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A cytoplasmic variant of the KH-type splicing regulatory protein serves as a decay-promoting factor for phosphoglycerate kinase 2 mRNA in murine male germ cells.

Xu M, McCarrey JR, Hecht NB - Nucleic Acids Res. (2008)

Bottom Line: KSRP, a protein of approximately 75 kDa, is widely expressed in somatic and germ cells where it is primarily nuclear.In addition to the approximately 75-kDa KSRP, a approximately 52-kD KSRP, t-KSRP, is present in the cytoplasm of a subpopulation of germ cells. t-KSRP binds directly to a 93-nt sequence (designated the F1 region) of the 3'-UTR of the Pgk2 mRNA and destabilizes Pgk2 mRNA constructs in testis extracts and in transfected cells.We conclude that this testicular variant of the multifunctional nucleic acid-binding protein, KSRP, serves as a decay-promoting factor for Pgk2 mRNA in male germ cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, Philadelphia, PA 6080, USA.

ABSTRACT
Phosphoglycerate kinase 2 (PGK2) is a germ cell-specific protein whose mRNA is translationally regulated in the mammalian testis. Using RNA affinity chromatography with the 3'-untranslated region (UTR) of Pgk2 mRNA and adult testis extracts, several associated proteins including a novel isoform of the AU-rich element RNA-binding protein and KH-type splicing regulatory protein (KSRP) were identified. KSRP, a protein of approximately 75 kDa, is widely expressed in somatic and germ cells where it is primarily nuclear. In addition to the approximately 75-kDa KSRP, a approximately 52-kD KSRP, t-KSRP, is present in the cytoplasm of a subpopulation of germ cells. t-KSRP binds directly to a 93-nt sequence (designated the F1 region) of the 3'-UTR of the Pgk2 mRNA and destabilizes Pgk2 mRNA constructs in testis extracts and in transfected cells. We conclude that this testicular variant of the multifunctional nucleic acid-binding protein, KSRP, serves as a decay-promoting factor for Pgk2 mRNA in male germ cells.

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t-KSRP destabilizes RNAs containing the 3′-UTR of Pgk2 mRNA. (A) t-KSRP destabilizes RNA constructs in testis extracts, and recombinant PTBP2 cannot prevent the destabilization. 5'-capped and 3'-polyadenylated radiolabeled F1 or F2 transcript was incubated at 30°C with testis extracts with or without recombinant t-KSRP (100 or 200 nM) or PTBP2 (200 nM). RNA was purified and analyzed by gel electrophoresis followed by autoradiography. Gels were quantitated by phosphorimaging. The amount of RNA at the beginning of the reaction was set at 100%. All the decay assays were performed at least twice. (B) F1 RNAs are degraded more rapidly in extracts from 17-day-old mice than from adults. Decay assays were performed as in (A) with prepuberal (extracts from testes of 17-day-old mice) or adult extracts except that incubation temperature was set at 25°C. (C) Depletion of KSRP from testis extracts leads to the stabilization of F1 RNA. Decay assays were performed as in (A) with cytoplasmic extracts from which KSRP was depleted.
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Figure 6: t-KSRP destabilizes RNAs containing the 3′-UTR of Pgk2 mRNA. (A) t-KSRP destabilizes RNA constructs in testis extracts, and recombinant PTBP2 cannot prevent the destabilization. 5'-capped and 3'-polyadenylated radiolabeled F1 or F2 transcript was incubated at 30°C with testis extracts with or without recombinant t-KSRP (100 or 200 nM) or PTBP2 (200 nM). RNA was purified and analyzed by gel electrophoresis followed by autoradiography. Gels were quantitated by phosphorimaging. The amount of RNA at the beginning of the reaction was set at 100%. All the decay assays were performed at least twice. (B) F1 RNAs are degraded more rapidly in extracts from 17-day-old mice than from adults. Decay assays were performed as in (A) with prepuberal (extracts from testes of 17-day-old mice) or adult extracts except that incubation temperature was set at 25°C. (C) Depletion of KSRP from testis extracts leads to the stabilization of F1 RNA. Decay assays were performed as in (A) with cytoplasmic extracts from which KSRP was depleted.

Mentions: To assess the role of t-KSRP in in vitro decay, 5′-capped [32P]-labeled F1 or F2 transcript was incubated with testis extracts and increasing amounts of recombinant t-KSRP (Figure 6A). Addition of 50 nM of t-KSRP leads to complete F1 RNA degradation within 20 min of incubation (Figure 6A, lane 2), although only a modest RNA reduction was seen in control testis extracts with up to 50 min of incubation (Figure 6A, lane 1). F1 RNA degradation increased when recombinant t-KSRP was increased to 100 nM (Figure 6A, lane 3). In contrast, no increased RNA degradation was seen when 100 nM t-KSRP was added to testis extracts containing the nonbinding F2 RNA (Figure 6A, compare lanes 6 and 7). As previously reported (9), addition of r-PTBP2 completely stabilized F1 RNA in incubations up to 50 min (Figure 6A, lane 5), but addition of up to 200 nM PTBP2 failed to prevent F1 RNA destabilization when exogenous t-KSRP was added (Figure 6A, lane 4). These in vitro decay assays indicate that t-KSRP promotes RNA degradation of a target mRNA and suggest t-KSRP exerts a dominant effect over PTBP2 in testis extracts under the assay conditions tested.Figure 6.


A cytoplasmic variant of the KH-type splicing regulatory protein serves as a decay-promoting factor for phosphoglycerate kinase 2 mRNA in murine male germ cells.

Xu M, McCarrey JR, Hecht NB - Nucleic Acids Res. (2008)

t-KSRP destabilizes RNAs containing the 3′-UTR of Pgk2 mRNA. (A) t-KSRP destabilizes RNA constructs in testis extracts, and recombinant PTBP2 cannot prevent the destabilization. 5'-capped and 3'-polyadenylated radiolabeled F1 or F2 transcript was incubated at 30°C with testis extracts with or without recombinant t-KSRP (100 or 200 nM) or PTBP2 (200 nM). RNA was purified and analyzed by gel electrophoresis followed by autoradiography. Gels were quantitated by phosphorimaging. The amount of RNA at the beginning of the reaction was set at 100%. All the decay assays were performed at least twice. (B) F1 RNAs are degraded more rapidly in extracts from 17-day-old mice than from adults. Decay assays were performed as in (A) with prepuberal (extracts from testes of 17-day-old mice) or adult extracts except that incubation temperature was set at 25°C. (C) Depletion of KSRP from testis extracts leads to the stabilization of F1 RNA. Decay assays were performed as in (A) with cytoplasmic extracts from which KSRP was depleted.
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Figure 6: t-KSRP destabilizes RNAs containing the 3′-UTR of Pgk2 mRNA. (A) t-KSRP destabilizes RNA constructs in testis extracts, and recombinant PTBP2 cannot prevent the destabilization. 5'-capped and 3'-polyadenylated radiolabeled F1 or F2 transcript was incubated at 30°C with testis extracts with or without recombinant t-KSRP (100 or 200 nM) or PTBP2 (200 nM). RNA was purified and analyzed by gel electrophoresis followed by autoradiography. Gels were quantitated by phosphorimaging. The amount of RNA at the beginning of the reaction was set at 100%. All the decay assays were performed at least twice. (B) F1 RNAs are degraded more rapidly in extracts from 17-day-old mice than from adults. Decay assays were performed as in (A) with prepuberal (extracts from testes of 17-day-old mice) or adult extracts except that incubation temperature was set at 25°C. (C) Depletion of KSRP from testis extracts leads to the stabilization of F1 RNA. Decay assays were performed as in (A) with cytoplasmic extracts from which KSRP was depleted.
Mentions: To assess the role of t-KSRP in in vitro decay, 5′-capped [32P]-labeled F1 or F2 transcript was incubated with testis extracts and increasing amounts of recombinant t-KSRP (Figure 6A). Addition of 50 nM of t-KSRP leads to complete F1 RNA degradation within 20 min of incubation (Figure 6A, lane 2), although only a modest RNA reduction was seen in control testis extracts with up to 50 min of incubation (Figure 6A, lane 1). F1 RNA degradation increased when recombinant t-KSRP was increased to 100 nM (Figure 6A, lane 3). In contrast, no increased RNA degradation was seen when 100 nM t-KSRP was added to testis extracts containing the nonbinding F2 RNA (Figure 6A, compare lanes 6 and 7). As previously reported (9), addition of r-PTBP2 completely stabilized F1 RNA in incubations up to 50 min (Figure 6A, lane 5), but addition of up to 200 nM PTBP2 failed to prevent F1 RNA destabilization when exogenous t-KSRP was added (Figure 6A, lane 4). These in vitro decay assays indicate that t-KSRP promotes RNA degradation of a target mRNA and suggest t-KSRP exerts a dominant effect over PTBP2 in testis extracts under the assay conditions tested.Figure 6.

Bottom Line: KSRP, a protein of approximately 75 kDa, is widely expressed in somatic and germ cells where it is primarily nuclear.In addition to the approximately 75-kDa KSRP, a approximately 52-kD KSRP, t-KSRP, is present in the cytoplasm of a subpopulation of germ cells. t-KSRP binds directly to a 93-nt sequence (designated the F1 region) of the 3'-UTR of the Pgk2 mRNA and destabilizes Pgk2 mRNA constructs in testis extracts and in transfected cells.We conclude that this testicular variant of the multifunctional nucleic acid-binding protein, KSRP, serves as a decay-promoting factor for Pgk2 mRNA in male germ cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, Philadelphia, PA 6080, USA.

ABSTRACT
Phosphoglycerate kinase 2 (PGK2) is a germ cell-specific protein whose mRNA is translationally regulated in the mammalian testis. Using RNA affinity chromatography with the 3'-untranslated region (UTR) of Pgk2 mRNA and adult testis extracts, several associated proteins including a novel isoform of the AU-rich element RNA-binding protein and KH-type splicing regulatory protein (KSRP) were identified. KSRP, a protein of approximately 75 kDa, is widely expressed in somatic and germ cells where it is primarily nuclear. In addition to the approximately 75-kDa KSRP, a approximately 52-kD KSRP, t-KSRP, is present in the cytoplasm of a subpopulation of germ cells. t-KSRP binds directly to a 93-nt sequence (designated the F1 region) of the 3'-UTR of the Pgk2 mRNA and destabilizes Pgk2 mRNA constructs in testis extracts and in transfected cells. We conclude that this testicular variant of the multifunctional nucleic acid-binding protein, KSRP, serves as a decay-promoting factor for Pgk2 mRNA in male germ cells.

Show MeSH
Related in: MedlinePlus