Ribonucleoprotein assembly defects correlate with spinal muscular atrophy severity and preferentially affect a subset of spliceosomal snRNPs - PubMed (original) (raw)

Ribonucleoprotein assembly defects correlate with spinal muscular atrophy severity and preferentially affect a subset of spliceosomal snRNPs

Francesca Gabanella et al. PLoS One. 2007.

Abstract

Spinal muscular atrophy (SMA) is a motor neuron disease caused by reduced levels of the survival motor neuron (SMN) protein. SMN together with Gemins2-8 and unrip proteins form a macromolecular complex that functions in the assembly of small nuclear ribonucleoproteins (snRNPs) of both the major and the minor splicing pathways. It is not known whether the levels of spliceosomal snRNPs are decreased in SMA. Here we analyzed the consequence of SMN deficiency on snRNP metabolism in the spinal cord of mouse models of SMA with differing phenotypic severities. We demonstrate that the expression of a subset of Gemin proteins and snRNP assembly activity are dramatically reduced in the spinal cord of severe SMA mice. Comparative analysis of different tissues highlights a similar decrease in SMN levels and a strong impairment of snRNP assembly in tissues of severe SMA mice, although the defect appears smaller in kidney than in neural tissue. We further show that the extent of reduction in both Gemin proteins expression and snRNP assembly activity in the spinal cord of SMA mice correlates with disease severity. Remarkably, defective SMN complex function in snRNP assembly causes a significant decrease in the levels of a subset of snRNPs and preferentially affects the accumulation of U11 snRNP--a component of the minor spliceosome--in tissues of severe SMA mice. Thus, impairment of a ubiquitous function of SMN changes the snRNP profile of SMA tissues by unevenly altering the normal proportion of endogenous snRNPs. These findings are consistent with the hypothesis that SMN deficiency affects the splicing machinery and in particular the minor splicing pathway of a rare class of introns in SMA.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Expression of SMN complex components in the spinal cord of severe SMA mice.

Western blot analysis of equal amounts of total proteins from the spinal cord of normal (SMN2 +/+;mSmn +/+), carrier (SMN2 +/+;mSmn +/−) and severe SMA (SMN2 +/+;mSmn −/−) mice at postnatal day 3. All mice harbored two copies of human SMN2 gene together with two, one or no copies of the mouse mSmn gene. Proteins were analyzed by SDS/PAGE and Western blot with antibodies against the proteins indicated on the left.

Figure 2. Analysis of SMN levels in different tissues of severe SMA mice.

(A) Western blot analysis of total proteins from spinal cord, brain or kidney of normal (SMN2 +/+;mSmn +/+), carrier (SMN2 +/+;mSmn +/−) and severe SMA (SMN2 +/+;mSmn −/−) mice at postnatal day 3. Extracts normalized per cell number (equal amounts of core histones) were analyzed by SDS/PAGE and Western blot with antibodies against the proteins indicated on the left. (B) Western blot analysis of equal amounts of whole tissue extracts (25 µg) from spinal cord, brain or kidney of normal (SMN2 +/+;mSmn +/+), carrier (SMN2 +/+;mSmn +/−) and severe SMA (SMN2 +/+;mSmn −/−) mice at postnatal day 3. Proteins were analyzed by SDS/PAGE and Western blot with antibodies against SMN and tubulin. (C) Analysis of SMN decrease in the spinal cord of severe SMA mice. Western blot analysis of the indicated serial dilutions (100 equals 50 µg of proteins) of whole tissue extracts from the spinal cord of normal (SMN2 +/+;mSmn +/+), carrier (SMN2 +/+;mSmn +/−) and severe SMA (SMN2 +/+;mSmn −/−) mice at postnatal day 3. Proteins were analyzed by SDS/PAGE and Western blot with antibodies against SMN and tubulin.

Figure 3. In vitro snRNP assembly activity in tissues of severe SMA mice.

(A) Equal amounts of whole tissue extracts (25 µg) from spinal cord, brain or kidney of normal (SMN2 +/+;mSmn +/+), carrier (SMN2 +/+;mSmn +/−) and severe SMA (SMN2 +/+;mSmn −/−) mice at postnatal day 3 were analyzed in snRNP assembly reactions with radioactive U1 snRNA followed by electrophoresis on native polyacrylamide gels. The position of U1 RNP complexes containing the Sm core is indicated on the left. (B) In vitro snRNP assembly reactions as in (A) were analyzed by immunoprecipitation with anti-Sm (Y12) antibodies followed by electrophoresis on denaturing polyacrylamide gels and autoradiography. (C) Quantification of snRNP assembly activity in whole tissue extracts from normal, carrier and SMA mice. The amount of U1 snRNA immunoprecipitated in snRNP assembly experiments as in (B) was quantified using a STORM 860 Phosphorimager (Molecular Dynamics) and expressed as a percentage of that in brain extracts of normal mice, which is set arbitrarily as 100%. The values from four independent experiments are presented as mean±SEM (*p<0.0001, **p<0.05). (D) For each tissue, the amount of U1 snRNA immunoprecipitated in snRNP assembly experiments with extracts from carrier (SMN2+/+;mSmn+/−) and SMA (SMN2+/+;mSmn−/−) mice is expressed as a percentage of that in the corresponding extract from normal (SMN2+/+;mSmn+/+) mice, which is set arbitrarily as 100%.

Figure 4. In vitro snRNP assembly defects correlate with SMA severity.

(A) Western blot analysis of equal amounts of whole tissue extracts (25 µg) from the spinal cord of normal (SMN2 +/+;mSmn +/+), carrier (SMN2 +/+;mSmn +/−), severe SMA (SMN2 +/+;mSmn −/−), SMNΔ7 SMA (SMN2 +/+ ;SMNΔ7 +/+;mSmn −/−), SMN(A2G) SMA (SMN2 +/+ _;_SMN(A2G)+/− ;mSmn −/−) and high copy SMN2 SMA (SMN2 +/−;SMN2(566) +/−;mSmn −/−) mice at postnatal day 3. Proteins were analyzed by SDS/PAGE and Western blot with antibodies against the proteins indicated on the left. (B) Representative snRNP assembly reactions carried out using in vitro transcribed radioactive U1 snRNA and 25 µg of whole spinal cord extracts as in (A) followed by immunoprecipitation with anti-Sm (Y12) antibodies. Immunoprecipitated U1 snRNAs were analyzed by electrophoresis on denaturing polyacrylamide gels and autoradiography. (C) Quantification of snRNP assembly activity in spinal cord extracts from control and SMA mice of different severity. Spinal cord extracts from at least five mice for each of the indicated genotypes were analyzed by snRNP assembly and immunoprecipitation experiments as in (B). The amount of immunoprecipitated U1 snRNA was quantified using a STORM 860 Phosphorimager (Molecular Dynamics). The values are presented as mean±SEM. SMN2 high refers to SMN2(566).

Figure 5. SMN levels and in vivo snRNP synthesis in human fibroblasts from type I SMA patients.

(A) SMN levels in type I SMA fibroblast cell lines. Western blot analysis of equal amounts of whole cell extracts (25 µg) from human fibroblast cell lines of a normal individual (GM03814) and four type I SMA patients (GM00232, GM09677, GM03813 and GM03815). Proteins were analyzed by SDS/PAGE and Western blot with antibodies against SMN and tubulin. (B) In vivo snRNP synthesis in type I SMA fibroblast cell lines. The human fibroblast cell lines in (A) were pulse-labeled in vivo for 6 h with [32P] phosphoric acid and equal amounts of extracts from these cells (200 µg) were then immunoprecipitated with anti-Sm (Y12) antibodies. The immunoprecipitated snRNAs were analyzed by electrophoresis on denaturing polyacrylamide gels and autoradiography. Known snRNAs are indicated on the left.

Figure 6. Analysis of endogenous snRNP levels in the spinal cord of severe SMA mice.

Equal amounts of whole tissue extracts (200 µg) from the spinal cord of normal (SMN2 +/+;mSmn +/+), carrier (SMN2 +/+; mSmn +/−) and severe SMA (SMN2 +/+;mSmn −/−) mice at postnatal day 3 were immunoprecipitated with anti-Sm (Y12) antibodies. Immunoprecipitated snRNAs were labeled at the 3′-end with [32P] pCp and analyzed by electrophoresis on denaturing polyacrylamide gels and autoradiography. Known snRNAs are indicated on the left. Note that the intensity of signal is not proportional to the abundance of individual snRNAs but reflects the efficiency with which they are labeled by T4 RNA ligase .

Figure 7. Quantification of endogenous snRNP levels in tissues of severe SMA mice.

Immunoprecipitation experiments with anti-Sm (Y12) antibodies from whole tissue extracts (200 µg) from either spinal cord, brain or kidney extracts of normal (SMN2 +/+;mSmn +/+), carrier (SMN2 +/+;mSmn +/−) and severe SMA (SMN2 +/+;mSmn −/−) mice at postnatal day 3 were carried out as in Figure 6. The relative amount of individual snRNAs immunoprecipitated from each tissue was quantified using a STORM 860 Phosphorimager (Molecular Dynamics) and expressed as a percentage of that in normal mice, which is set arbitrarily as 100%. The values from four independent experiments are presented as mean±SEM (*p<0.05). Normal (black bars); carrier (dark grey bars); severe SMA (light grey bars).

Figure 8. Analysis of endogenous U1 and U11 snRNA levels in the spinal cord of severe SMA mice.

(A) Northern blot analysis of four increasing concentrations (0.5, 1, 2 and 4 µg) of total RNA from whole spinal cord extracts of five normal (SMN2 +/+;mSmn +/+) and five severe SMA (SMN2+/+;mSmn−/−) mice at postnatal day 3 with probes specific for the RNAs indicated on the left. (B) The intensity of signal of individual RNAs at each concentration was quantified using a STORM 860 Phosphorimager (Molecular Dynamics) and values in the severe SMA mice were expressed as a percentage of the corresponding ones in normal mice, which are set arbitrarily as 100%. The values are presented as mean±SEM. Normal (black bars); severe SMA (light grey bars).

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Ribonucleoprotein assembly defects correlate with spinal muscular atrophy severity and preferentially affect a subset of spliceosomal snRNPs - PubMed (original) (raw)