A conserved pseudouridine modification in eukaryotic U2 snRNA induces a change in branch-site architecture

Abstract

The removal of noncoding sequences (introns) from eukaryotic precursor mRNA is catalyzed by the spliceosome, a dynamic assembly involving specific and sequential RNA-RNA and RNA-protein interactions. An essential RNA-RNA pairing between the U2 small nuclear (sn)RNA and a complementary consensus sequence of the intron, called the branch site, results in positioning of the 29OH of an unpaired intron adenosine residue to initiate nucleophilic attack in the first step of splicing. To understand the structural features that facilitate recognition and chemical activity of the branch site, duplexes representing the paired U2 snRNA and intron sequences from Saccharomyces cerevisiae were examined by solution NMR spectroscopy. Oligomers were synthesized with pseudouridine (c) at a conserved site on the U2 snRNA strand (opposite an A-A dinucleotide on the intron strand, one of which forms the branch site) and with uridine, the unmodified analog. Data from NMR spectra of nonexchangeable protons demonstrated A-form helical backbone geometry and continuous base stacking throughout the unmodified molecule. Incorporation of c at the conserved position, however, was accompanied by marked deviation from helical parameters and an extrahelical orientation for the unpaired adenosine. Incorporation of c also stabilized the branch-site interaction, contributing -0.7 kcal/mol to duplex DG8 37 . These findings suggest that the presence of this conserved U2 snRNA pseudouridine induces a change in the structure and stability of the branch-site sequence, and imply that the extrahelical orientation of the branch-site adenosine may facilitate recognition of this base during splicing.

Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg2+ Binding Site is Located Close to the Stacked Branch Adenosine

“…[36,37] NMR structural analyses have shown that this Ψ is responsible for an extrahelical positioning of the branch adenosine in the isolated branch helix. [35,38] Although no Ψ has been identified in group II introns to date, the relationship with the spliceosome makes a bulged-out adenosine in context of the whole group II intron also tempting to propose. However, in the light of the many differences between the two systems and the here presented solution structure of an active D6 construct, the occurrence of a helically stacked branch adenosine should be considered more closely.…”

Mitochondrial Myopathy and Sideroblastic Anemia (MLASA)

“…Pseudouridine is known to affect the structure of tRNAs, affecting base-stacking (5) in the anticodon loop, and when it appears in stems or the anticodon, it strengthens base-pairing (6,(32)(33)(34). When modeled with RNA oligomers, the presence of ⌿ in U2 small nuclear RNA causes a change in the structure of the lariat branch point of pre-messenger RNA (35,36). A functional requirement for ⌿ in U2 small nuclear RNA has also been shown in reconstitution of splicing systems (37)(38)(39).…”

Pseudouridines in spliceosomal snRNAs

“…NMR measurements indicated that these effects then propagated throughout the helix, indicating that pseudouridine has an intrinsic ability to stabilize RNA-RNA base-pairing interactions. In the context of the base-pairing interaction between the pre-mRNA branch site and the U2 branch site recognition region, Newby and Greenbaum have shown that pseudouridine indeed enhances pairing affinity (Newby and Greenbaum, 2001). Taken together, it is conceivable that all these features of pseudouridine, including its ability to alter structure, increase base-stacking, and improve RNA-RNA base-pairing, contribute to the function of splicesomal snRNAs in splicing.…”