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Presynaptic localization of Smn and hnRNP R in axon terminals of embryonic and postnatal mouse motoneurons.

Dombert B, Sivadasan R, Simon CM, Jablonka S, Sendtner M - PLoS ONE (2014)

Bottom Line: Smn and hnRNP R are localized in close proximity to each other in axons and axon terminals both in vitro and in vivo.We also provide new evidence for a direct interaction of Smn and hnRNP R in vitro and in vivo, particularly in the cytosol of motoneurons.These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute for Clinical Neurobiology, University Hospital Wuerzburg, Wuerzburg, Germany.

ABSTRACT
Spinal muscular atrophy (SMA) is caused by deficiency of the ubiquitously expressed survival motoneuron (SMN) protein. SMN is crucial component of a complex for the assembly of spliceosomal small nuclear ribonucleoprotein (snRNP) particles. Other cellular functions of SMN are less characterized so far. SMA predominantly affects lower motoneurons, but the cellular basis for this relative specificity is still unknown. In contrast to nonneuronal cells where the protein is mainly localized in perinuclear regions and the nucleus, Smn is also present in dendrites, axons and axonal growth cones of isolated motoneurons in vitro. However, this distribution has not been shown in vivo and it is not clear whether Smn and hnRNP R are also present in presynaptic axon terminals of motoneurons in postnatal mice. Smn also associates with components not included in the classical SMN complex like RNA-binding proteins FUS, TDP43, HuD and hnRNP R which are involved in RNA processing, subcellular localization and translation. We show here that Smn and hnRNP R are present in presynaptic compartments at neuromuscular endplates of embryonic and postnatal mice. Smn and hnRNP R are localized in close proximity to each other in axons and axon terminals both in vitro and in vivo. We also provide new evidence for a direct interaction of Smn and hnRNP R in vitro and in vivo, particularly in the cytosol of motoneurons. These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis.

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Coimmunoprecipitation of Smn and hnRNP R in primary motoneurons and native spinal cord.(A) 1 000 000 primary motoneurons were cultured for 7DIV on laminin-111. Cytosolic and soluble nuclear fractions were subjected to a pull-down with either Smn or hnRNP R antibodies, respectively. Coprecipitation of hnRNP R or Smn, respectively, was determined revealing an interaction of Smn and hnRNP R, particularly in the cytosolic fraction of embryonic mouse motoneurons (eluate lane). Smn was not detectable in the soluble nuclear fraction of motoneurons. HnRNP R was found both in nuclear and cytosolic extracts. For immunoprecipitation experiments a C-terminal antibody directed against hnRNP R (Abcam) was used [29]. Supernatants still contained some Smn or hnRNP R protein, respectively, suggesting that the interaction appears not to be exclusive as demonstrated by immunofluorescence colocalization analysis. No signal was obtained in the washing solution. Successful fractionation was controlled by α tubulin (cytosol) and histone H3 (nucleus) (right panel). (B) Fractionation of spinal cord tissue from E18 mouse embryos revealed a similar result as shown in (A). In the cytosolic fraction hnRNP R IP pulled-down Smn protein and vice versa. Nuclear Smn was not detected in the soluble, but in the corresponding insoluble nuclear fraction (right panel, lower blot). In contrast, nuclear hnRNP R was not found in the insoluble nuclear fraction. Cytosolic and nuclear extracts were validated by α tubulin and histone H3. (C) HEK293T cells were cultured and cytosolic and soluble nuclear fractions were prepared. Smn and hnRNP R were detected in cytosolic extracts as well as in soluble nuclear fractions. The pull down of Smn and hnRNP R, respectively, was successful (eluate lane, IP), but hnRNP R or Smn, respectively, could not be coprecipitated, neither from cytosolic nor from nuclear extracts. Successful fractionation was verified by GAPDH (cytosolic) and histone H3 (nucleus) (right panel).
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pone-0110846-g004: Coimmunoprecipitation of Smn and hnRNP R in primary motoneurons and native spinal cord.(A) 1 000 000 primary motoneurons were cultured for 7DIV on laminin-111. Cytosolic and soluble nuclear fractions were subjected to a pull-down with either Smn or hnRNP R antibodies, respectively. Coprecipitation of hnRNP R or Smn, respectively, was determined revealing an interaction of Smn and hnRNP R, particularly in the cytosolic fraction of embryonic mouse motoneurons (eluate lane). Smn was not detectable in the soluble nuclear fraction of motoneurons. HnRNP R was found both in nuclear and cytosolic extracts. For immunoprecipitation experiments a C-terminal antibody directed against hnRNP R (Abcam) was used [29]. Supernatants still contained some Smn or hnRNP R protein, respectively, suggesting that the interaction appears not to be exclusive as demonstrated by immunofluorescence colocalization analysis. No signal was obtained in the washing solution. Successful fractionation was controlled by α tubulin (cytosol) and histone H3 (nucleus) (right panel). (B) Fractionation of spinal cord tissue from E18 mouse embryos revealed a similar result as shown in (A). In the cytosolic fraction hnRNP R IP pulled-down Smn protein and vice versa. Nuclear Smn was not detected in the soluble, but in the corresponding insoluble nuclear fraction (right panel, lower blot). In contrast, nuclear hnRNP R was not found in the insoluble nuclear fraction. Cytosolic and nuclear extracts were validated by α tubulin and histone H3. (C) HEK293T cells were cultured and cytosolic and soluble nuclear fractions were prepared. Smn and hnRNP R were detected in cytosolic extracts as well as in soluble nuclear fractions. The pull down of Smn and hnRNP R, respectively, was successful (eluate lane, IP), but hnRNP R or Smn, respectively, could not be coprecipitated, neither from cytosolic nor from nuclear extracts. Successful fractionation was verified by GAPDH (cytosolic) and histone H3 (nucleus) (right panel).

Mentions: We proceeded to examine whether the interaction of hnRNP R and Smn differs between cellular compartments (Fig. 4) using cytosolic and nuclear fractions from isolated motoneurons (Fig. 4A), E18 spinal cord (Fig. 4B) and HEK293T cells (Fig. 4C). Motoneurons were cultured for 7DIV on laminin-111 since the relative proportion of cytosolic hnRNP R and the degree of overlap with Smn protein was highest at this time point as described above. Antibodies against histone H3 were used as marker for the nuclear fraction, and antibodies against α tubulin and GAPDH for the cytosolic fraction (Fig. 4A–C, right panels). HnRNP R was found both in the soluble nuclear and in the cytosolic fraction. Intriguingly, interaction of Smn and hnRNP R was predominantly detected in cytosolic compartments of cultured motoneurons (Fig. 4A) and spinal cord extracts (Fig. 4B). Pulldown of hnRNP R coprecipitated Smn and vice versa.


Presynaptic localization of Smn and hnRNP R in axon terminals of embryonic and postnatal mouse motoneurons.

Dombert B, Sivadasan R, Simon CM, Jablonka S, Sendtner M - PLoS ONE (2014)

Coimmunoprecipitation of Smn and hnRNP R in primary motoneurons and native spinal cord.(A) 1 000 000 primary motoneurons were cultured for 7DIV on laminin-111. Cytosolic and soluble nuclear fractions were subjected to a pull-down with either Smn or hnRNP R antibodies, respectively. Coprecipitation of hnRNP R or Smn, respectively, was determined revealing an interaction of Smn and hnRNP R, particularly in the cytosolic fraction of embryonic mouse motoneurons (eluate lane). Smn was not detectable in the soluble nuclear fraction of motoneurons. HnRNP R was found both in nuclear and cytosolic extracts. For immunoprecipitation experiments a C-terminal antibody directed against hnRNP R (Abcam) was used [29]. Supernatants still contained some Smn or hnRNP R protein, respectively, suggesting that the interaction appears not to be exclusive as demonstrated by immunofluorescence colocalization analysis. No signal was obtained in the washing solution. Successful fractionation was controlled by α tubulin (cytosol) and histone H3 (nucleus) (right panel). (B) Fractionation of spinal cord tissue from E18 mouse embryos revealed a similar result as shown in (A). In the cytosolic fraction hnRNP R IP pulled-down Smn protein and vice versa. Nuclear Smn was not detected in the soluble, but in the corresponding insoluble nuclear fraction (right panel, lower blot). In contrast, nuclear hnRNP R was not found in the insoluble nuclear fraction. Cytosolic and nuclear extracts were validated by α tubulin and histone H3. (C) HEK293T cells were cultured and cytosolic and soluble nuclear fractions were prepared. Smn and hnRNP R were detected in cytosolic extracts as well as in soluble nuclear fractions. The pull down of Smn and hnRNP R, respectively, was successful (eluate lane, IP), but hnRNP R or Smn, respectively, could not be coprecipitated, neither from cytosolic nor from nuclear extracts. Successful fractionation was verified by GAPDH (cytosolic) and histone H3 (nucleus) (right panel).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4206449&req=5

pone-0110846-g004: Coimmunoprecipitation of Smn and hnRNP R in primary motoneurons and native spinal cord.(A) 1 000 000 primary motoneurons were cultured for 7DIV on laminin-111. Cytosolic and soluble nuclear fractions were subjected to a pull-down with either Smn or hnRNP R antibodies, respectively. Coprecipitation of hnRNP R or Smn, respectively, was determined revealing an interaction of Smn and hnRNP R, particularly in the cytosolic fraction of embryonic mouse motoneurons (eluate lane). Smn was not detectable in the soluble nuclear fraction of motoneurons. HnRNP R was found both in nuclear and cytosolic extracts. For immunoprecipitation experiments a C-terminal antibody directed against hnRNP R (Abcam) was used [29]. Supernatants still contained some Smn or hnRNP R protein, respectively, suggesting that the interaction appears not to be exclusive as demonstrated by immunofluorescence colocalization analysis. No signal was obtained in the washing solution. Successful fractionation was controlled by α tubulin (cytosol) and histone H3 (nucleus) (right panel). (B) Fractionation of spinal cord tissue from E18 mouse embryos revealed a similar result as shown in (A). In the cytosolic fraction hnRNP R IP pulled-down Smn protein and vice versa. Nuclear Smn was not detected in the soluble, but in the corresponding insoluble nuclear fraction (right panel, lower blot). In contrast, nuclear hnRNP R was not found in the insoluble nuclear fraction. Cytosolic and nuclear extracts were validated by α tubulin and histone H3. (C) HEK293T cells were cultured and cytosolic and soluble nuclear fractions were prepared. Smn and hnRNP R were detected in cytosolic extracts as well as in soluble nuclear fractions. The pull down of Smn and hnRNP R, respectively, was successful (eluate lane, IP), but hnRNP R or Smn, respectively, could not be coprecipitated, neither from cytosolic nor from nuclear extracts. Successful fractionation was verified by GAPDH (cytosolic) and histone H3 (nucleus) (right panel).
Mentions: We proceeded to examine whether the interaction of hnRNP R and Smn differs between cellular compartments (Fig. 4) using cytosolic and nuclear fractions from isolated motoneurons (Fig. 4A), E18 spinal cord (Fig. 4B) and HEK293T cells (Fig. 4C). Motoneurons were cultured for 7DIV on laminin-111 since the relative proportion of cytosolic hnRNP R and the degree of overlap with Smn protein was highest at this time point as described above. Antibodies against histone H3 were used as marker for the nuclear fraction, and antibodies against α tubulin and GAPDH for the cytosolic fraction (Fig. 4A–C, right panels). HnRNP R was found both in the soluble nuclear and in the cytosolic fraction. Intriguingly, interaction of Smn and hnRNP R was predominantly detected in cytosolic compartments of cultured motoneurons (Fig. 4A) and spinal cord extracts (Fig. 4B). Pulldown of hnRNP R coprecipitated Smn and vice versa.

Bottom Line: Smn and hnRNP R are localized in close proximity to each other in axons and axon terminals both in vitro and in vivo.We also provide new evidence for a direct interaction of Smn and hnRNP R in vitro and in vivo, particularly in the cytosol of motoneurons.These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute for Clinical Neurobiology, University Hospital Wuerzburg, Wuerzburg, Germany.

ABSTRACT
Spinal muscular atrophy (SMA) is caused by deficiency of the ubiquitously expressed survival motoneuron (SMN) protein. SMN is crucial component of a complex for the assembly of spliceosomal small nuclear ribonucleoprotein (snRNP) particles. Other cellular functions of SMN are less characterized so far. SMA predominantly affects lower motoneurons, but the cellular basis for this relative specificity is still unknown. In contrast to nonneuronal cells where the protein is mainly localized in perinuclear regions and the nucleus, Smn is also present in dendrites, axons and axonal growth cones of isolated motoneurons in vitro. However, this distribution has not been shown in vivo and it is not clear whether Smn and hnRNP R are also present in presynaptic axon terminals of motoneurons in postnatal mice. Smn also associates with components not included in the classical SMN complex like RNA-binding proteins FUS, TDP43, HuD and hnRNP R which are involved in RNA processing, subcellular localization and translation. We show here that Smn and hnRNP R are present in presynaptic compartments at neuromuscular endplates of embryonic and postnatal mice. Smn and hnRNP R are localized in close proximity to each other in axons and axon terminals both in vitro and in vivo. We also provide new evidence for a direct interaction of Smn and hnRNP R in vitro and in vivo, particularly in the cytosol of motoneurons. These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis.

Show MeSH
Related in: MedlinePlus