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The assembly of spliceosomal U snRNPs depends on the coordinated action of PRMT5 and SMN complexes in vivo. These trans-acting factors enable the faithful delivery of seven Sm proteins onto snRNA and the formation of the common core of snRNPs. To gain mechanistic insight into their mode of action, we reconstituted the assembly machinery from recombinant sources. We uncover a stepwise and ordered formation of distinct Sm protein complexes on the PRMT5 complex, which is facilitated by the assembly chaperone pICln. Upon completion, the formed pICln-Sm units are displaced by new pICln-Sm protein substrates and transferred onto the SMN complex. The latter acts as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to prevent mis-assembly and to ensure the transfer of Sm proteins to cognate RNA. Investigation of mutant SMN complexes provided insight into the contribution of individual proteins to these activities. The biochemical reconstitution presented here provides a basis for a detailed molecular dissection of the U snRNP assembly reaction., (© 2015 The Authors.)

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 For further resources related to this article, please visit the WIREs website., (Copyright © 2011 John Wiley & Sons, Ltd.)

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.