophy and Alzheimer’s disease, where spliceosome disruption can lead to cryptic RNA splicing. Other neurodegenerative conditions, such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia, and Parkinson’s disease, also show dysregulated alternative splicing. The brain is particularly sensitive to splicing defects, possibly due to its complex cellular diversity and high reliance on alternative splicing.