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A snRNP's ordered path to maturity
Abstract and Figures
The U5 snRNP (small ribonucleoprotein) contains several functionally crucial splicing factors that form an extensive interaction network both in the snRNP and within the spliceosome. In this issue of Genes & Development, Weber and colleagues (pp. 1601-1612) shed light on the dynamic assembly of this critical spliceosomal component and elucidate the molecular interactions underlying the ordered addition of Brr2, a pivotal spliceosomal helicase, to the U5 snRNP.
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... RNA splicing is a critical posttranscriptional regulatory mechanism, and ∼60% of noninfectious human diseases arise from splicing disorders; thus, the study of pre-mRNA splicing mechanisms has a high priority for human health (37). Splicing involves highly dynamic, organized, and exceptional compositional and structural rearrangements within the spliceosome during complex assembly, catalytic activation, and disassembly (38). Therefore, any mutations in the genes involved in RNA splicing machinery may give rise to serious diseases (37). ...
Significance
Protein arginine methyltransferase 5 (PRMT5) is involved in various developmental processes by globally regulating pre-mRNA splicing of diverse genes, but the underlying mechanism remains elusive. Here we demonstrate for the first time, to our knowledge, that Arabidopsis PRMT5 promotes the recruitment of the NineTeen Complex and splicing factors in the catalytic reactions to the spliceosome, thus promoting global pre-mRNA splicing. Our findings uncover a key molecular mechanism for PRMT5 in the regulation of pre-mRNA splicing, which fills a major gap in understanding of the role for PRMT5 in spliceosome assembly. Due to the conservation of PRMT5 in plants and animals, our finding is likely a fundamental molecular mechanism applicable to all eukaryotes, thereby shedding light on PRMT5 functions and spliceosome activation in animals.
Little is known about how particle-specific proteins are assembled on spliceosomal small nuclear ribonucleoproteins (snRNPs). Brr2p is a U5 snRNP-specific RNA helicase required for spliceosome catalytic activation and disassembly. In yeast, the Aar2 protein is part of a cytoplasmic precursor U5 snRNP that lacks Brr2p and is replaced by Brr2p in the nucleus. Here we show that Aar2p and Brr2p bind to different domains in the C-terminal region of Prp8p; Aar2p interacts with the RNaseH domain, whereas Brr2p interacts with the Jab1/MPN domain. These domains are connected by a long, flexible linker, but the Aar2p-RNaseH complex sequesters the Jab1/MPN domain, thereby preventing binding by Brr2p. Aar2p is phosphorylated in vivo, and a phospho-mimetic S253E mutation in Aar2p leads to disruption of the Aar2p-Prp8p complex in favor of the Brr2p-Prp8p complex. We propose a model in which Aar2p acts as a phosphorylation-controlled U5 snRNP assembly factor that regulates the incorporation of the particle-specific Brr2p. The purpose of this regulation may be to safeguard against nonspecific RNA binding to Prp8p and/or premature activation of Brr2p activity.
Prp8 is the largest and most highly conserved protein of the spliceosome, encoded by all sequenced eukaryotic genomes but missing from prokaryotes and viruses. Despite all evidence that Prp8 is an integral part of the spliceosomal catalytic center, much remains to be learned about its molecular functions and evolutionary origin. By analyzing sequence and structure similarities between Prp8 and other protein domains, we show that its N-terminal region contains a putative bromodomain. The central conserved domain of Prp8 is related to the catalytic domain of reverse transcriptases (RTs) and is most similar to homologous enzymes encoded by prokaryotic retroelements. However, putative catalytic residues in this RT domain are only partially conserved and may not be sufficient for the nucleotidyltransferase activity. The RT domain is followed by an uncharacterized sequence region with relatives found in fungal RT-like proteins. This part of Prp8 is predicted to adopt an α-helical structure and may be functionally equivalent to diverse maturase/X domains of retroelements and to the thumb domain of retroviral RTs. Together with a previously identified C-terminal domain that has an RNaseH-like fold, our results suggest evolutionary connections between Prp8 and ancient mobile elements. Prp8 may have evolved by acquiring nucleic acid-binding domains from inactivated retroelements, and their present-day role may be in maintaining proper conformation of the bound RNA cofactors and substrates of the splicing reaction. This is only the second example-the other one being telomerase-of the RT recruitment from a genomic parasite to serve an essential cellular function.
RNA helicases are involved in many cellular processes. Pre-mRNA splicing requires eight different DExD/H-box RNA helicases, which facilitate spliceosome assembly and remodelling of the intricate network of RNA rearrangements that are central to the splicing process. Brr2p, one of the spliceosomal RNA helicases, stands out through its unusual domain architecture. In the present review we highlight the advances made by recent structural and biochemical studies that have important implications for the mechanism and regulation of Brr2p activity. We also discuss the involvement of human Brr2 in retinitis pigmentosa, a degenerative eye disease, and how its functions in splicing might connect to the molecular pathology of the disease.
The spliceosome, the ribonucleoprotein assembly that removes the intervening sequences from pre-mRNAs through splicing, is one of the most complex cellular machines. In humans it is composed of -150 proteins and five RNAs (snRNAs). One of the snRNAs, U6, contains sequences analogous to all the RNA elements that form the active site of the group II introns, ribozymes that perform a splicing reaction mechanistically identical to spliceosomal splicing. Interestingly, U6 is the only snRNA that is indispensable for splicing and in vitro, in complex with another snRNA, it can catalyze a primordial splicing reaction in the absence of all other spliceosomal factors. On the other hand, discovery of an RNase H-like domain in a spliceosomal protein that is closely associated with splice sites suggests that proteins may be involved in formation of the active site. Thus, whether the spliceosome is an RNA or RNA-protein catalyst remains uncertain.
Precursor-messenger RNA (pre-mRNA) splicing encompasses two sequential transesterification reactions in distinct active sites of the spliceosome that are transiently established by the interplay of small nuclear (sn) RNAs and spliceosomal proteins. Protein Prp8 is an active site component but the molecular mechanisms, by which it might facilitate splicing catalysis, are unknown. We have determined crystal structures of corresponding portions of yeast and human Prp8 that interact with functional regions of the pre-mRNA, revealing a phylogenetically conserved RNase H fold, augmented by Prp8-specific elements. Comparisons to RNase H–substrate complexes suggested how an RNA encompassing a 5 0-splice site (SS) could bind relative to Prp8 residues, which on mutation, suppress splice defects in pre-mRNAs and snRNAs. A truncated RNase H-like active centre lies next to a known contact region of the 5 0 SS and directed mutagenesis confirmed that this centre is a functional hotspot. These data suggest that Prp8 employs an RNase H domain to help assemble and stabilize the spliceosomal catalytic core, coordinate the activities of other splicing factors and possibly participate in chemical catalysis of 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.
Almost all primary transcripts in higher eukaryotes undergo several splicing events and alternative splicing is a major factor in generating proteomic diversity. Thus, the spliceosome, the ribonucleoprotein assembly that performs splicing, is a highly critical cellular machine and as expected, a very complex one. Indeed, the spliceosome is one of the largest, if not the largest, molecular machine in the cell with over 150 different components in human. A large fraction of the spliceosomal proteome is organized into small nuclear ribonucleoprotein particles by associating with one of the small nuclear RNAs, and the function of many spliceosomal proteins revolve around their association or interaction with the spliceosomal RNAs or the substrate pre-messenger RNAs. In addition to the complex web of protein-RNA interactions, an equally complex network of protein-protein interactions exists in the spliceosome, which includes a number of large, conserved proteins with critical functions in the spliceosomal catalytic core. These include the largest conserved nuclear protein, Prp8, which plays a critical role in spliceosomal function in a hitherto unknown manner. Taken together, the large spliceosomal proteome and its dynamic nature has made it a highly challenging system to study, and at the same time, provides an exciting example of the evolution of a proteome around a backbone of primordial RNAs likely dating from the RNA World.
Proteomic studies have shown that spliceosomal complexes are massive, dynamic ribonucleoprotein assemblies that undergo extensive remodelling and exchange of components as spliceosomes are constructed, activated and recycled. Cryo-electron microscopy has revealed the overall shape of several spliceosomal complexes and the locations of key splicing factors. Over the last two years X-ray crystallography has produced the first detailed structure of a spliceosomal snRNP-the U1 snRNP from human-giving us important new insights into snRNP assembly and 5' splice site recognition. High-resolution structures of domains from the essential Brr2 and Prp8 splicing factors have shed new light on the mechanism of spliceosome activation and the interactions between Prp8 and the spliceosome's RNA core.
Ribonucleoproteins (RNPs) mediate key cellular functions such as gene expression and its regulation. Whereas most RNP enzymes are stable in composition and harbor preformed active sites, the spliceosome, which removes noncoding introns from precursor messenger RNAs (pre-mRNAs), follows fundamentally different strategies. In order to provide both accuracy to the recognition of reactive splice sites in the pre-mRNA and flexibility to the choice of splice sites during alternative splicing, the spliceosome exhibits exceptional compositional and structural dynamics that are exploited during substrate-dependent complex assembly, catalytic activation, and active site remodeling.