Stem-loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly

Abstract

The pairing of 5' and 3' splice sites across an intron is a critical step in spliceosome formation and its regulation. Interactions that bring the two splice sites together during spliceosome assembly must occur with a high degree of specificity and fidelity to allow expression of functional mRNAs and make particular alternative splicing choices. Here, we report a new interaction between stem-loop 4 (SL4) of the U1 snRNA, which recognizes the 5' splice site, and a component of the U2 small nuclear ribonucleoprotein particle (snRNP) complex, which assembles across the intron at the 3' splice site. Using a U1 snRNP complementation assay, we found that SL4 is essential for splicing in vivo. The addition of free U1-SL4 to a splicing reaction in vitro inhibits splicing and blocks complex assembly prior to formation of the prespliceosomal A complex, indicating a requirement for a SL4 contact in spliceosome assembly. To characterize the interactions of this RNA structure, we used a combination of stable isotope labeling by amino acids in cell culture (SILAC), biotin/Neutravidin affinity pull-down, and mass spectrometry. We show that U1-SL4 interacts with the SF3A1 protein of the U2 snRNP. We found that this interaction between the U1 snRNA and SF3A1 occurs within prespliceosomal complexes assembled on the pre-mRNA. Thus, SL4 of the U1 snRNA is important for splicing, and its interaction with SF3A1 mediates contact between the 5' and 3' splice site complexes within the assembling spliceosome.

Keywords: RNA–protein interaction; alternative splicing; gene expression; pre-mRNA splicing; ribonucleoprotein; snRNA; spliceosome.

Figure 1.

Suppressor U1 snRNAs can rescue splicing. (A) Schematic representation of three-exon/two-intron Dup51 and Dup51p reporters. (B) Base-pairing of the wild-type and mutant 5′ splice sites with the 5′ end of U1 and U1-5a snRNAs. (C) Primer extension analysis of the Dup51 minigenes after cotransfection with control pcDNA or U1 expression plasmids. The mRNA products are indicated at the right and quantified in the graph below. (D) Primer extension analysis with oligonucleotide U1_7–26 showing expression of the wild-type U1 and variant U1-5a and U1-5g snRNAs.

Figure 2.

SL4 of U1 snRNA is important for U1 function. (A) Schematic of the secondary structures of SL4 region of the wild-type and mutant U1 snRNAs. Structures predicted to have the lowest ΔG are shown (Zuker 2003). (B) Primer extension analysis of the Dup51 minigene transcripts after cotransfection with control or U1 plasmids. The mRNA products are indicated at the right and quantified in the graph below. (C) Primer extension analysis with oligonucleotide U1_7–26 showing expression of the endogenous wild-type U1 and variant U1-5a snRNAs.

Figure 3.

Free U1-SL4 inhibits pre-mRNA splicing in vitro. (A) In vitro splicing of the AdML transcript in HeLa nuclear extract in the absence (lane 1) or presence of 0.5, 1.0, 2.5, 5.0, 7.5, and 10 μM wild-type (lanes 2–7) and mutant (lanes 8–13) short U1-SL4 RNA competitors. Prior to addition of the pre-mRNA, the splicing reactions were preincubated with the SL4 RNAs for 20 min at 4°C. The RNA splicing products and intermediates are diagrammed at the right. (B) Splicing activity is plotted as a function of SL4 concentration (in micromolar). Splicing was measured as the percent intensity of the intron lariat product in the presence of wild-type (A, lanes 2–7) and mutant (lanes 8–13) SL4 RNAs relative to the control reaction lacking SL4 (lane 1). (C) Analysis of ATP-dependent spliceosomal complexes in the presence of the short U1-SL4 RNA competitors. Transcripts were incubated in HeLa nuclear extract under splicing conditions with ATP in the absence (lanes 1–4) or presence of wild-type (lanes 5–8) or mutant (lanes 9–12) SL4 for the indicated times and separated by 2% native agarose gel. (D) Analysis of ATP-independent spliceosomal complexes in the presence of U1-SL4 RNA competitors. Transcripts were incubated in HeLa nuclear extract under splicing conditions without ATP in the absence (lanes 1–4) or presence of wild-type (lanes 5–8) or mutant (lanes 9–12) SL4 RNA for the indicated times and separated by 1.5% native agarose gel. Positions of the H, E, A, B, and C complexes are indicated.

Figure 4.

Identification of U1-SL4-interacting proteins. (A) Protocol for SILAC, RNA affinity purification, and MS. (B) Graph of SILAC ratios versus protein. The inset shows the ratios for U2 snRNP-specific proteins.

Figure 5.

SF3A complex proteins interact with the wild-type U1-SL4. (A) Salt-dependent disassembly of the U2 snRNP. (B) snRNA analysis of the complexes that bind to the biotinylated wild-type and mutant U1-SL4 RNAs after preincubation of HeLa nuclear extract with 0, 250, 500, and 1000 mM NaCl. Positions of the spliceosomal snRNAs are indicated at the left. (C) Western analyses of the proteins present in the wild-type and mutant U1-SL4 RNA complexes.

Figure 6.

Interaction between SF3A1 and U1 snRNA occurs in prespliceosomal complexes. UV cross-linking and immunoprecipitation identified a direct contact between the wild-type SL4 RNA and SF3A1 protein. (A) ³²P-labeled wild-type and mutant SL4 RNAs were incubated in HeLa nuclear extracts in splicing conditions. After incubation, the reactions were UV cross-linked and immunoprecipitated with antibodies against U1 70k (lanes 2,6), SF3A1 (lanes 3,7), and SF3A3 (lanes 4,8) proteins, followed by analysis by SDS-PAGE. (B) Splicing reactions containing ATP in the presence (lanes 5–7) or absence (lanes 2–4) of AdML pre-mRNA were fractionated on glycerol density gradients (see Supplemental Fig. S6). Gradient fractions from the 21S peak containing the pre-mRNA and U1 and U2 snRNA were pooled, UV cross-linked, and immunoprecipitated with anti-SF3A1 (lanes 7–10) or anti-His (lane 6) antibodies. Equivalent fractions from gradients lacking pre-mRNA were also immunoprecipitated with anti-SF3A1 (lane 4) or anti-His (lane 3) antibodies. Total RNA from the fractions (shown in lanes 2,5) and the immunoprecipitated complexes was extracted and analyzed. (Lanes 8–10) The identity of the U1 and U4 snRNAs was confirmed by RNase H cleavage in the presence of U1 oligos (U1_1–15 and U1_64–75) and U4 oligos (U4_2–16 and U4_66–85). (Lane 1) Total RNA from nuclear extract was used as a marker for the U snRNAs. The positions of the U snRNAs and pre-mRNA are indicated.

Figure 7.

SL4 mutations that affect function also affect binding of the SF3A1 protein. (A) Immunoblot of the SF3A1, PTBP1, and U1 70k proteins bound to immobilized SL4 RNAs after incubation in HeLa nuclear extract under splicing conditions. SL4 variants included the wild-type human sequence, the M10 and M10h mutants, and the Drosophila SL4 (Dm). The input lane contains 1.5% of the total reaction. Lanes 2–5 contain 20% of the protein eluted from each RNA. (B) Quantification of the amount of bound SF3A1 and PTBP1 proteins expressed as the ratio of bound protein to that bound by the wild-type SL4.

Figure 8.

Model for the role of SL4 of U1 snRNA in splicing assembly. Interaction of the SL4 with the U2 snRNP-specific SF3A1 protein occurs during pairing of splice sites prior to the formation of the prespliceosomal A complex. The presence of free U1-SL4 blocks this interaction and subsequent A complex formation. U2 is shown engaged at the branch point, as seen in the prespliceosomal A complex.