The Drosophila U2 snRNP protein U2A' has an essential function that is SNF/U2B" independent

Abstract

Recruitment of the U2 snRNP to the pre-mRNA is an essential step in spliceosome assembly. Although the protein components of the U2 snRNP have been identified, their individual contributions to function are poorly defined. In vitro studies with the Drosophila and human proteins suggest that two of the U2 snRNP-specific proteins, U2A' and U2B", function exclusively as a dimer. In Drosophila the presence of the U2B" counterpart, Sans-Fille (SNF), in the U2 snRNP is dispensable for viability, suggesting that SNF is not necessary for U2 snRNP function in vivo. With the identification of a single U2A'-like protein in the Drosophila genome, we can now investigate the relationship between SNF and its putative binding partner in vivo. Here we show that Drosophila U2A' protein interacts with SNF in vivo and, like its human counterpart, is U2 snRNP specific. Unexpectedly, however, we find that loss of function causes lethality, suggesting that U2A', but not SNF, is critical for U2 snRNP function. Moreover, although we demonstrate that several domains in the SNF protein are important for the interaction with the Drosophila U2A' protein, including a redundant domain at the normally dispensable C-terminus, we find that U2A' does not require heterodimer formation for either its vital function or U2 snRNP assembly. Thus together these data demonstrate that in Drosophila U2A' has an essential function that is unrelated to its role as the partner protein of SNF/U2B".

Figure 1

The Drosophila U2A′ ortholog is U2 snRNP specific. (A) Amino acid sequence alignment of the U2A′ protein from human and Drosophila. Identical residues are boxed in gray. The positions of the six LRRs are indicated by black lines above the sequence. The position of the l(2)43Ee¹ mutation is indicated by an arrow. (B) Comparison of the human and Drosophila protein structures. The percent amino acid identity for the different domains is indicated. (C) dU2A′ is a U2 snRNP protein. snRNP incorporation was tested by immunoprecipitation of either SNF or dU2A′ from whole fly extracts followed by northern blotting to detect U1 and U2 snRNAs in the RNA extracted from the precipitated fractions.

Figure 2

Impact of snf mutations on dU2A′/SNF heterodimer formation in vivo. (A) Schematic of the wild-type SNF protein showing the locations of mutations discussed in this study. SNF contains two RRM domains separated by a short linker region. As indicated, the sequence of the N- and C-terminal RRM motifs share significant sequence identity with both the human U1A and U2B″ proteins. The positions of the mutations used in this study are indicated by arrows. (B) Amino acid sequence alignment of the N-terminal RRM from SNF and human U1A and U2B″ proteins. Identical amino acids are boxed in gray. The critical residues for differential interactions with the human U2A′ protein are boxed in black. The relevant position of the point mutations and the position of the snf^ΔKpn truncation are also indicated by arrows. (C) dU2A′ co-immunoprecipitates with wild-type SNF and SNF^E21D, but not SNF^5MER. dU2A′ was immunoprecipitated from protein extracts made from either wild-type or mutant adults followed by western blotting with an antibody against SNF protein. Mutant extracts were made from homozygous y w snf^J210 animals which carried two autosomal copies of the appropriate P[w⁺, snf] mutant snf minigene construct. Because snf^J210 is a deletion of the entire open reading frame, all SNF protein is due to expression from the transgenic copy of snf. As a control for the amount of SNF protein expected, 20% of each protein extract used in the co-immunoprecipitation experiment is shown in the lane marked Input.

Figure 3

Several domains of SNF mediate the interaction with dU2A′ in yeast. dU2A′ was co-expressed in the yeast two-hybrid assay with wild-type SNF or different SNF mutant constructs. The positions of the mutants used in this study are indicated in Figure 2B. Positive interactions were tested by assaying the ability of the transformed yeast to grow on selective media after 3 days or by liquid culture assay for β-galactosidase activity.

Figure 4

Assembly of dU2A′ into U2 snRNPs takes place in several different snf mutant backgrounds, including mutations that interfere with heterodimer formation. The ability of dU2A′ to incorporate into U2 snRNPs was tested by immunoprecipitation of dU2A′ from protein extracts made from either wild-type or homozygous mutant adults, followed by northern blotting to detect U2 snRNAs, as described in Materials and Methods. The positions of the mutants used in this study are indicated in Figure 2B. Mutant protein extracts were made from homozygous y w snf^J210 animals which also carried two autosomal copies of the appropriate P[w⁺, snf] mutant transgene.