HSURs compete with U snRNAs for Sm core assembly. A total of 10,000 cpm of [³²P]UTP-labeled U4 snRNA was incubated with buffer (IN), HeLa cytoplasmic extract (−), or HeLa cytoplasmic extract plus increasing amounts (50, 250, or 500 nM) of nonradioactive U1, U4, HSUR1, or HSUR4 for 1 h at 30°C. Assembly reaction products were analyzed by electrophoresis on 6% native polyacrylamide gels and quantitated by using a phosphorimager. Sm cores and free RNAs are each indicated by brackets, and relative percentages of assembly are indicated below each lane.

HSUR5 colocalizes with SMN and U snRNAs. (a) Indirect immunofluorescence of SMN protein with anti-SMN monoclonal antibody (2B1) and a FITC-conjugated secondary antibody showing localization in nuclear gems. (b) In situ hybridization of HSUR5 with a biotinylated antisense probe and an antibiotin secondary antibody conjugated to Texas red. (c) Combined image of panels a and b showing colocalization of HSUR5 in nuclear gems. (d) Indirect immunofluorescence of Sm proteins with anti-Sm monoclonal antibody (Y12) and an FITC-conjugated secondary antibody showing nuclear speckles. (e) Same as panel b. (f) Combined image of panels d and e showing colocalization of HSUR5 with Sm proteins. (g) In situ hybridization of endogenous U1 snRNA with an FITC-conjugated antisense probe. (h) Same as panel b. (i) Combined image of panels g and h showing colocalization of U1 snRNA with HSUR5.

HSURs assemble SMN-dependent Sm cores in vitro. (A) [³²P]UTP-labeled HSUR1, HSUR3, HSUR4, and HSUR5 were incubated with buffer (−), HeLa mock-depleted extracts (CE), or SMN complex-depleted HeLa extracts (ΔSMN) for 1 h at 30°C. Anti-Sm monoclonal antibody (Y12) was added to supershift Sm cores assembled in CE (+Y12). The assembly reaction products were analyzed by electrophoresis on 6% native polyacrylamide gels and autoradiography. Sm cores and free RNAs are each indicated by brackets. (B) The HeLa cytoplasmic extracts used in Fig. 2A were immunodepleted by using control (SP2/0) antibody (Mock) or anti-SMN (2B1) monoclonal antibody (ΔSMN). Subsequently, proteins were resolved on 4 to 12% gradient polyacrylamide gels and immunodepletion of SMN complex components was confirmed by Western blotting.

SMN complex purified with Sm proteins is sufficient for Sm core assembly on HSURs. (A) Native SMN complexes (SMN) or nonspecific proteins (Control) were purified from Flag-Gemin2 cells or the parental HeLa cells, respectively, under low-salt conditions as described in Materials and Methods. Flag-purified proteins were eluted with 3× Flag peptides, resolved by electrophoresis on 4 to 12% gradient polyacrylamide gels, and Western blotted for components of the SMN complex. (B) [³²P]UTP-labeled U1 snRNA, U4 snRNA, HSUR1, HSUR3, and HSUR4 were incubated with buffer (−) or with Flag-purified SMN complex (+) isolated under low-salt conditions for 1 h at 30°C. Assembly reaction products were analyzed by electrophoresis on 6% native polyacrylamide gels and autoradiography. Sm cores and free RNAs are each indicated by brackets.

Reduction of SMN in vivo leads to decreased Sm core assembly on HSURs. (A) HSUR3, HSUR4, HSUR5, and HSUR4ΔSm were transcribed in the presence of [³²P]UTP and incubated with buffer (−), cytoplasmic extract derived from S5 cells grown under wild-type SMN conditions (CE + 10 ng of tetracycline [tet]/ml) or from S5 cells grown under low SMN conditions (CE + 18 ng of tetracycline/ml) as described in Materials and Methods. After 1 h of incubation at 30°C, the assembly reaction products were analyzed by electrophoresis on 6% native polyacrylamide gels and autoradiography. Sm cores and free RNAs are each indicated by brackets. (B) The S5 cell extracts used in Fig. 3A were run on 4 to 12% gradient polyacrylamide gels and Western blotted to confirm the in vivo reduction of SMN.

HSURs bind to the SMN complex with high affinity. (A) A nitrocellulose filter-binding assay was used to determine the affinities of HSUR1 and HSUR4 to the SMN complex. Flag-purified SMN complexes were incubated with various amounts of HSUR1 or HSUR4. A plot of the fraction of SMN complex saturation as a function of RNA concentration is shown. Error bars represent the standard deviations from at least four independent experiments for each RNA. (B) U4 snRNA was transcribed in the presence of [³²P]UTP, and 10,000 cpm was mixed with increasing concentrations of nonradioactive U1 snRNA, HSUR1, HSUR3, and HSUR4 (10, 50, or 250 nM) or no competitor (−) and immediately incubated with purified SMN complexes for 1 h at 4°C. Bound RNAs were isolated and analyzed by electrophoresis on 7 M urea-8% polyacrylamide gels. Total represents 10% of input.

The SMN complex binds directly to HSURs in vitro and associates with HSURs in vivo. (A) Native SMN complexes (SMN) were purified under high-salt conditions from stable cell lines expressing Flag-Gemin2 (as described in Materials and Methods) and were analyzed by electrophoresis on 4 to 12% gradient polyacrylamide gels and by silver staining. Immunoprecipitation with anti-Flag antibody from the parental HeLa cell line was used as a control (Control). Gemin6 and Gemin7 are not shown. The total amount of SMN complex shown in this gel was used for direct RNA-binding experiments. (B) [³²P]UTP-labeled HSUR1, HSUR3, HSUR4, or HSUR5 was mixed with SL1A3 RNA (a mutant of SL1 of U1 snRNA that does not efficiently bind to the SMN complex) and incubated for 1 h at 4°C with Flag-purified SMN complex (SMN complex) or nonspecific proteins purified from HeLa cells (Control). Bound RNAs were washed, isolated, and analyzed by electrophoresis on 7 M urea-8% polyacrylamide gels and autoradiography. Total represents 20% of input. (C) HeLa cells stably expressing Flag-Gemin2 were transiently transfected with empty vector (Vector) or with a 7.4-kb fragment of genomic HVS DNA that contains the genes for HSURs 1 to 5 (HSUR DNA). After 48 h, cell extracts were made and subjected to immunoprecipitation with either anti-Sm (Y12) or anti-Flag monoclonal antibodies, and RNAs were purified by phenol-chloroform extraction and ethanol precipitation. Immunoprecipitated RNAs were reverse transcribed (+RT), and the cDNAs were amplified by using primers for U1 snRNA and HSURs 1 to 5. RT in the absence of transcriptase was performed as a negative control (−RT). cDNAs were run on 1.5% agarose gels, stained with ethidium bromide, and visualized by UV light. Total RNA represents 10% of input.