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. 2007 Jul 18:8:28.
doi: 10.1186/1471-2121-8-28.

Absence of gemin5 from SMN complexes in nuclear Cajal bodies

Le thi Hao  1 , Heidi R Fuller, Le Thanh Lam, Thanh T Le, Arthur H M Burghes, Glenn E Morris
Affiliations

Absence of gemin5 from SMN complexes in nuclear Cajal bodies

Le thi Hao et al. BMC Cell Biol. .

Abstract

Background: Spinal muscular atrophy is caused by reduced levels of the survival of motor neurons (SMN) protein. SMN is found in large complexes with Sm proteins and at least eight other proteins, including seven "gemins". These complexes are involved in the assembly of snRNPs in the cytoplasm and their transport into the nucleus, but the precise roles of the individual protein components are largely unknown.

Results: We have investigated the subcellular distribution of gemins using novel antibodies against gemins 3-7, and existing mAbs against SMN, gemin2, unrip, fibrillarin and profilin II. Most gemins were equally distributed between nuclear and cytoplasmic fractions of HeLa cells, but gemin5 and unrip were more abundant in the cytoplasm. In a cytoplasmic extract obtained by mild disruption of HeLa cells, nearly all the SMN and gemins 2-4 were in large complexes, but most of the gemin5 sedimented separately with a lower S value. Most of the unrip sedimented with gemins 6 and 7 near the top of the sucrose density gradients, separate from both SMN and gemin5. Anti-SMN mAbs pulled down gemin5 from cytoplasmic extracts, but not from nuclear extracts, and gemin5 did not co-sediment with large SMN complexes in nuclear extracts. These data suggest that gemin5 is easily detached from SMN-gemin complexes in the nucleus. By immuno-histochemistry, gemin5 was rarely detectable in nuclear gems/Cajal bodies, although it was accessible to antibody and easily detectable when present. This suggests that gemin5 is normally absent from SMN complexes in these nuclear storage sites.

Conclusion: We conclude that SMN complexes usually exist without gemin5 in nuclear gems/Cajal bodies. Gemin5 is believed to be involved in capturing snRNA into SMN complexes in the cytoplasm for transport into the nucleus. We hypothesize that gemin5, though present in the nucleus, is no longer needed for SMN complex function during the time these complexes are stored in gems/Cajal bodies.

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Figures

Figure 1
Figure 1
Characterization of gemin antibodies by western blots of total HeLa cell proteins. An SDS extract of HeLa cells was run as a horizontal strip alongside Mr markers on SDS-PAGE and transferred to nitrocellulose membranes by electroblotting. 7% acrylamide was used for (a)-(c) with prestained Sigma markers and 14% for (d) and (e) with prestained SeeBlue markers. The blot was either cut into vertical lanes or, in (c) only, used directly on a 28-lane miniblotter apparatus. In each case, the main antigen band is shown as a broken arrow. (a) gemin3 antiserum stains a single protein of about 102 kD (97 kD gemin4 is shown as a marker). (b) the panel of 6 gemin4 mAbs all stain a band of 97 kD, but 3 of them (GEM4A, C and F) cross-react with several higher Mr proteins. Non-specific bands (*) are due to the secondary antibody system. (c) the 12 mAbs against the gemin5 N-terminal region stain a single band consistent with the 167 kD expected for gemin5 and the 6 mAbs against the C-terminal region stain the same band, together with a ladder of smaller bands that may be degradation products. (d) The 7 gemin6 mAbs all stain a band of 16 kD, but 2 of them cross-react with a 32 kD band. (e) All 7 gemin7 mAbs stain a single band of 15 kD.
Figure 2
Figure 2
Gemins 3, 4, 6 and 7 colocalise with SMN in nuclear gems/CBs, but gems/CBs in most cells lack gemin5. HeLa cells grown on coverslips were fixed with 1% formalin in PBS and permeabilized with 1% Triton X-100. Gemins 4, 5, 6 and 7 were identified using GEM4E, GEM5Q, GEM6F and GEM7E, respectively in a double label with SMN rabbit antiserum. Typical fields are shown, except that an unusual field containing several cells with gemin5-positive gems/CBs was chosen for GEM5Q. The anti-gemin3 rabbit antiserum was double-labelled with MANSMA1 mAb against SMN. Alexa fluor 546 goat anti-mouse IgG (red) and Alexa fluor 488 goat anti-rabbit IgG (green) were used as second antibodies. DAPI (blue) was used to counterstain the nuclei. (See text for white and green arrows).
Figure 3
Figure 3
In Hela PV cells, which have separate gems and CBs, gemin5 colocalizes with gems, not CBs, in those rare cells that do have gemin5-positive nuclear bodies. HeLa strain PV cells grown on coverslips were fixed with 1% formalin in PBS and permeabilized with 1% Triton X-100. (A) Separation of gems (red: MANSMA1 mAb and Alexa-546 anti-mouse Ig) and CBs (green: rabbit anti-coilin and FITC anti-rabbit Ig) in HeLa PV, counterstained with DAPI (blue). Rare gemin5-positive nuclear bodies were double-labelled with (B) mAb GEM5P and Alexa-546 anti-mouse Ig and (C) rabbit anti-SMN and FITC anti-rabbit Ig to show colocalization of SMN and gemin5 (white arrows). (D) is an overlay from a double label with GEM5P (red) and rabbit anti-coilin (green) with a DAPI counterstain (blue), showing no colocalization.
Figure 4
Figure 4
Overexpression of SMN in HeLa cells stimulates formation of gems/CBs containing other gemins, except gemin 5. Gemin2 (A), gemin 4 (B), gemin6 (D) and 7 (E) co-localise with SMN in HeLa cells stably transfected with human SMN1 in pcDNA3 [49]. Gemin5 (C) does not co-localise with SMN. The same mAbs as in Fig. 2 were used, plus MANSIP1A for gemin2. Double label of SMN mAb MANSMA1 with gemin3 rabbit antiserum (F), unrip rabbit antiserum (G) and fibrillarin human auto-antiserum (H), or SMN antiserum with p80 mAb (I) were performed on the HeLa cell line stably transfected with SMN1. Alexa fluor 546 goat anti-mouse IgG (red) was used to detect mouse mAbs and Alexa fluor 488 goat anti-rabbit IgG (green) was used to detect rabbit antibodies. FITC-conjugated goat anti-human IgG (Chemicon) was used to detect fibrillarin. DAPI (blue) was used to counterstain the nuclei.
Figure 5
Figure 5
All gemins, except gemin5, are proportionately reduced in SMN-deficient human skin fibroblasts. Total SDS extracts of the two skin fibroblast cell lines (Coriell cell lines, GM08333, control and GM03813, SMA) were subjected to SDS-PAGE on adjacent lanes of a 7% or 14% acrylamide gel (cf. Fig. 1), followed by western blotting with the same antibodies used in Fig. 2 plus MANSIP1A for gemin2. β-actin was used as a control for equal loading of the gel lanes. Microdensitometry was used to express protein levels in SMA fibroblasts as a percentage of those in control fibroblasts.
Figure 6
Figure 6
Nuclear gems/CBs and cytoplasmic levels of most gemins are greatly reduced in SMN-deficient human skin fibroblasts, but cytoplasmic gemin5 levels remain high. Alexa fluor 546 goat anti-(mouse IgG) (red) was used to detect SMN and gemin 2, 4, 5, 6 and 7 primary mAbs (the same mAbs as in Fig. 2). TRITC donkey anti-(rabbit Ig) was used to detect anti-gemin3 rabbit antibodies. DAPI (blue) was used to counterstain the nuclei.
Figure 7
Figure 7
Distribution of gemins and other proteins between cytoplasmic and nuclear fractions of HeLa and Ntera2 cells. HeLa C and Ntera C are cytoplasmic fractions, HeLa N and Ntera N are nuclear fractions. After extraction with cytoplasmic buffer (see Methods), the whole pellet was boiled in SDS sample buffer to obtain the "nuclear" extract. Samples were loaded as serial dilutions (from left to right, 1/4, 1/2 and 1) for more accurate microdensitometry quantitation. γ tubulin and lamin A/C were used as controls for cross-contamination between nuclear and cytoplasmic fractions.
Figure 8
Figure 8
Distribution of SMN and gemins between large, fast-sedimenting SMN complexes and slower sedimenting fractions. HeLa total, cytoplasmic and nuclear extracts were prepared and analyzed by centrifugation on 15–30% sucrose density gradients. Thirty 1 ml fractions were collected from each gradient and the protein contents were concentrated into 0.1 ml using Strataclean resin. Every other fraction (1, 3, 5, etc) was subjected to SDS-PAGE for western blotting using appropriate antibodies. The size markers were immunoglobulin (7S), catalase (11S) and thyroglobulin (19S).
Figure 9
Figure 9
(A) Co-immunoprecipitation with MANSMA1 anti-SMN mAb shows that SMN and gemin5 exist as complexes in cytoplasmic extracts, but not in nuclear extracts (arrows show lanes to be compared). SMN and gemin4 controls are pulled down from both extracts equally. (B) Addition of KCl to the cytoplasmic extract to the same concentration as nuclear extraction buffer did not disrupt the SMN-gemin5 interaction. (C) All gemins in SMN complexes were accessible to appropriate antibodies. All anti-gemin antibodies pulled down SMN from total RIPA extracts of HeLa cells, except GEM5G which only recognizes denatured gemin5 on western blots (this also acts as a negative control). Antibodies were (left to right; see Table 1): No mAb (input control), GEM6B, GEM7B, GEM5G, GEM5P, GEM4C, GEM4D, MANSIP1B, MANSIP1A, rabbit anti-gemin3 and MANSMA1. In each case, 0.05 ml of Dynabead magnetic beads (Dynal, Oslo, Cat. No. 100.41), with anti-mouse Ig (or anti-rabbit Ig) attached covalently, were incubated with 0.1 ml of undiluted mAb culture supernatant (or 1/100 dilution antiserum) for 1 h at 4°C, washed 3× with PBS containing 0.1% BSA, and then incubated for 16 h at 4°C with 0.08 ml of HeLa extract (also sampled as "input"). After removing the "unbound" extract, the beads were washed 5× with PBS and boiled in 0.02 ml of SDS sample buffer. Gels (10% or 12.5% polyacrylamide) were loaded with 0.01 ml of SDS extract for SDS-PAGE and western blotting with anti-SMN mAb MANSMA12, as described in Methods. All lanes loaded with SDS extracts of beads contain a 50 KDa band of mouse Ig heavy chain which reacts with the HRP anti-mouse Ig used to develop the blot (band is absent from input and when rabbit antisera are used on beads).
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