Dynamic stacking of an expected branch point adenosine in duplexes containing pseudouridine-modified or unmodified U2 snRNA sites

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Highlights

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    A syn-guanosine precedes the expected branch site of a splice site RNA duplex.

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    A conserved U2 snRNA pseudouridine lacks detectable effects on the RNA conformation.

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    The expected branch site adenosine pairs with pseudouridine within this RNA duplex.

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    Other factors/contexts may extrude the branch point adenosine for pre-mRNA splicing.

Abstract

The pre-mRNA branch point sequence (BPS) anneals with a pseudouridine-modified region of the U2 small nuclear (sn)RNA, and offers a 2′ hydroxyl group of a bulged adenosine as the nucleophile for the first catalytic step of pre-mRNA splicing. To increase our structural understanding of branch site selection, we characterized a duplex containing a BPS sequence that is common among multicellular eukaryotes (5′-UACUGAC-3′) and the complementary U2 snRNA site using NMR. A major conformation of the expected branch site adenosine stacked within the duplex and paired with the conserved pseudouridine of the U2 snRNA strand. In contrast, the guanosine preceding the branch site appeared flexible and had weak contacts with the surrounding nucleotides. Pseudouridine-modified and unmodified U2 snRNA–BPS-containing duplexes remained structurally similar. These results highlight the importance of auxiliary factors to achieve the active bulged conformation of the branch site nucleophile for the first step of pre-mRNA splicing.

Introduction

The highly regulated process of pre-mRNA splicing offers an important source of transcript diversity [1] and is often dysregulated in cancers and hematologic malignancies [2]. The spliceosome assembles through the actions of protein co-factors coupled with dynamic base-pairing among small nuclear (sn)RNAs and specific pre-mRNA sites. At the active site for catalysis, a post-transcriptionally modified region of the U2 snRNA forms a short, base-paired duplex with the branch point sequence (BPS) of pre-mRNA introns [3]. The pre-mRNA splicing reaction then proceeds in two sequential catalytic steps: First, an adenosine within the BPS typically offers a 2′ hydroxyl group for nucleophilic attack on the 5′ splice site [4]. The products of this reaction are a 2′-5′-linkage at the “branch site” adenosine and 3′-hydroxyl group of the upstream 5′-exon. Second, this 3′ hydroxyl of the exon attacks the downstream 3′ splice site, which releases the 2′–5′ branched intron lariat from the spliced exon-exon junction of the mRNA.

Despite emerging views of spliceosome intermediates, selection of the BPS nucleophile remains only partially understood. The BPS of human introns are relatively degenerate (YUNAY where Y is pyrimidine and N any nucleotide) compared to Saccharomyces cerevisiae BPS (5′-UACUAAC-3′) [5,6]. In principle, either the branch site or preceding nucleotide could base-pair with a highly conserved pseudouridine (Ψ35) in the complementary region of the U2 snRNA, leaving the other nucleotide unpaired. The second adenosine (underlined) typically serves as the branch site nucleophile, yet several findings indicate that the branch site choice has some plasticity. In human nuclear extracts, branch site mutations typically activate a nearby cryptic branch site rather than preventing pre-mRNA splicing [7,8], and substitution of the canonical branch site with deoxy-adenosine induces efficient branching from the preceding nucleotide [4]. Branch site selection appears to be more stringent in yeast than humans, since pyrimidine mutations of the branch site adenosine in S. cerevisiae reduce splicing efficiency at the mutated nucleotide [9]. Nevertheless, systematic use of an orthogonal U2 snRNA – BPS system [10] demonstrates that a “bulged” conformation of the branch site adenosine is as an important criterion to produce the intron lariat in both S. cerevisiae and humans.

The potential for base-pairing between Ψ35 and either the branch site adenosine or preceding nucleotide raises the question of how the “bulged” conformation of the nucleophile is selected for the first step of splicing. The Ψ35-modification (compared to unmodified U) promotes bulging of the branch site adenosine in NMR characterization of a duplex containing the yeast consensus U2 snRNA and “AA”-containing BPS sequences [11]. On the other hand, crystal structures containing pseudouridine-modified U2 snRNA duplexes with “GA”- or “AA”-containing BPS sequences capture either the expected branch site or the preceding nucleotide in an extrahelical position [12]. Here, we investigate the solution conformation of a “GA”-containing BPS – U2 snRNA duplex, which is a common sequence among splice sites of multicellular eukaryotes. Our results show that a predominant conformation of the expected branch site adenosine (underlined) of a 5′-UACUGAC-3′ BPS pairs with the pseudouridine in the U2 snRNA-containing duplex, whereas the preceding guanosine appears to be unstacked. Comparison of pseudouridylated or unmodified duplexes further shows the conserved Ψ35-modification of the U2 snRNA makes few detectable changes in the BPS conformation of this sequence context.

Section snippets

NMR sample preparation

HPLC-purified RNA oligonucleotides (BPS, 5′-GCUACUGACGA; U2, 5′-CGUAGUAGCA, or Ψ-U2, 5′-CGΨAGUAGCA) (GE Healthcare Dharmacon, Inc.) were dissolved in a filter-sterilized solution of 10 mM potassium phosphate, 0.25 mM ethylenediaminetetraacetic acid at pH 6.8 in RNase-free 90% water/10% D2O. The strands were mixed in equal molarity at a concentration of 1.1 mM, annealed by incubating at 60 °C for 15 min followed by gradual cooling to 4 °C, and transferred to a thin-walled Shigemi tube (Allison

NMR characterization of “GA”-Containing BPS – U2 snRNA duplexes

We chose a “GA”-containing sequence with G preceding the expected branch site (A8) (sequence inset in Fig. 1a) for NMR studies of a typical BPS of multicellular organisms [5]. We annealed the BPS with a complementary U2 snRNA region either including a conserved pseudouridine modification or for comparison, the unmodified counterpart (Ψ35 or U35 in 5′-GΨAGUAG-3′ or 5′-GUAGUAG-3′, numbered 34–39). Flanking GC base-pairs and 3′-A overhangs increased duplex stability for NMR measurements [16]. The

Discussion

Our NMR characterization of an isolated, “GA”-containing BPS – U2 snRNA duplex shows that a major conformation of expected branch site adenosine stacks within the duplex and pairs with Ψ35 of the U2 snRNA strand, whereas the preceding guanosine is primarily extrahelical. This base orientation and pairing appears independent of the Ψ35-modification in the U2 snRNA strand. The reason for the contrast between this “GA” branch point and an “AA” branch point sequence, for which pseudouridine

Acknowledgments

This work was supported by the National Institutes of Health grants R01GM117005 and R01GM070503. We thank Dr. Samuel E. Butcher for guidance with NMR sample preparation.

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    • Cited by (0)

    1

    Present address: Hauptman-Woodward Medical Research Institute, Buffalo, NY 14203, USA.

    2

    Present address: Regeneron Pharmaceuticals, Inc. Rensselaer, NY 12144, USA.

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