Structural basis for snRNA recognition by the double-WD40 repeat domain of Gemin5

Abstract

Assembly of the spliceosomal small nuclear ribonucleoparticle (snRNP) core requires the participation of the multisubunit SMN (survival of motor neuron) complex, which contains SMN and several Gemin proteins. The SMN and Gemin2 subunits directly bind Sm proteins, and Gemin5 is required for snRNP biogenesis and has been implicated in snRNA recognition. The RNA sequence required for snRNP assembly includes the Sm site and an adjacent 3 ′ stem-loop, but a precise understanding of Gemin5's RNA-binding specificity is lacking. Here we show that the N-terminal half of Gemin5, which is composed of two juxtaposed seven-bladed WD40 repeat domains, recognizes the Sm site. The tandem WD40 repeat domains are rigidly held together to form a contiguous RNA-binding surface. RNA-contacting residues are located mostly on loops between β strands on the apical surface of the WD40 domains. Structural and biochemical analyses show that base-stacking interactions involving four aromatic residues and hydrogen bonding by a pair of arginines are crucial for specific recognition of the Sm sequence. We also show that an adenine immediately 5 ′ to the Sm site is required for efficient binding and that Gemin5 can bind short RNA oligos in an alternative mode. Our results provide mechanistic understandings of Gemin5's snRNA-binding specificity as well as valuable insights into the molecular mechanism of RNA binding by WD40 repeat proteins in general.

Impact of RNA–Protein Interaction Modes on Translation Control: The Versatile Multidomain Protein Gemin5

“…Gemin5 is an abundant protein of about 170 kDa, which contains different functional domains (Figure b). The N‐terminal half of Gemin5 (residues 1–739) is composed of two juxtaposed seven‐bladed WD40 domains, which form a contiguous RNA‐binding surface . The recognition of the Sm site and the m 7 G cap of pre‐snRNAs takes place by two binding sites via the respective base‐specific interactions .…”

Negative Cooperativity between Gemin2 and RNA provides Insights into RNA Selection and the SMN Complex’s Release in snRNP Assembly

“…The structures of the 6S complex (human 5Sm plus Drosophila pICln), the 8S complex (6S plus Drosophila Gemin2/SMN-N-terminal domain) and the later phase human SMN(26-62)/Gemin2/5Sm complex (hereafter we will refer to it as the 7S complex for brevity because it is equivalent to the 7S complex reported earlier which contains additional segments of SMN(12)) provide detailed insights into the mechanisms of the first phase, the transition from the first phase to the second phase, and the initial state of the second phase (14,15). Just recently, the structures of the complexes comprised of Gemin5's N-terminal WD domain and RNA provide details of the recognition of RNA by Gemin5 (32)(33)(34).…”

“…Gemin4 usually forms a complex with Gemin3, but its role is unknown (28). Gemin5 is the component to initially bind pre-snRNAs and deliver them to the rest of the SMN complex for assembly into the Sm core (29), and is currently considered to be the protein conferring the RNA assembly specificity by direct recognition of the snRNP code (30)(31)(32)(33)(34)(35).…”

Structural Insights into Negative Cooperativity between Gemin2 and RNA in Sm-class snRNP Assembly

“…The final structure has improved quality as indicated by the reduction of R and Rfree from 25.3% and 33.2% to 21.7% and 29.5% respectively ( Supplementary Table S1). The final model (PDB code 5XJL) contains SmD1 (residues 1-81), SmD2 (residues 21-77 and 89-117), SmF (residues 3-76), SmE (residues 14-90), SmG (residues 8-52 and 55-72), Gemin2 (residues 22-31, 48-73, 79-124, 135-151, and 174-278), and SMN (residues [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51]. Ramachandran plot shows 95.8% of the dihedral angles in favored region, 2.6% in additional allowed region, and 1.6 (9 out of 576) in disallowed region ( Supplementary Table S1).…”

“…Gemin5 is the component to initially bind precursor (pre)-snRNAs and deliver them to the rest of the SMN complex for assembly into the Sm core [35]. Although it had long been considered as the protein conferring the RNA assembly specificity by direct recognition of the snRNP code [36][37][38][39][40][41], our recent study indicates that it is Gemin2 that plays the role of enhancing RNA assembly specificity by an unusual way, via a negative cooperativity with RNA in binding to 5Sm (https://doi.org/10.1101/312124).…”