Among the many macromolecular machines involved in eukaryotic gene expression the spliceosome remains one of the most challenging for Tacalcitol structural biologists. Together these tools are rapidly changing our structural appreciation of this amazingly dynamic machine. Introduction In all organisms gene expression requires coordinate action of multiple macromolecular machines many with multi-megadalton (MDa) molecular weights. Whereas high-resolution crystal structures have revealed the overall architecture and detailed inner workings of many such machines (e.g. ribosomes and Tacalcitol RNA polymerases) one elusive “structure of desire” [1] is the spliceosome. Weighing in at over 3 MDa the spliceosome is the ribonucleoprotein (RNP) complex responsible for excision of intragenic regions (introns; Box 1) from eukaryotic RNA polymerase II transcripts (precursors to messenger RNAs; pre-mRNAs). The spliceosome must be at once highly accurate – a single nucleotide shift in the site of splicing (splice site; SS) within an open reading frame will result in a non-functional mRNA – and highly malleable to permit alternative splicing the process by which expressed regions (exons) are spliced together in different plans enabling synthesis of many different Tacalcitol protein isoforms from a single gene. The proliferation of alternate splicing is the primary reason why organismal complexity is not tightly linked to gene number in the eukaryotic lineage[2 3 To achieve the right balance between precision and malleability the spliceosome contains scores of individual parts many of which are structurally disordered. Working in a highly orchestrated manner these parts perform incredible feats of molecular gymnastics with each round of splicing. These extremes of complexity and dynamics are no doubt to blame for the spliceosome’s recalcitrance to crystallize despite intense efforts by multiple labs over many years. Nonetheless significant progress is now being made by combining crystal structures of smaller pieces with EM reconstructions of larger assemblages. As detailed in the upcoming review [4] answer of several high-resolution structures made up of pieces of Prp8 the Tacalcitol massive and highly-conserved protein at heart of the spliceosome is usually rapidly transforming our understanding of Itgb7 the catalytic core. In this review we will focus instead on recent progress in understanding spliceosome evolutionary and structural dynamics. Evolutionary Dynamics One of the defining features of pre-mRNA splicing is the sheer number of components that must come and go to accurately identify and excise each new intron (Fig 1A). In budding yeast this includes five small nuclear RNAs (snRNAs) and ~100 different proteins whereas mammals utilize nine unique snRNAs and over 300 different proteins [5 6 . Metazoans have more spliceosomal snRNAs Tacalcitol because they contain not one but two spliceosomes: the more abundant “major spliceosome” responsible for removing 99.5% of introns and the “minor spliceosome” excising the other 0.5% [7] (Fig 1B). A long-standing question regarding the function of these minor introns was recently resolved by Younis et al. [8] who showed that under normal growth conditions their splicing is limited by quick decay of the key minor snRNA U6atac. In the presence of stress Tacalcitol however U6atac is usually stabilized allowing splicing of preexisting minor intron-containing transcripts which can then be rapidly translated to help alleviate the stress. Fig 1 An updated spliceosome assembly cycle The presence of two spliceosomes is usually thought to reflect a long ago merging of two eukaryotic genomes that experienced diverged and separately evolved for prior untold generations. By the time of the merge so many mutations had accumulated in the individual lineages that the two machineries were no longer fully compatible. Nonetheless the major and minor spliceosomes do share some key components and much can be learned about core spliceosome structure from comparing their commonalities and differences (Fig 1B). The largest common component is usually U5 snRNP which contains U5 snRNA and 14-15 stably-bound proteins[6]. Of all the snRNAs U5 has the highest percentage of internal secondary structure and is the least accessible to RNase digestion or nucleotide modification reagents[9] consistent with it being almost entirely coated with proteins. The only region of U5 snRNA making intermolecular RNA-RNA interactions is usually a U-rich loop that contacts exonic nucleotides to either side of the intron and is thought to help align the exons to facilitate both actions of.