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RNA binding protein Caprin-2 is a pivotal regulator of the central osmotic defense response.

Konopacka A, Greenwood M, Loh SY, Paton J, Murphy D - Elife (2015)

Bottom Line: In a recapitulated in vitro system, we confirm that Caprin-2 over-expression enhances AVP mRNA abundance and poly(A) tail length.Importantly, we show that Caprin-2 knockdown in the hypothalamus decreases urine output and fluid intake, and increases urine osmolality, urine sodium concentration, and plasma AVP levels.Thus Caprin-2 controls physiological mechanisms that are essential for the body's response to osmotic stress.

View Article: PubMed Central - PubMed

Affiliation: School of Clinical Sciences, University of Bristol, Bristol, United Kingdom.

ABSTRACT
In response to an osmotic challenge, the synthesis of the antidiuretic hormone arginine vasopressin (AVP) increases in the hypothalamus, and this is accompanied by extension of the 3' poly(A) tail of the AVP mRNA, and the up-regulation of the expression of RNA binding protein Caprin-2. Here we show that Caprin-2 binds to AVP mRNAs, and that lentiviral mediated shRNA knockdown of Caprin-2 in the osmotically stimulated hypothalamus shortens the AVP mRNA poly(A) tail at the same time as reducing transcript abundance. In a recapitulated in vitro system, we confirm that Caprin-2 over-expression enhances AVP mRNA abundance and poly(A) tail length. Importantly, we show that Caprin-2 knockdown in the hypothalamus decreases urine output and fluid intake, and increases urine osmolality, urine sodium concentration, and plasma AVP levels. Thus Caprin-2 controls physiological mechanisms that are essential for the body's response to osmotic stress.

No MeSH data available.


Caprin-2 binds to the AVP mRNA in the SON and PVN.Binding of AVP by Caprin-2 protein in the SON and PVN of euhydrated (EU) and salt-loaded (SL) rats determined by RNA immunoprecipitation assay. (A) In the RNA immunoprecipitation assay, SON or PVN tissue punches from EU or SL rats were first exposed to formaldehyde in order to covalently cross-link RNA with associated proteins. Cell extracts were then incubated with antibodies recognizing Caprin-2. Following immunoprecipitation, and hence enrichment of specific complexes, cross-links were reversed and extracted RNA was subject to qRT-PCR to detect AVP mRNA sequences. (B) Effects of salt-loading on the amount of Caprin-2 binding to AVP mRNA in the SON (1.2 ± 0.36 EU vs 2.05 ± 0.20 SL, n = 5, p = 0.072) and PVN (1.03 ± 0.13 EU vs 3.6 ± 0.92; n = 5, p = 0.0243). (C) Salt-loading has no effect on the amount of Caprin-2 binding to Rpl19 mRNA in the SON and PVN. *p ≤ 0.05.DOI:http://dx.doi.org/10.7554/eLife.09656.008
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fig5: Caprin-2 binds to the AVP mRNA in the SON and PVN.Binding of AVP by Caprin-2 protein in the SON and PVN of euhydrated (EU) and salt-loaded (SL) rats determined by RNA immunoprecipitation assay. (A) In the RNA immunoprecipitation assay, SON or PVN tissue punches from EU or SL rats were first exposed to formaldehyde in order to covalently cross-link RNA with associated proteins. Cell extracts were then incubated with antibodies recognizing Caprin-2. Following immunoprecipitation, and hence enrichment of specific complexes, cross-links were reversed and extracted RNA was subject to qRT-PCR to detect AVP mRNA sequences. (B) Effects of salt-loading on the amount of Caprin-2 binding to AVP mRNA in the SON (1.2 ± 0.36 EU vs 2.05 ± 0.20 SL, n = 5, p = 0.072) and PVN (1.03 ± 0.13 EU vs 3.6 ± 0.92; n = 5, p = 0.0243). (C) Salt-loading has no effect on the amount of Caprin-2 binding to Rpl19 mRNA in the SON and PVN. *p ≤ 0.05.DOI:http://dx.doi.org/10.7554/eLife.09656.008

Mentions: Previous studies have shown that Caprin-2 is an RNA binding protein (Shiina and Tokunaga, 2010). We thus tested the hypothesis that in the rat SON and PVN, Caprin-2 might bind to the AVP mRNA. We performed an RNA immunoprecipitation assay on extracts from the SON and PVN of EU and SL rats using anti-Caprin-2 antibodies, followed by qRT-PCR (Figure 5A). The level of Caprin-2-AVP mRNA binding was quantified by comparing it to the signal detected with the non-specific binding control IgG performed simultaneously for each individual sample. In all samples incubated with Caprin-2 antibody, we found AVP mRNA at levels 1–2 orders of magnitude higher than in samples incubated with non-specific IgG. AVP mRNA levels in the Caprin-2-enriched extracts from EU and SL SON were respectively 49.44 ± 10.77 (n = 5, p = 0.002) and 23.77 ± 4.22 (n = 5, p = 0.0006) times higher, as compared to extracts incubated with non-specific IgG. In the EU and SL PVN, these values were respectively, 91.92 ± 24.25 (n = 4, p = 0.0037) and 108 ± 11.74 (n = 5, p ≤ 0.0001). Binding to the control Rpl19 mRNA was negligible (not shown).10.7554/eLife.09656.008Figure 5.Caprin-2 binds to the AVP mRNA in the SON and PVN.


RNA binding protein Caprin-2 is a pivotal regulator of the central osmotic defense response.

Konopacka A, Greenwood M, Loh SY, Paton J, Murphy D - Elife (2015)

Caprin-2 binds to the AVP mRNA in the SON and PVN.Binding of AVP by Caprin-2 protein in the SON and PVN of euhydrated (EU) and salt-loaded (SL) rats determined by RNA immunoprecipitation assay. (A) In the RNA immunoprecipitation assay, SON or PVN tissue punches from EU or SL rats were first exposed to formaldehyde in order to covalently cross-link RNA with associated proteins. Cell extracts were then incubated with antibodies recognizing Caprin-2. Following immunoprecipitation, and hence enrichment of specific complexes, cross-links were reversed and extracted RNA was subject to qRT-PCR to detect AVP mRNA sequences. (B) Effects of salt-loading on the amount of Caprin-2 binding to AVP mRNA in the SON (1.2 ± 0.36 EU vs 2.05 ± 0.20 SL, n = 5, p = 0.072) and PVN (1.03 ± 0.13 EU vs 3.6 ± 0.92; n = 5, p = 0.0243). (C) Salt-loading has no effect on the amount of Caprin-2 binding to Rpl19 mRNA in the SON and PVN. *p ≤ 0.05.DOI:http://dx.doi.org/10.7554/eLife.09656.008
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fig5: Caprin-2 binds to the AVP mRNA in the SON and PVN.Binding of AVP by Caprin-2 protein in the SON and PVN of euhydrated (EU) and salt-loaded (SL) rats determined by RNA immunoprecipitation assay. (A) In the RNA immunoprecipitation assay, SON or PVN tissue punches from EU or SL rats were first exposed to formaldehyde in order to covalently cross-link RNA with associated proteins. Cell extracts were then incubated with antibodies recognizing Caprin-2. Following immunoprecipitation, and hence enrichment of specific complexes, cross-links were reversed and extracted RNA was subject to qRT-PCR to detect AVP mRNA sequences. (B) Effects of salt-loading on the amount of Caprin-2 binding to AVP mRNA in the SON (1.2 ± 0.36 EU vs 2.05 ± 0.20 SL, n = 5, p = 0.072) and PVN (1.03 ± 0.13 EU vs 3.6 ± 0.92; n = 5, p = 0.0243). (C) Salt-loading has no effect on the amount of Caprin-2 binding to Rpl19 mRNA in the SON and PVN. *p ≤ 0.05.DOI:http://dx.doi.org/10.7554/eLife.09656.008
Mentions: Previous studies have shown that Caprin-2 is an RNA binding protein (Shiina and Tokunaga, 2010). We thus tested the hypothesis that in the rat SON and PVN, Caprin-2 might bind to the AVP mRNA. We performed an RNA immunoprecipitation assay on extracts from the SON and PVN of EU and SL rats using anti-Caprin-2 antibodies, followed by qRT-PCR (Figure 5A). The level of Caprin-2-AVP mRNA binding was quantified by comparing it to the signal detected with the non-specific binding control IgG performed simultaneously for each individual sample. In all samples incubated with Caprin-2 antibody, we found AVP mRNA at levels 1–2 orders of magnitude higher than in samples incubated with non-specific IgG. AVP mRNA levels in the Caprin-2-enriched extracts from EU and SL SON were respectively 49.44 ± 10.77 (n = 5, p = 0.002) and 23.77 ± 4.22 (n = 5, p = 0.0006) times higher, as compared to extracts incubated with non-specific IgG. In the EU and SL PVN, these values were respectively, 91.92 ± 24.25 (n = 4, p = 0.0037) and 108 ± 11.74 (n = 5, p ≤ 0.0001). Binding to the control Rpl19 mRNA was negligible (not shown).10.7554/eLife.09656.008Figure 5.Caprin-2 binds to the AVP mRNA in the SON and PVN.

Bottom Line: In a recapitulated in vitro system, we confirm that Caprin-2 over-expression enhances AVP mRNA abundance and poly(A) tail length.Importantly, we show that Caprin-2 knockdown in the hypothalamus decreases urine output and fluid intake, and increases urine osmolality, urine sodium concentration, and plasma AVP levels.Thus Caprin-2 controls physiological mechanisms that are essential for the body's response to osmotic stress.

View Article: PubMed Central - PubMed

Affiliation: School of Clinical Sciences, University of Bristol, Bristol, United Kingdom.

ABSTRACT
In response to an osmotic challenge, the synthesis of the antidiuretic hormone arginine vasopressin (AVP) increases in the hypothalamus, and this is accompanied by extension of the 3' poly(A) tail of the AVP mRNA, and the up-regulation of the expression of RNA binding protein Caprin-2. Here we show that Caprin-2 binds to AVP mRNAs, and that lentiviral mediated shRNA knockdown of Caprin-2 in the osmotically stimulated hypothalamus shortens the AVP mRNA poly(A) tail at the same time as reducing transcript abundance. In a recapitulated in vitro system, we confirm that Caprin-2 over-expression enhances AVP mRNA abundance and poly(A) tail length. Importantly, we show that Caprin-2 knockdown in the hypothalamus decreases urine output and fluid intake, and increases urine osmolality, urine sodium concentration, and plasma AVP levels. Thus Caprin-2 controls physiological mechanisms that are essential for the body's response to osmotic stress.

No MeSH data available.