SL1 of U1 snRNA inhibits the assembly of Sm core in vitro. HeLa extracts were added (+) to nonradioactive SL1 or SL1A3 and incubated for 30 min on ice. Subsequently, [³²P]UTP-labeled U1, U2, U4, or U5 snRNA was added to the preincubated extracts and the mixtures were further incubated for 30 min at 30°C; as a control, the extracts were also mixed with [³²P]UTP-labeled U1, U2, U4, or U5 snRNA, respectively, in the absence of nonradioactive RNAs (lanes 2, 6, 10, and 14). The formation of Sm cores was analyzed by electrophoresis on native 6% polyacrylamide gels and autoradiography.

The assembly of U11 snRNP is mediated by the SMN complex. (A) The SMN complexes were purified and incubated with [³²P]UTP-labeled U11 snRNA. [³²P]UTP-labeled SL1A3 was used as an internal control (Control). Immunoprecipitations were performed using anti-Flag antibodies, and immunoprecipitated RNAs were analyzed by electrophoresis on 7 M urea-8% polyacrylamide gels. Total, 10% input. (B) U11 snRNA was transcribed in the presence of [³²P]UTP and incubated with buffer (−), HeLa extracts (CE), or SMN complex-depleted HeLa extracts (ΔSMN) for 30 min at 30°C. Additional assembly reactions were performed using HeLa extracts in the presence of Y12 monoclonal antibody for antibody supershifting (Y12). The products were analyzed by electrophoresis on 6% native polyacrylamide gels and autoradiography.

U snRNAs bind with high affinity to the SMN complex. (A) The affinity of U4 snRNA to the SMN complex was determined by a nitrocellulose filter binding assay. The SMN complex was incubated under equilibrium conditions with increasing amounts of U4 snRNA. A plot of the fraction of SMN complex saturation as a function of U4 snRNA concentration is shown. Error bars represent standard deviations from three independent experiments. (B) Purified SMN complexes were preincubated with nonradioactive U1, U2, U4, or U5 snRNA at a concentration of 10, 50, or 250 nM for 30 min at 4°C; subsequently, 10,000 cpm of [³²P]UTP-labeled U1, U2, U4, or U5 snRNA, respectively, was added to the preincubated mixtures and further incubated for 1 h at 4°C. Bound RNAs were isolated and analyzed by electrophoresis on 7 M urea-8% polyacrylamide gels. Total, 10% input.

The correct arrangement of the SMN complex-binding domain and Sm site is required for snRNP assembly. (A) U1Swap RNA was constructed by swapping SL1 (nucleotides 17 to 47) and SL4 (nucleotides 140 to 164) of U1 snRNA. SL1 of U1 snRNA is highlighted in red, and SL4 is highlighted in blue. (B) U1, U1A3, U1 Swap, and U1A3 Swap RNAs were transcribed in the presence of [³²P]UTP and incubated with buffer (−), HeLa extracts (CE), or the SMN complex-depleted HeLa extracts (ΔSMN) for 30 min at 30°C. Additional assembly reactions were performed using HeLa extracts in the presence of Y12 monoclonal antibody for antibody supershifting (Y12). The products were analyzed by electrophoresis on 6% native polyacrylamide gels and autoradiography. Assembled snRNPs are indicated by brackets. (C) The SMN complexes were purified and incubated with [³²P]UTP-labeled U1, U1A3, U1 Swap, and U1A3 Swap RNAs. Immunoprecipitations were performed using anti-Flag antibodies, and immunoprecipitated RNAs were analyzed by electrophoresis on 7 M urea-8% polyacrylamide gels. Total, 10% input. (D) snRNP TPs were purified as described previously by Raker et al. (47). Purified TPs were mixed with [³²P]UTP-labeled U1, U1A3, U1 Swap, and U1A3 Swap RNAs and further incubated for 30 min at 30°C. Assembled snRNPs were analyzed by electrophoresis on 6% native polyacrylamide gels and autoradiography.

The SMN complex-binding domains and Sm sequences of U snRNAs are required for the formation of Sm cores in cell extracts. (A) Mini snRNA fragments from each U snRNA mapped by minimal binding analysis using alkaline hydrolysis were transcribed in the presence of [³²P]UTP and mixed with radiolabeled U6 as an internal control. RNA mixtures were incubated with the SMN complex or nonspecific proteins purified from HeLa cells (Control) for 1 h. The bound RNAs were isolated and analyzed by electrophoresis on 7 M urea-8% polyacrylamide gels. Total, 10% input; IPs, immunoprecipitations. (B) U2SL4 + 5, U4SL2 + 3 and U5SL2ext were transcribed in the presence of [³²P]UTP and incubated with buffer (−), HeLa extracts (CE), the SMN complex-depleted HeLa extracts (ΔSMN), or HeLa extracts preincubated with anti-Sm (Y12) monoclonal antibody (+Y12) for 30 min at 30°C. After assembly reactions, the products were analyzed by electrophoresis on 6% native polyacrylamide gels and autoradiography. Sm cores and free RNAs are indicated by brackets.

Mapping of U snRNA domains binding to the SMN complex. (A) The SMN complex-binding domain of Xenopus laevis U2 snRNA. The 5′ (5′-P*)- and 3′ (3′-P*)-end-labeled U2 snRNA was subjected to limited alkaline hydrolysis in the presence of tRNA (10 μg). The resulting hydrolyzed RNA ladders were incubated with the SMN complex. The RNA fragments bound to the SMN complex were isolated and analyzed by electrophoresis on 7 M urea-8% acrylamide gels. RNase T1-digested RNA ladders of the same RNAs were used as size markers. Solid red arrows indicate the largest extent of the SMN complex-binding domains. Open red arrows indicate the smallest possible binding fragments. Total, 5% input; control, binding in control purification. (B) The SMN complex-binding domain of chicken U4 snRNA. The same experiment was performed using 5′ and 3′-end-labeled U4 snRNAs as described for panel A. (C) The SMN complex-binding domain of X. laevis U5 snRNA. The same experiment was performed using 5′- and 3′-end-labeled U5 snRNAs as described for panel A. (D) The secondary structure of X. laevis U2 snRNA and its domain for SMN complex binding. The region denoted by the gray box (from nucleotide 100 to the 3′ end) is sufficient for the interaction with the SMN complex and is designated U2SL4 + 5. (E) The secondary structure of chicken U4 snRNAs and its domain for SMN complex binding. The region denoted by the gray box (nucleotide 77 to the 3′ end) is sufficient for the interaction with the SMN complex and is designated U4SL2 + 3. (F) The secondary structure of X. laevis U5 snRNA and its domain for SMN complex binding. The region denoted by the gray box (nucleotide 55 to the 3′ end) is sufficient for the interaction with the SMN complex and is designated U5SL2ext.

The SMN complex associates with major spliceosomal U snRNAs in vivo and in vitro. (A) [³²P]UTP-labeled U1, U2, U4, or U5 snRNA was mixed with U6 snRNA, and each RNA mixture was injected into the cytoplasm of Xenopus oocytes. After incubation for 3 h, oocytes were homogenized and immunoprecipitations were carried out using anti-SMN (2B1) monoclonal antibody and control (SP2/0) antibody. The RNAs were isolated and analyzed by electrophoresis on 7 M urea-8% polyacrylamide gels. Lanes labeled “Total” represent 10% of input. (B) Native SMN complexes were purified from stable cell lines expressing Flag-Gemin2 (as described in Materials and Methods) and were analyzed by sodium dodecyl sulfate-12.5% PAGE and silver staining. Immunoprecipitation using anti-Flag antibody from the parental HeLa cell line (Tet ON) was carried out as a control (Control). Components of the SMN complex are indicated on the basis of molecular weight and Western blotting (data not shown). Half of the total amount of the SMN complex shown in this gel was used for direct RNA-binding experiments. (C) The same mixtures of RNAs used as described for panel A were added to the Flag-purified SMN complex as shown in Fig. 1B (SMN complex) and incubated for 1 h. Subsequently, bound RNAs were isolated after washing and analyzed by electrophoresis on 7 M urea-8% polyacrylamide gels.