Modified nucleotides at the 5' end of human U2 snRNA are required for spliceosomal E-complex formation

Abstract

U2 snRNA, a key player in nuclear pre-mRNA splicing, contains a 5'-terminal m3G cap and many internal modifications. The latter were shown in vertebrates to be generally required for U2 function in splicing, but precisely which residues are essential and their role in snRNP and/or spliceosome assembly is presently not clear. Here, we investigated the roles of individual modified nucleotides of HeLa U2 snRNA in pre-mRNA splicing, using a two-step in vitro reconstitution/complementation assay. We show that the three pseudouridines and five 2'O-methyl groups within the first 20 nucleotides of U2 snRNA, but not the m3G cap, are required for efficient pre-mRNA splicing. Individual pseudouridines were not essential, but had cumulative effects on U2 function. In contrast, four of five 2'O-methylations (at positions 1, 2, 12, and 19) were individually required for splicing. The in vitro assembly of 17S U2 snRNPs was not dependent on the presence of modified U2 residues. However, individual internal modifications were required for the formation of the ATP-independent early spliceosomal E complex. Our data strongly suggest that modifications within the first 20 nucleotides of U2 play an important role in facilitating the interaction of U2 with U1 snRNP and/or other factors within the E complex.

FIGURE 1.

Primary and secondary structure of human U2 snRNA. (m₃G) N^2,2,7-trimethylguanosine; (m) 2′O-methyl; (Ψ) pseudouridine; (m6) N⁶-methyl. The Sm site and branchpoint recognition sequences are indicated. Stem loops are numbered with roman numerals.

FIGURE 2.

Internal modifications in the first 24 nucleotides of U2 snRNAs are required for splicing. (A) The sequence composition of the chemically synthesized oligonucleotides corresponding to the first 24 nucleotides of U2 snRNA. The following notation is used: ΔΨm, replacement of all pseudouridines (Ψ) by uridines (U) and deletion of all 2′O-methylations (2′O-Me); (Ψm) retention of all Ψ and 2′O-Me; (ΔΨ) replacement of all Ψs by U; (ΔΨn) replacement of Ψ at position n with U; (Δm) deletion of all 2′O-Me; (Δmn) deletion of 2′O-Me at position n. The oligonucleotides were ligated to in vitro-transcribed U2 starting at G25. For comparison, sequences of purified HeLa U2 and U2 transcript are shown. (B) Internal modifications within the first 24 nucleotides of U2 snRNA are required for efficient complementation of splicing. Splicing complementation of U2 snRNAs differing in the number and type of internal modifications was assayed in the two-step reconstitution system by monitoring splicing of ³²P-labeled pre-mRNA (lane 1). Untreated (lane 2), mock-depleted (lane 3), and U2-depleted (lane 4) nuclear extract are shown as controls. (Lanes 5–18) Complementation of U2-depleted nuclear extract with U2 snRNPs reconstituted with the U2 snRNA indicated above each lane. RNA was analyzed by denaturing PAGE and visualized by autoradiography.

FIGURE 3.

Chimaeric U2 snRNAs are not pseudouridylated and remain stable during reconstitution and splicing in the HeLa cell nuclear extract. (A) Chimaeric U2 RNAs Ψm (lanes 1,2) and ΔΨm (lanes 3,4) bearing internal 5′[³²P]U labels downstream of G25 were treated with nuclease P1 before (lanes 1,3) or after reconstitution and splicing (lanes 2,4) and analyzed by TLC on PEI plates. The position of uridine 5′-monophosphate (pU) is indicated. Pseudouridine 5′-monophos-phate (pΨ) would be expected at the position indicated by square brackets. (B) Internal RNA modifications do not differentially affect the stability of U2 snRNP in nuclear extract. Equivalent amounts of four different 3′ end-labeled chimaeric U2 snRNAs (indicated above the panels) were analyzed before (top) and after (bottom) an in vitro splicing complementation reaction, using unlabeled pre-mRNA.

FIGURE 4.

Internal modifications within the first 24 nucleotides of U2 snRNA are required for efficient spliceosome assembly. Analysis of A-complex (A) and E-complex (B) formation with chimaeric U2 snRNAs. Untreated (lane 1), mock-depleted (lane 2), and U2-depleted (lane 3) nuclear extract are shown as controls. (Lanes 4–17, each panel) Complementation of the U2-depleted nuclear extract with U2 snRNPs reconstituted with the U2 snRNA indicated above each lane. Positions of the H, A, and E complexes are indicated. The ATP depletion of nuclear extract for the E-complex assay (B) was complete, as no A complex could be detected when control samples were analyzed in the presence of heparin (data not shown). Complexes were analyzed on native agarose gels and visualized by autoradiography.

FIGURE 5.

17S U2 snRNP assembly is not dependent on the presence of modifications in U2 snRNA. (A) Both HeLa U2 snRNA (top) and U2 transcript (bottom) assemble into a 17S U2 snRNP in vitro. The 3′ end-labeled RNAs were used for two-step reconstitution and RNPs were analyzed on a 10%–30% glycerol gradient. The RNA content of the odd fractions was analyzed by denaturing PAGE and visualized by autoradiography. Native 17S U2 snRNP, isolated according to Will et al. (2002), exhibited an identical sedimentation behavior (data not shown). (B,C) Immunoprecipitation assays of U2 snRNPs reconstituted with 3′ end-labeled HeLa U2 snRNA (B, lanes 1–6, C, lanes 1–7) or U2 transcript (B, lanes 7–12, C, lanes 8–14). The antibody used is indicated above each lane. (NIS) Nonimmune serum of the antibody in the preceeding lane. Antibodies denoted with * were affinity purified. Major (B) and substoichiometric (C) components of U2 snRNP were analyzed.