Negative cooperativity between Gemin2 and RNA provides insights into RNA selection and the SMN complex's release in snRNP assembly

Abstract

The assembly of snRNP cores, in which seven Sm proteins, D1/D2/F/E/G/D3/B, form a ring around the nonameric Sm site of snRNAs, is the early step of spliceosome formation and essential to eukaryotes. It is mediated by the PMRT5 and SMN complexes sequentially in vivo. SMN deficiency causes neurodegenerative disease spinal muscular atrophy (SMA). How the SMN complex assembles snRNP cores is largely unknown, especially how the SMN complex achieves high RNA assembly specificity and how it is released. Here we show, using crystallographic and biochemical approaches, that Gemin2 of the SMN complex enhances RNA specificity of SmD1/D2/F/E/G via a negative cooperativity between Gemin2 and RNA in binding SmD1/D2/F/E/G. Gemin2, independent of its N-tail, constrains the horseshoe-shaped SmD1/D2/F/E/G from outside in a physiologically relevant, narrow state, enabling high RNA specificity. Moreover, the assembly of RNAs inside widens SmD1/D2/F/E/G, causes the release of Gemin2/SMN allosterically and allows SmD3/B to join. The assembly of SmD3/B further facilitates the release of Gemin2/SMN. This is the first to show negative cooperativity in snRNP assembly, which provides insights into RNA selection and the SMN complex's release. These findings reveal a basic mechanism of snRNP core assembly and facilitate pathogenesis studies of SMA.

Mechanism of assembly of snRNP cores assisted by ICln and the SMN complex in fission yeast

“…In contrast, although human SmF/E/G has been known to form a hexamer, 4 it still has a small, but significant fraction of trimer when characterized by GFC (10%–40% of total SmF/E/G). 6 , 18 The mixture of D1/D2 and F/E/G was eluted with two peaks at positions similar to those of individual F/E/G and D1/D2 ( Figure 1 G), and the SDS-PAGE/CBB staining showed that the bands of D1/D2 and F/E/G had little overlap. This indicates that although there is a stronger interaction between D1/D2 and F/E/G than the other pairs, they could not form a stable D1/D2/F/E/G pentamer or higher oligomers.…”

“… 6 , 16 Gemin2 is a central player for it is not only the acceptor of 5Sm, 17 but also enhances the specificity of RNA binding to 5Sm and mediates the release of the SMN complex from Sm cores. 18 In addition to the tudor domain in the middle, SMN has two highly conserved regions: an N-terminal Gemin2-binding helix, which binds the C-terminal helical domain of Gemin2, 17 , 19 and a C-terminal self-oligomerized YG box, 20 which interacts with Gemin8. 21 Gemin8 further interacts with Gemin6/7 and Unrip.…”

“… 26 Although it had been considered as the main factor determining the RNA specificity in Sm core assembly previously, 27 , 28 , 29 , 30 recent discovery of the role of Gemin2 in RNA selection challenged this idea. 18 Moreover, Gemin5 also plays roles in other processes. 31 …”

Biology of the mRNA Splicing Machinery and Its Dysregulation in Cancer Providing Therapeutic Opportunities

“…Of note, pICln was postulated to not only function as structural chaperone in the formation of the Sm protein ring, but also to prevent the formation of aggregates by unassembled Sm proteins [ 41 ]. In the final steps of the Sm ring assembly, the 6S complex and SmD3-SmB/B’ complex release their pICln subunits and are loaded onto the SMN complex through interactions with Gemin2, where the final heptameric Sm ring is formed [ 44 , 45 ]. Interestingly, Sm proteins are not the only proteins that adopt the Sm fold, as this structural arrangement was also observed for Gemin6 and Gemin7 in the SMN complex.…”

In vitro methylation of the U7 snRNP subunits Lsm11 and SmE by the PRMT5/MEP50/pICln methylosome

“…Among these components, Gemin5 recognizes spliceosomal snRNAs as correct assembly targets by simultaneously binding to their monomethylated cap structure and Sm site ( Battle et al 2006b ; Lau et al 2009 ; Yong et al 2010 ; Wahl and Fischer 2016 ). Gemin2 binds and stabilizes the SmD1/D2/E/F/G pentamer, making extensive contacts with all five subunits ( Zhang et al 2011 ; Grimm et al 2013 ; Yi et al 2020 ). This step likely occurs after the departure of pICln from the 6S intermediate and prior to the delivery of a spliceosomal snRNA by Gemin5.…”