The survival of motor neurons protein determines the capacity for snRNP assembly: biochemical deficiency in spinal muscular atrophy

Abstract

Reduction of the survival of motor neurons (SMN) protein levels causes the motor neuron degenerative disease spinal muscular atrophy, the severity of which correlates with the extent of reduction in SMN. SMN, together with Gemins 2 to 7, forms a complex that functions in the assembly of small nuclear ribonucleoprotein particles (snRNPs). Complete depletion of the SMN complex from cell extracts abolishes snRNP assembly, the formation of heptameric Sm cores on snRNAs. However, what effect, if any, reduction of SMN protein levels, as occurs in spinal muscular atrophy patients, has on the capacity of cells to produce snRNPs is not known. To address this, we developed a sensitive and quantitative assay for snRNP assembly, the formation of high-salt- and heparin-resistant stable Sm cores, that is strictly dependent on the SMN complex. We show that the extent of Sm core assembly is directly proportional to the amount of SMN protein in cell extracts. Consistent with this, pulse-labeling experiments demonstrate a significant reduction in the rate of snRNP biogenesis in low-SMN cells. Furthermore, extracts of cells from spinal muscular atrophy patients have a lower capacity for snRNP assembly that corresponds directly to the reduced amount of SMN. Thus, SMN determines the capacity for snRNP biogenesis, and our findings provide evidence for a measurable deficiency in a biochemical activity in cells from patients with spinal muscular atrophy.

FIG. 1.

Analysis of in vitro-assembled snRNPs by the mobility gel shift assay and by the magnetic beads assay. (A) [³²P]UTP-labeled U1, U1ΔSm, U1A3, U4, U4ΔSm, U5, or U5ΔSm snRNA was mixed with HeLa cytoplasmic extracts (CE) containing 25 μg total proteins (+ lanes) or with buffer only (− lanes) for in vitro assembly of Sm cores. The reaction mixtures were analyzed by electrophoresis on 6% native polyacrylamide gels. The brackets on the right indicate the positions of assembled Sm cores and the free RNAs. The complex that results from the binding of the U1-specific protein U1A to stem-loop 2 of the U1 snRNA is marked on the left. The band indicated by an asterisk is likely to be U1A/Sm core complexes. (B) The same reaction mixtures as used in panel A were immunoprecipitated by Y12 bound to protein A-Sepharose beads. RNAs were isolated from the bound fractions and analyzed by electrophoresis on 7 M urea-8% polyacrylamide gels. The RNAs that migrate at different positions on the gel are indicated on the left. (C) SnRNAs were produced by in vitro transcription in the presence of biotin-UTP instead of [³²P]UTP, and similar assembly reactions as in A were carried out with these biotinylated RNAs or without any RNA using either HeLa cytoplasmic extracts (+ lanes) or buffer (− lanes). The amount of the Sm cores assembled on these RNAs was assessed by the magnetic beads assay as depicted in Fig. 2. The error bars, which for some data points are too small to be seen on the figure, represent standard deviations from three independent experiments.

FIG. 2.

Schematic depiction of the magnetic bead assay procedure for the detection of in vitro-assembled snRNPs.

FIG. 3.

Specific parameters that affect snRNP assembly in vitro. In vitro assembly reactions were performed using standard conditions except one of the following parameters. Sm core assembly on U4 (A, B, C, and D) and U4ΔSm (B, C, and D) was examined by the magnetic beads assay and graphed against ATP or AMP-PNP concentration (A), the snRNA concentration (B), the amount of HeLa cytoplasmic extract (C), or the assembly reaction time (D). Error bars, which are too small to be visible for some of the data points, represent standard deviations from two independent experiments.

FIG. 4.

Reduced snRNP assembly correlates with decreased SMN protein levels in extracts from chicken S5 cells. (A) Cytoplasmic extracts were prepared from S5 cells cultured at 10, 12, 14, 16, or 18 ng/ml tetracycline. SMN, SmB/B′, and Y14 proteins in each of these extracts containing 20 μg total proteins were detected by quantitative Li-Cor Western blotting analysis. (B) The intensity of each protein band in panel A was analyzed using the Odyssey infrared imaging system. SMN and SmB/B′ proteins levels in extracts of various tetracycline concentrations were normalized using the Y14 signals as an internal control. The relative SMN and SmB/B′ protein levels at 18 ng/ml tetracycline were set to 1. The relative protein levels at other tetracycline concentrations were calculated as ratios to those at 18 ng/ml tetracycline. (C) SnRNP assembly capacities of the same extracts as in A on U4 snRNA were examined by the magnetic bead assay. The relative assembly activity of extract at 18 ng/ml tetracycline was set to 1. The relative assembly activities of the other extracts were calculated as ratios to that at 18 ng/ml tetracycline. (D) The relative assembly activity at each tetracycline concentration as in panel C was plotted on the y axis against the corresponding relative SMN protein level as in panel B on the x axis. The data points fitted best to a linear graph (R² = 0.9911).

FIG. 5.

Cells with low SMN accumulate snRNPs more slowly. (A) To determine the rate of snRNP biogenesis in vivo, S5 cells cultured at 10 or 18 ng/ml tetracycline were pulse-labeled with 25 μCi/ml [³H]uridine for one hour. Total RNAs from 10% of the labeled cells and snRNAs isolated by immunoprecipitations with Y12 from the remaining cells were analyzed on 7 M urea-8% polyacrylamide gel. Known RNAs are indicated on the left. (B) To measure the level of snRNPs in vivo at steady state, Y12 immunoprecipitations were performed on S5 cells cultured at 10 or 18 ng/ml tetracycline. The immunoprecipitated RNAs were isolated, radioactively labeled at the 3′ end, and analyzed on 7 M urea-8% polyacrylamide gel. Known RNAs are indicated on the left.

FIG. 6.

Cells of SMA patients are deficient in their capacity for snRNP assembly. (A) Cytoplasmic extracts were prepared from two lymphoblast cell lines. One was established from an SMA type I patient (GM10684) and the other from an age- and gender-matched individual (GM12497) as a control. SMN, Gemins 2 to 5, and SmB/B′ proteins were detected by quantitative Li-Cor Western blotting analysis, using Y14 as a loading control. (B) The intensity of each of the protein bands in panel A was analyzed using the Odyssey infrared imaging system. The SMN protein level in each of the cell extract was normalized using Y14 signal as an internal control. The relative level of SMN protein in the cell extract of an SMA patient (GM10684) was calculated as the percentage of that of the control (GM12497). Similarly, the relative assembly activity in cell extracts of the patient (GM10684) was calculated as a percentage of the control (GM12497). (C) Cytoplasmic extracts from SMA patient fibroblast cell lines were assayed by the magnetic bead assay. SnRNP assembly activities in cell extracts of the controls (GM00498, GM08333, and GM03814) were set to be 100%. Assembly activities in cell extracts of the SMA type I patients (GM09677, GM00232, GM03813, and GM03815) were expressed as percentages of their respective controls.