A crystal structure of a collaborative RNA regulatory complex reveals mechanisms to refine target specificity

Abstract

In the Caenorhabditis elegans germline, fem-3 Binding Factor (FBF) partners with LST-1 to maintain stem cells. A crystal structure of an FBF-2/LST-1/RNA complex revealed that FBF-2 recognizes a short RNA motif different from the characteristic 9-nt FBF binding element, and compact motif recognition coincided with curvature changes in the FBF-2 scaffold. Previously, we engineered FBF-2 to favor recognition of shorter RNA motifs without curvature change (Bhat et al., 2019). In vitro selection of RNAs bound by FBF-2 suggested sequence specificity in the central region of the compact element. This bias, reflected in the crystal structure, was validated in RNA-binding assays. FBF-2 has the intrinsic ability to bind to this shorter motif. LST-1 weakens FBF-2 binding affinity for short and long motifs, which may increase target selectivity. Our findings highlight the role of FBF scaffold flexibility in RNA recognition and suggest a new mechanism by which protein partners refine target site selection.

Diverse Roles of PUF Proteins in Germline Stem and Progenitor Cell Development in C. elegans

“…Mutational analysis of Drosophila Pumilio revealed the amino acids mediating contacts with the mRNA and protein partners (Zamore et al, 1997;Wharton et al, 1998;Wharton, 1999, 2001). Subsequent structural studies of Drosophila, C. elegans, and mammalian PUFs extended the genetic and biochemical data and have identified the TRMs that contact RNA on the concave surface as well as the sites on the convex surface that interact with protein partners (Edwards et al, 2001;Wang et al, 2002Wang et al, , 2009Campbell et al, 2014;Bhat et al, 2019;Qiu et al, 2019). Differences in the PUF RNA-binding domains result in distinct RNA motifs bound by PUF homologs.…”

“…Using SEQRS (in vitro selection, high-throughput sequencing of RNA, and sequence specificity landscapes), analysis of RNA-binding preference of FBF-2 PUF domain bound to a 150-amino-acid LST-1 fragment FIGURE 3 | Modification of FBF biological activity though interactions with protein partners. (A) On its own, FBF PUF domain binds to target mRNAs containing a canonical 9-nt motif (Wang et al, 2009;Bhat et al, 2019;Qiu et al, 2019). (B) FBF PUF domain's RNA-binding specificity can be influenced by interactions with protein partners such as CPB-1 (Menichelli et al, 2013) and LST-1 (Qiu et al, 2019).…”

“…(A) On its own, FBF PUF domain binds to target mRNAs containing a canonical 9-nt motif (Wang et al, 2009;Bhat et al, 2019;Qiu et al, 2019). (B) FBF PUF domain's RNA-binding specificity can be influenced by interactions with protein partners such as CPB-1 (Menichelli et al, 2013) and LST-1 (Qiu et al, 2019). (C) FBFs can repress target mRNAs by recruiting deadenylase complex (Goldstrohm et al, 2006;Suh et al, 2009).…”

“…(D) FBFs can promote mRNA polyadenylation by interacting with the poly(A) polymerase complex (Eckmann et al, 2002;Suh et al, 2009). containing one of FBF-binding sites revealed a distinct RNAbinding specificity of the FBF-2/LST-1 complex (Qiu et al, 2019). Crystal structure of FBF-2 in complex with a 24-amino-acid fragment of LST-1 and an 8-nucleotide RNA oligo isolated by in vitro selection showed that FBF-2 PUF domain changes its RNA-binding mode to 1:1 association of PUM repeats R4-R5 with GA in positions four and five (Qiu et al, 2019).…”

Principles of mRNA control by human PUM proteins elucidated from multi-modal experiments and integrative data analysis

“…Furthermore, the sequence specificity by PUM-HD across species can be predicted from the identity of these three key amino acids across the helical repeats in any given PUM-HD [9]. Thus, there are slight differences in the exact set of sequences recognized by the PUM-HD of different PUF family members and, in addition, interactions with protein partners can alter sequence preference [10–12].…”