Introduction

The splicing mechanism

Introns must be spliced out to achieve mature RNA. This mechanism must be achieved accurately and efficiently to ensure the maintenance of the original protein coding potential of the gene transcribed. The mechanism involves a stepwise recruitment of five snRNPs and associated proteins which catalyse two trans-esterification reactions. 

Figure 1 illustrates the cycle of the splicing mechanism – adapted from Lodish et al. Molecular Biology of the cell. 1.       U1 snRNP binds to the 5’ splice site of the intron, and U2 binds to the branch site (BS) creating a “Complex A.” ATP is required to initiate this binding interaction. 2.       U4/U6.U5 tri-snRNP associated with Complex A creating an inactive “Complex B,” which is also known as a mega-dalton complex. 3.       For a catalytically active Complex B, major structural rearrangements must occur: U1 and U4 are displaced from the complex through the action of RNA helicases Prp28, Brr2, Prp2. This displacement allows for the integration of NTC’s and NTC-associated proteins. The disruption of base-pairing between the U4/U6 complex initiates U6 to create an internal stem loop (ISL). U6 and U2 undergo additional base pairing to create catalytically essential Helices 1a and 1b. An “active Complex B” is now formed. The changes in composition were initially analysed through mass spectrometry. 4.       The helix 1b mentioned contains an essential, invariant AGC triad required for the splicing mechanism. The first step of splicing can now occur, whereby the 5’SS is cleaved and the intron is ligated through a trans-esterification reaction to the BS adenosine, creating a lariat,  “Complex C.” ATP is required for the formation of this lariat structure. 5.       Complex C is remodelled through the action of RNA helicase Prp16, which assists in further rearrangement of the catalytic centre of the spliceosome, allowing for exon ligation through a second trans-esterification reaction, fuelled by ATP. 6.       C complex can now be released from the  complex, and disassembled 7.       The snRNPs are thought to then reassemble for a new round of splicing.  The structure of the splicing complex The specific conformation of the catalytic core of the spliceosome is vital to create catalytically active components such as the ISL.  It allows for the integration of additional proteins, which also assist in further remodelling of the spliceosome, and promote RNA-RNA interactions: In yeast, the NTC (“the nine complex “) is composed of 8 core proteins, formed by the scaffold protein Prp19 and a number of associated splicing factors. Their abundance greatly increases during the transition towards can active B complex. Abundance of NTC increases greatly on formation of B complex, suggesting its involved in transition to the active state.  NTC-associated proteins appear to have a more dynamic association with the spliceosome. Cwc2 is the only associated protein found to have RNA binding domains ( RRM and ZnF), so therefore also must have a role in the actively. Cwc2 also cross-links with the snRNP U6, all of which is explored later.  The interactions with these proteins is not fully understood, however creation of a crystal structure of Cwc2, managed to shed some light into this unfamiliar arrangement of proteins.