Precursor-mRNA splicing is catalyzed by an extraordinarily large and highly dynamic macromolecular assemblage termed the spliceosome. Detailed biochemical and structural study of the spliceosome presents a formidable challenge, but there has recently been significant progress made on this front highlighted by the crystal structure of a 10-subunit human U1 snRNP. This review provides an overview of our current understanding of the architecture of the spliceosome and the RNA-protein complexes integral to its function, the U snRNPs.
SUMMARY There is significant demand for experimental approaches to aid protein localization in electron microscopy micrographs and ultimately in three-dimensional reconstructions of macromolecular assemblies. We report preparation and use of a reagent consisting of tris-nitrilotriacetic acid (tris-NTA) conjugated with a monofunctional gold nanoparticle (AuNPtris-NTA) for site-specific, non-covalent labeling of protein termini fused to a histidine-tag (His-tag). Multivalent binding of tris-NTA to a His-tag via complexed Ni(II) ions results in subnanomolar affinity and a defined 1:1 stoichiometry. Precise localization of AuNPtris-NTA labeled proteins by electron microscopy is further ensured by the reagent’s short conformationally restricted linker. We have employed AuNPtris-NTA to localize His-tagged proteins in an oligomeric ATPase and in the bacterial 50S ribosomal subunit. AuNPtris-NTA can specifically bind to the target proteins in these assemblies and is clearly discernible. Our new labeling reagent should find broad application in non-covalent site-specific labeling of protein termini to pinpoint their location in macromolecular assemblies.
The removal of noncoding regions or introns and the splicing together of the protein‐coding regions or exons from precursor messenger ribonucleic acid (pre‐mRNA) transcripts is fundamental to metzoan cell development, as well as its maintenance. The nuclear process of pre‐mRNA splicing is a complex phenomenon catalysed by the ‘spliceosome’. The spliceosome consists of greater than a hundred protein and RNA molecules. Integral to the spliceosome are five RNA–protein complexes, the U1, U2, U4, U5 and U6 small nuclear ribonucleoproteins (snRNPs). The numerous proteins and the U snRNPs that make up the spliceosome come on and off during the spliceosome's reaction cycle. This article places an emphasis on our current understanding of the dynamics, composition and structure of the spliceosome. Key Concepts: Specific sequences defining the boundaries between a protein‐coding gene's noncoding regions or introns and its protein‐coding regions or exons are recognised by components of the pre‐mRNA splicing ‘machinery’ – the spliceosome. The spliceosome is composed of a large number of RNA and protein molecules. The protein subunits have diverse domain structures, whereas the RNA subunits form hydrogen bonding or base‐pairing interactions with critical sequences in a pre‐mRNA as well as with each other. The spliceosome reaction cycle involves an ordered assembly onto a pre‐mRNA transcript to ultimately catalyse two trans ‐esterification reactions that result in the removal or excision of an intron and the splicing together of two exons. Integral to the function of the spliceosome are five U snRNPs, each of which has a single RNA and 3–12 protein constituents including a homologous set of proteins (Sm or LSm) that forms a structural unit critical to the biogensis and integrity of each U snRNP. The U1, U2, U4 and U5 snRNPs all have a common core structure that consists of seven Sm proteins which assemble around a short U‐rich single‐stranded RNA sequence (the Sm site) present in the U1, U2, U4 and U5 snRNAs to form a heptameric ring with the single‐stranded RNA Sm site leafing through the central hole. Metazoan U1 snRNP functions to initiate the assembly of the spliceosome onto a pre‐mRNA transcript; its single RNA subunit (U1 snRNA) forms a cruciform‐like structure onto which the Sm proteins assemble to form the Sm core, while its three additional U‐specific proteins (U1‐A, U1‐70K, and U1‐C) participate in particle specific functions – including recognition of a pre‐mRNA transcripts 5' splice site sequence.
Electron microscopy (EM) is a leading technique to elucidate structure of macromolecular assemblies. Obtaining high‐resolution information by EM is often hindered by sample compositional and conformational heterogeneity. It is challenging and error prone to build an informative model into a low‐resolution 3‐dimensional reconstruction. Thus, there is demand to expand upon current approaches used for accurate protein localization to aid model building. We report use of a labeling reagent consisting of tris‐nitrilotriacetic acid (tris‐NTA) conjugated with a monofunctionalized gold nanoparticle that site‐specifically labels protein termini fused to a His‐tag. Multivalent binding of tris‐NTA to a His‐tag via complexed Ni(II) ion results into subnanomolar affinity and a defined 1:1 stoichiometry. Precise localization of a protein is further ensured by the reagents conformationally restricted linker and monofunctionalized small Au nanoparticle. We have employed the label to localize His‐tagged proteins in a T. thermophiles ATPase, RuvB, and in the E. coli 50S ribosomal subunit. Our mono‐tris‐NTA‐Au conjugate specifically binds to the target protein in these assemblies, exhibiting stoichiometric labeling. Our new Au labeling reagent improves upon current labeling approaches and should have extensive applicability in sites‐pecific labeling of His‐tag protein termini in macromolecular assemblies.
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