Spliceosome

A spliceosome is a large and complex molecular machine found primarily within the nucleus of eukaryotic cells. The spliceosome is assembled from small nuclear RNAs (snRNA) and approximately 80 proteins. The spliceosome removes introns from a transcribed pre-mRNA, a type of primary transcript. This process is generally referred to as splicing. An analogy is a film editor, who selectively cuts out irrelevant or incorrect material (equivalent to the introns) from the initial film and sends the cleaned-up version to the director for the final cut. A spliceosome is a large and complex molecular machine found primarily within the nucleus of eukaryotic cells. The spliceosome is assembled from small nuclear RNAs (snRNA) and approximately 80 proteins. The spliceosome removes introns from a transcribed pre-mRNA, a type of primary transcript. This process is generally referred to as splicing. An analogy is a film editor, who selectively cuts out irrelevant or incorrect material (equivalent to the introns) from the initial film and sends the cleaned-up version to the director for the final cut. In 1977, work by the Sharp and Roberts labs revealed that genes of higher organisms are 'split' or present in several distinct segments along the DNA molecule. The coding regions of the gene are separated by non-coding DNA that is not involved in protein expression. The split gene structure was found when adenoviral mRNAs were hybridized to endonuclease cleavage fragments of single stranded viral DNA. It was observed that the mRNAs of the mRNA-DNA hybrids contained 5' and 3' tails of non-hydrogen bonded regions. When larger fragments of viral DNAs were used, forked structures of looped out DNA were observed when hybridized to the viral mRNAs. It was realized that the looped out regions, the introns, are excised from the precursor mRNAs in a process Sharp named 'splicing'. The split gene structure was subsequently found to be common to most eukaryotic genes. Phillip Sharp and Richard J. Roberts were awarded the 1993 Nobel Prize in Physiology or Medicine for their discovery of introns and the splicing process. Each spliceosome is composed of five small nuclear RNAs (snRNA) and a range of associated protein factors. When these small RNAs are combined with the protein factors, they make RNA-protein complexes called snRNPs (small nuclear ribonucleo proteins, pronounced 'snurps').The snRNAs that make up the major spliceosome are named U1, U2, U4, U5, and U6, so-called because they are rich in uridine, and participate in several RNA-RNA and RNA-protein interactions. The canonical assembly of the spliceosome occurs anew on each pre-mRNA (also known as heterogeneous nuclear RNA). The pre-mRNA contains specific sequence elements that are recognized and utilized during spliceosome assembly. These include the 5' end splice site, the branch point sequence, the polypyrimidine tract, and the 3' end splice site. The spliceosome catalyzes the removal of introns, and the ligation of the flanking exons. Introns typically have a GU nucleotide sequence at the 5' end splice site, and an AG at the 3' end splice site. The 3' splice site can be further defined by a variable length of polypyrimidines, called the polypyrimidine tract (PPT), which serves the dual function of recruiting factors to the 3' splice site and possibly recruiting factors to the branch point sequence (BPS). The BPS contains the conserved Adenosine required for the first step of splicing. New evidence derived from the first crystal structure of a group II intron suggests that the spliceosome is actually a ribozyme, and that it uses a two–metal ion mechanism for catalysis. In addition, many proteins exhibit a zinc-binding motif, which underscores the importance of zinc metal in the splicing mechanism. The first molecular-resolution reconstruction of U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) complex was reported in 2016. Cryo-EM has been applied extensively by Shi et al to elucidate the near-/atomic structure of spliceosome in both yeast and humans. The molecular framework of spliceosome at near-atomic-resolution demonstrates Spp42 component of U5 snRNP forms a central scaffold and anchors the catalytic center in yeast. The atomic structure of the human spliceosome illustrates the step II component Slu7 adopts an extended structure, poised for selection of the 3'-splice site. All five metals (assigned as Mg2+) in the yeast complex are preserved in the human complex. Alternative splicing (the re-combination of different exons) is a major source of genetic diversity in eukaryotes. Splice variants have been used to account for the relatively small number of protein coding genes in the human genome. For years the estimate widely varied, with top estimates reaching 100,000 protein coding genes, but now, due to the Human Genome Project, the figure is believed to be closer to 20,000. One particular Drosophila gene (Dscam, the Drosophila homolog of the human Down syndrome cell adhesion molecule DSCAM) can be alternatively spliced into 38,000 different mRNA. The model for formation of the spliceosome active site involves an ordered, stepwise assembly of discrete snRNP particles on the pre-mRNA substrate. The first recognition of pre-mRNAs involves U1 snRNP binding to the 5' end splice site of the pre-mRNA and other non-snRNP associated factors to form the commitment complex, or early (E) complex in mammals. The commitment complex is an ATP-independent complex that commits the pre-mRNA to the splicing pathway. U2 snRNP is recruited to the branch region through interactions with the E complex component U2AF (U2 snRNP auxiliary factor) and possibly U1 snRNP. In an ATP-dependent reaction, U2 snRNP becomes tightly associated with the branch point sequence (BPS) to form complex A. A duplex formed between U2 snRNP and the pre-mRNA branch region bulges out the branch adenosine specifying it as the nucleophile for the first transesterification.