The Spliceosome: Design Principles of a Dynamic RNP Machine

The collection of components required to carry out the intricate processes involved in generating and maintaining a living, breathing and, sometimes, thinking organism is staggeringly complex. Where do all of the parts come from? Early estimates stated that about 100,000 genes would be required to make up a mammal; however, the actual number is less than one-quarter of that, barely four times the number of genes in budding yeast. It is now clear that the 'missing' information is in large part provided by alternative splicing, the process by which multiple different functional messenger RNAs, and therefore proteins, can be synthesized from a single gene.

/pdf/expansion-of-the-eukaryotic-proteome-by-alternative-splicing-44nppu5fec.pdf

Expansion of the eukaryotic proteome by alternative splicing

Pre-mRNA splicing is catalyzed by the spliceosome, a multimegadalton ribonucleoprotein (RNP) complex comprised of five snRNPs and numerous proteins. Intricate RNA-RNA and RNP networks, which serve to align the reactive groups of the pre-mRNA for catalysis, are formed and repeatedly rearranged during spliceosome assembly and catalysis. Both the conformation and composition of the spliceosome are highly dynamic, affording the splicing machinery its accuracy and flexibility, and these remarkable dynamics are largely conserved between yeast and metazoans. Because of its dynamic and complex nature, obtaining structural information about the spliceosome represents a major challenge. Electron microscopy has revealed the general morphology of several spliceosomal complexes and their snRNP subunits, and also the spatial arrangement of some of their components. X-ray and NMR studies have provided high resolution structure information about spliceosomal proteins alone or complexed with one or more binding partners. The extensive interplay of RNA and proteins in aligning the pre-mRNA's reactive groups, and the presence of both RNA and protein at the core of the splicing machinery, suggest that the spliceosome is an RNP enzyme. However, elucidation of the precise nature of the spliceosome's active site, awaits the generation of a high-resolution structure of its RNP core.

/pdf/spliceosome-structure-and-function-3j278at609.pdf

Spliceosome structure and function.

Hypertension 66 (20.3%) 24 (24.2%) 30 (16.3%) NS Diabetes 20 (6.2%) 7 (7.1%) 10 (5.4%) NS Excess weight 78 (24%) 27 (27.3%) 44 (23.9%) NS Smokers 64 (19.7%) 17 (17.2%) 35 (19.0%) NS Age >50 years 137 (42.2%) 54 (54.5%) 67 (36.4%) <0.02 Kidney disease 7 (2.2%) 1 (1%) 5 (2.7%) NS Family history, DM 102 (31.4%) 28 (28.3%) 66 (35.9%) NS

Conflict of interest statement. None declared.

The nucleolus is a distinct subnuclear compartment that was first observed more than 200 years ago. Nucleoli assemble around the tandemly repeated ribosomal DNA gene clusters and 28S, 18S and 5.8S ribosomal RNAs (rRNAs) are transcribed as a single precursor, which is processed and assembled with the 5S rRNA into ribosome subunits. Although the nucleolus is primarily associated with ribosome biogenesis, several lines of evidence now show that it has additional functions. Some of these functions, such as regulation of mitosis, cell-cycle progression and proliferation, many forms of stress response and biogenesis of multiple ribonucleoprotein particles, will be discussed, as will the relation of the nucleolus to human diseases.

The multifunctional nucleolus

I. INTRODUCTION The RNA world pictures a time when RNA structures catalyzed many reactions, almost certainly including RNA splicing. The self-splicing reactions of group I and group II introns may be direct remnants of the RNA world. Both of these catalytic structures can facilitate cis -splicing, where the two exons are initially part of a contiguous RNA, and trans -splicing, where the two exons are initially on two separate RNAs. In the latter case, the partial intron sequences flanking the two exons pair through sequence complementarity to form a catalytic structure joining the associated exons. Many have speculated that the exon-intron structure was common in the genetic material in the RNA world. In fact, exons have been hypothesized as having originally evolved as units of sequences which encoded functional protein domains that were assembled by RNA splicing into large multidomain proteins with more complex functions. In this period, it is highly likely that RNA splicing was the product of the action of group I- and group II-like catalytic structures, and peptide-bond formation was catalyzed by progenitors of ribosomal RNA. As the potential to generate specific proteins evolved, some of these proteins probably bound to sequences within the catalytic introns to facilitate the rate of splicing. Eventually, the situation must have evolved to a stage where a fragment of RNA derived from a catalytic group I- or group II-like intron was transcribed in abundance and began to associate in trans with many different self-splicing introns to facilitate splicing. Perhaps the RNA fragment promoted...

13 Splicing of Precursors to mRNA by the Spliceosome

https://rnajc.ucsf.edu/sites/rnajc.ucsf.edu/files/2012_mRNA_stability.pdf

Single-Molecule mRNA Decay Measurements Reveal Promoter- Regulated mRNA Stability in Yeast

Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa protein (p14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this protein by microsequencing a 14 kDa protein isolated from U2-type spliceosomes. This protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded protein precipitated the 14 kDa protein cross-linked to the branch adenosine, confirming the identity of the p14 cDNA. A combination of immunoblotting, protein microsequencing and immunoprecipitation revealed that p14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2- and U12-type pre-mRNAs, respectively. p14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immuno precipitations indicated that p14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.

/pdf/a-novel-u2-and-u11-u12-snrnp-protein-that-associates-with-1s8deq3iin.pdf

A novel U2 and U11/U12 snRNP protein that associates with the pre-mRNA branch site

"Nought may endure but mutability": spliceosome dynamics and the regulation of splicing.

Publisher Summary This chapter discusses the joining of RNAs by splinted ligation method. T4 DNA ligase is often the method of choice for joining two or more RNA molecules. The chapter also presents procedures for carrying out such ligation reactions. The first procedure gives generic conditions for RNA–RNA ligations using T4 DNA ligase. The two basic steps are hybridization of the RNA substrates to a bridging complementary (cDNA) oligonucleotide template and then incubation with T4 DNA ligase. This protocol is readily adaptable and can be used to make pico- to nano-molar amounts of linear ligated products. The second procedure gives a detailed protocol for the synthesis and confirmation of circular RNAs. The key for generating circles is to carry out the hybridization at low RNA and bridging cDNA oligonucleotide concentrations to favor the formation of circular monomers over linear dimers. The third and fourth procedures are for creating RNAs that contain a single internal radioactive label of high specific activity. The main differences between these protocols are the amounts of radioactivity used and whether the ligase reaction immediately follows the phosphorylation reaction without changing buffers and tubes.

Charles C. Query

Papers

13 Splicing of Precursors to mRNA by the Spliceosome

Single-Molecule mRNA Decay Measurements Reveal Promoter- Regulated mRNA Stability in Yeast

A novel U2 and U11/U12 snRNP protein that associates with the pre-mRNA branch site

"Nought may endure but mutability": spliceosome dynamics and the regulation of splicing.

Joining of RNAs by splinted ligation.