Downloaded from rnajournal.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Post-transcriptional modifications modulate conformational dynamics in human U2–U6 snRNA complex KRISHANTHI S. KARUNATILAKA1 and DAVID RUEDA1,2,3,4 1Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA 2Department of Medicine, Section of Virology, Imperial College London, London W12 0NN, United Kingdom 3Single Molecule Imaging Group, MRC Clinical Sciences Center, Imperial College London, London W12 0NN, United Kingdom ABSTRACT The spliceosome catalyzes precursor-mRNA splicing in all eukaryotes. It consists of over 100 proteins and five small nuclear RNAs (snRNAs), including U2 and U6 snRNAs, which are essential for catalysis. Human and yeast snRNAs share structural similarities despite the fact that human snRNAs contain numerous post-transcriptional modifications. Although functions for these modifications have been proposed, their exact roles are still not well understood. To help elucidate these roles in pre-mRNA splicing, we have used single-molecule fluorescence to characterize the effect of several post-transcriptional modifications in U2 snRNA on the conformation and dynamics of the U2–U6 complex in vitro. Consistent with yeast, the human U2–U6 complex reveals the presence of a magnesium-dependent dynamic equilibrium among three conformations. Interestingly, our data show that modifications in human U2 stem I modulate the dynamic equilibrium of the U2–U6 complex by stabilizing the four-helix structure. However, the small magnitude of this effect suggests that post-transcriptional modifications in human snRNAs may have a primary role in the mediation of specific RNA–protein interactions in vivo. Keywords: splicing; human U2–U6 snRNAs; single-molecule FRET; structural dynamics; post-transcriptional modifications INTRODUCTION have suggested that only U2 and U6 are critical for catalysis (Parker et al. 1987; Lesser and Guthrie 1993; O’Keefe et al. Precursor mRNA (pre-mRNA) splicing is an essential process 1996; Segault et al. 1999; Valadkhan and Manley 2001, of eukaryotic gene expression, during which noncoding in- 2003). Furthermore, a recent crystal structure of a large tron sequences are removed from primary transcripts to Prp8 fragment (a U5 snRNP protein) further supports form mature mRNA. It consists of two transesterification RNA-based catalysis (Galej et al. 2013). Although the U2– steps that resemble analogous steps in group II intron splicing U6 complex is essential for pre-mRNA splicing, its structure (Newman 1998; Sontheimer et al. 1999; Gordon et al. 2000). and role in catalysis are still not well understood (Madhani Splicing regulation is critical to maintain proper cellular func- and Guthrie 1992; Wolff and Bindereif 1993; Sun and tion, and defects in splicing have been linked to neurodegen- Manley 1995; Sashital et al. 2004; Mefford and Staley 2009). erative disorders and various cancers (Kalnina et al. 2005; The U6 snRNA contains two highly conserved sequences Licatalosi and Darnell 2006; Pettigrew and Brown 2008). ′ that are involved in catalysis (Fig. 1): the 5 splice site recog- Splicing is highly conserved in all eukaryotes (from yeast to nition sequence (ACAGAGA box) and the AGC triad (Lesser humans) and is catalyzed by the spliceosome, a dynamic ri- and Guthrie 1993; Hilliker and Staley 2004). In addition, it bonucleoprotein (RNP) complex assembled from five small also contains the highly conserved U74 (analogous to U80 nuclear RNAs (U1, U2, U4, U5, and U6 snRNAs) and a large in yeast) in the U6 intramolecular stem–loop (U6 ISL), which number of proteins (Stark and Luhrmann 2006; Ritchie et al. is involved in metal ion binding and coordination for splicing 2009; Wahl et al. 2009). In addition to catalyzing pre-mRNA (Huppler et al. 2002; Valadkhan et al. 2009). Based on in vivo splicing, the spliceosome plays an important central role in genetic and in vitro NMR studies (Madhani and Guthrie alternative splicing regulation (Saltzman et al. 2011). Follow- 1992; Sun and Manley 1995; Sashital et al. 2004) two different ing a highly orchestrated assembly, the activated spliceosome secondary structures for the yeast U2–U6 (yU2–U6) snRNA maintains only the U2, U5, and U6 snRNPs and releases U1 and U4 (Wahl et al. 2009). However, several lines of evidence © 2013 Karunatilaka and Rueda This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publica- 4Corresponding author tion date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 E-mail [email protected] months, it is available under a Creative Commons License (Attribution- Article published online ahead of print. Article and publication date are at NonCommercial 3.0 Unported), as described at http://creativecommons. http://www.rnajournal.org/cgi/doi/10.1261/rna.041806.113. org/licenses/by-nc/3.0/. RNA 20:1–8; Published by Cold Spring Harbor Laboratory Press for the RNA Society 1 Downloaded from rnajournal.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Karunatilaka and Rueda from humans can adopt primarily the four-helix junction structure but also the three-helix junction (Zhao et al. 2013). Although the primary sequences of U2 and U6 are highly conserved from yeast to humans (Brow and Guthrie 1988), the human snRNAs contain numerous post-transcriptional modifications such as 2′-O-methyl groups and pseudouri- dines (Massenet et al. 1998; Yu et al. 1998; Sashital et al. 2007). The majority of these conserved modifications are dis- tributed in functionally important regions of the snRNAs, where they can affect pre-mRNA splicing by regulating RNA–RNA and RNA–protein interactions (Newby et al. 2002b; Karijolich and Yu 2010). Out of the five snRNAs in humans, U2 is the most highly modified, containing 10 2′-O-methylated residues and 13 pseudouridines (Fig. 1A; Donmez et al. 2004). Recent NMR and UV melting studies have shown that although the human and yeast U2 stem I structures are nearly isosteric in vitro, post-transcriptional FIGURE 1. hU2–U6 snRNA complex and single-molecule experimen- tal setup. (A) Proposed four-helix structure of the hU2–U6 complex modifications in human U2 significantly increase its stability with FRET donor-D (Cy3, blue), FRET acceptor-A (Cy5, red), and bio- (Sashital et al. 2007). Since the formation of helix Ib requires tin-B (gray). Highly conserved regions of U6 snRNA (ACAGAGA, AGC the melting of base pairs in stem I, the increased stability of triad, and U74) are shown in red. (B) Proposed three-helix structure of the hU2–U6 complex containing helix Ib. (C) Total internal reflection the modified U2 stem I may hinder formation of Helix Ib fluoresence (TIRF)-based single-molecule experimental setup. Fluro- in human U2–U6 (hU2–U6). phore-labeled RNA was surface immobilized via a biotin-streptavidin Here, we have used single-molecule Förster resonance en- linkage and excited using a 532-nm laser beam. Fluoresence intensities ergy tranfer (smFRET) to characterize the structural dynam- of donor (blue) and acceptor (red) fluorophores are collected through – the objective and detected using a CCD camera. ics of a protein-free U2 U6 from humans (Fig. 1A,C). smFRET experiments help dissect RNA folding pathways by revealing the presence of transient folding intermediates complex have been proposed: a three- and a four-helix junc- that can be hidden in ensemble-averaged experiments and tion (Fig. 1A,B). The three-helix junction (Fig. 1B) comprises by providing real-time dynamic information (Rueda and the so-called Helix Ib that forms between the highly con- Walter 2005; Aleman et al. 2008; Karunatilaka and Rueda served AGC triad in U6 and three residues in U2 snRNA 2009; Zhao and Rueda 2009). Our data show that, similarly (Madhani and Guthrie 1992; Mefford and Staley 2009). A re- to the yeast complex, the hU2–U6 also adopts at least three cent NMR and SAXS study on the yU2–U6 complex has pro- conformations in dynamic equilibrium, and that Mg2+ ions vided further evidence for the existence of a three-helix modulate their relative stability. Furthermore, post-tran- junction in solution (Burke et al. 2012). The resulting struc- scriptional modifications in the U2 stem I stabilize the tural model positions the pre-mRNA recognition sites, the four-helix structure. However, the relatively small energetic AGC triad, and U80 on the same face of the molecule. magnitude of this effect raises the interesting possibility In the four-helix junction (Fig. 1A), the AGC triad forms that post-transcriptional modifications in U2 play a more im- three intramolecular base pairs within U6 snRNA that extend portant role in protein-specific recognition in vivo. the U6 ISL (Sashital et al. 2004). Interestingly, compensa- tory mutational genetic studies have shown that Helix Ib res- RESULTS idues from both U2 and U6 participate in intramolecular rather than intermolecular base-pairing in humans, suggest- hU2–U6 snRNA adopts three dynamic ing that, in humans, the U2–U6 complex forms the four-helix conformations junction (Wolff and Bindereif 1993; Sun and Manley 1995). Since then, several ensemble-averaged studies have provided To characterize the structural dynamics of the unmodified evidence that support a hypothesis in which the U2–U6 com- hU2–U6 complex, we have used a previously characterized plex can adopt multiple conformations corresponding to dif- labeling strategy (Fig. 1A,C; Guo et al. 2009). The U6 strand ferent activation states of the spliceosome (Rhode et al. 2006; was labeled with a FRET pair (Cy3, Cy5) at the 5′ end and at Sashital et al. 2007). Recently, we have shown by single-mol- uracil 64, respectively, and a 3′-biotin for surface immobiliza- ecule fluorescence that a protein-free U2–U6 snRNA complex tion. This labeling scheme enables monitoring of the dynam- from yeast can adopt at least three conformations, consistent ics of helix III relative to the U6 ISL, a motion that is thought with the previously proposed structures (Guo et al.