Ashwin Chari

An Assembly Chaperone Collaborates with the SMN Complex to Generate Spliceosomal SnRNPs

Spliceosomal small nuclear ribonucleoproteins (snRNPs) are essential components of the nuclear pre-mRNA processing machinery. A hallmark of these particles is a ring-shaped core domain generated by the binding of Sm proteins onto snRNA. PRMT5 and SMN complexes mediate the formation of the core domain in vivo. Here, we have elucidated the mechanism of this reaction by both biochemical and structural studies. We show that pICln, a component of the PRMT5 complex, induces the formation of an otherwise unstable higher-order Sm protein unit. In this state, the Sm proteins are kinetically trapped, preventing their association with snRNA. The SMN complex subsequently binds to these Sm protein units, dissociates pICln, and catalyzes ring closure on snRNA. Our data identify pICln as an assembly chaperone and the SMN complex as a catalyst of spliceosomal snRNP formation. The mode of action of this combined chaperone/catalyst system is reminiscent of the mechanism employed by DNA clamp loaders.

Translation and replication of hepatitis C virus genomic RNA depends on ancient cellular proteins that control mRNA fates.

Inevitably, viruses depend on host factors for their multiplication. Here, we show that hepatitis C virus (HCV) RNA translation and replication depends on Rck/p54, LSm1, and PatL1, which regulate the fate of cellular mRNAs from translation to degradation in the 5′-3′-deadenylation-dependent mRNA decay pathway. The requirement of these proteins for efficient HCV RNA translation was linked to the 5′ and 3′ untranslated regions (UTRs) of the viral genome. Furthermore, LSm1–7 complexes specifically interacted with essential cis-acting HCV RNA elements located in the UTRs. These results bridge HCV life cycle requirements and highly conserved host proteins of cellular mRNA decay. The previously described role of these proteins in the replication of 2 other positive-strand RNA viruses, the plant brome mosaic virus and the bacteriophage Qß, pinpoint a weak spot that may be exploited to generate broad-spectrum antiviral drugs.

A Comprehensive Interaction Map of the Human Survival of Motor Neuron (SMN) Complex

Assembly of the Sm-class of U-rich small nuclear ribonucleoprotein particles (U snRNPs) is a process facilitated by the macromolecular survival of motor neuron (SMN) complex. This entity promotes the binding of a set of factors, termed LSm/Sm proteins, onto snRNA to form the core structure of these particles. Nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2-8 and unrip have been identified as the major components of the SMN complex. So far, however, only little is known about the architecture of this complex and the contribution of individual components to its function. Here, we present a comprehensive interaction map of all core components of the SMN complex based upon in vivo and in vitro methods. Our studies reveal a modular composition of the SMN complex with the three proteins SMN, Gemin8, and Gemin7 in its center. Onto this central building block the other components are bound via multiple interactions. Furthermore, by employing a novel assay, we were able to reconstitute the SMN complex from individual components and confirm the interaction map. Interestingly, SMN protein carrying an SMA-causing mutation was severely impaired in formation of the SMN complex. Finally, we show that the peripheral component Gemin5 contributes an essential activity to the SMN complex, most likely the transfer of Sm proteins onto the U snRNA. Collectively, the data presented here provide a basis for the detailed mechanistic and structural analysis of the assembly machinery of U snRNPs.

Biogenesis of spliceosomal small nuclear ribonucleoproteins

Virtually, all eukaryotic mRNAs are synthesized as precursor molecules that need to be extensively processed in order to serve as a blueprint for proteins. The three most prevalent processing steps are the capping reaction at the 5′‐end, the removal of intervening sequences by splicing, and the formation of poly (A)‐tails at the 3′‐end of the message by polyadenylation. A large number of proteins and small nuclear ribonucleoprotein complexes (snRNPs) interact with the mRNA and enable the different maturation steps. This chapter focuses on the biogenesis of snRNPs, the major components of the pre‐mRNA splicing machinery (spliceosome). A large body of evidence has revealed an intricate and segmented pathway for the formation of snRNPs that involves nucleo‐cytoplasmic transport events and elaborates assembly strategies. We summarize the knowledge about the different steps with an emphasis on trans‐acting factors of snRNP maturation of higher eukaryotes. WIREs RNA 2011 2 718–731 DOI: 10.1002/wrna.87

Deciphering the assembly pathway of Sm-class U snRNPs.

The assembly of the Sm‐class of uridine‐rich small nuclear ribonucleoproteins (U snRNPs), albeit spontaneous in vitro, has recently been shown to be dependent on the aid of a large number of assisting factors in vivo. These factors are organized in two interacting units termed survival motor neuron (SMN)‐ and protein arginine methyltransferase 5 (PRMT5)‐complexes, respectively. While the PRMT5‐complex acts early in the assembly pathway by activating common proteins of U snRNPs, the SMN‐complex functions to join proteins and RNA in a highly ordered, apparently regulated manner. Here, we summarize recent progress in the understanding of this process and discuss the influence exerted by the aforementioned trans‐acting factors.

The role of RNP biogenesis in spinal muscular atrophy.

Mutations that affect pre-mRNA processing are the cause for many genetic diseases. Most such mutations target cis-acting regulatory sequences in a given transcript, thus preventing its proper maturation. Only recently however, mutations in trans-acting factors involved in pre-mRNA processing have likewise been linked to disease. One prominent example is spinal muscular atrophy (SMA), a monogenic, neuromuscular disorder caused by reduced levels of functional survival motor neuron (SMN) protein. This ubiquitous factor is part of a complex that mediates the formation of spliceosomal snRNPs. The detailed biochemical investigation of SMN under normal conditions and in SMA has provided clues how mutations in factors with general functions elicit tissue-specific phenotypes.

Molecular architecture of the Saccharomyces cerevisiae activated spliceosome.

The activated spliceosome (Bact) is in a catalytically inactive state and is remodeled into a catalytically active machine by the RNA helicase Prp2, but the mechanism is unclear. Here, we describe a 3D electron cryomicroscopy structure of the Saccharomyces cerevisiae Bact complex at 5.8-angstrom resolution. Our model reveals that in Bact, the catalytic U2/U6 RNA-Prp8 ribonucleoprotein core is already established, and the 5′ splice site (ss) is oriented for step 1 catalysis but occluded by protein. The first-step nucleophile—the branchsite adenosine—is sequestered within the Hsh155 HEAT domain and is held 50 angstroms away from the 5′ss. Our structure suggests that Prp2 adenosine triphosphatase–mediated remodeling leads to conformational changes in Hsh155’s HEAT domain that liberate the first-step reactants for catalysis.

Evolution of an RNP assembly system: A minimal SMN complex facilitates formation of UsnRNPs in Drosophila melanogaster

In vertebrates, assembly of spliceosomal uridine-rich small nuclear ribonucleoproteins (UsnRNPs) is mediated by the SMN complex, a macromolecular entity composed of the proteins SMN and Gemins 2–8. Here we have studied the evolution of this machinery using complete genome assemblies of multiple model organisms. The SMN complex has gained complexity in evolution by a blockwise addition of Gemins onto an ancestral core complex composed of SMN and Gemin2. In contrast to this overall evolutionary trend to more complexity in metazoans, orthologs of most Gemins are missing in dipterans. In accordance with these bioinformatic data a previously undescribed biochemical purification strategy elucidated that the dipteran Drosophila melanogaster contains an SMN complex of remarkable simplicity. Surprisingly, this minimal complex not only mediates the assembly reaction in a manner very similar to its vertebrate counterpart, but also prevents misassembly onto nontarget RNAs. Our data suggest that only a minority of Gemins are required for the assembly reaction per se, whereas others may serve additional functions in the context of UsnRNP biogenesis. The evolution of the SMN complex is an interesting example of how the simplification of a biochemical process contributes to genome compaction.

Structural Basis of Assembly Chaperone- Mediated snRNP Formation

Small nuclear ribonucleoproteins (snRNPs) represent key constituents of major and minor spliceosomes. snRNPs contain a common core, composed of seven Sm proteins bound to snRNA, which forms in a step-wise and factor-mediated reaction. The assembly chaperone pICln initially mediates the formation of an otherwise unstable pentameric Sm protein unit. This so-called 6S complex docks subsequently onto the SMN complex, which removes pICln and enables the transfer of pre-assembled Sm proteins onto snRNA. X-ray crystallography and electron microscopy was used to investigate the structural basis of snRNP assembly. The 6S complex structure identifies pICln as an Sm protein mimic, which enables the topological organization of the Sm pentamer in a closed ring. A second structure of 6S bound to the SMN complex components SMN and Gemin2 uncovers a plausible mechanism of pICln elimination and Sm protein activation for snRNA binding. Our studies reveal how assembly factors facilitate formation of RNA-protein complexes in vivo.

The inhibition mechanism of human 20S proteasomes enables next-generation inhibitor design

Insights into proteasome inhibition Proteasomes are large protein complexes that degrade and remove proteins to maintain proper cellular physiology and growth. Proteasomes are a validated target for anticancer therapy, but drug design has been hampered by poor understanding of how inhibitors interact with the active site. Schrader et al. succeeded in crystallizing various proteasome-inhibitor complexes. They subsequently obtained crystal structures for the native human proteasome and eight different inhibitor complexes at resolutions between 1.9 and 2.1 Å. The inhibitors sampled include drugs that are approved or in trial for cancer treatment. Science, this issue p. 594 High-resolution structures of human 20S proteasomes reveal chemical principles for next-generation drug design. The proteasome is a validated target for anticancer therapy, and proteasome inhibition is employed in the clinic for the treatment of tumors and hematological malignancies. Here, we describe crystal structures of the native human 20S proteasome and its complexes with inhibitors, which either are drugs approved for cancer treatment or are in clinical trials. The structure of the native human 20S proteasome was determined at an unprecedented resolution of 1.8 angstroms. Additionally, six inhibitor-proteasome complex structures were elucidated at resolutions between 1.9 and 2.1 angstroms. Collectively, the high-resolution structures provide new insights into the catalytic mechanisms of inhibition and necessitate a revised description of the proteasome active site. Knowledge about inhibition mechanisms provides insights into peptide hydrolysis and can guide strategies for the development of next-generation proteasome-based cancer therapeutics.