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Bombyx E75 isoforms display stage- and tissue-specific responses to 20-hydroxyecdysone.

Li K, Guo E, Hossain MS, Li Q, Cao Y, Tian L, Deng X, Li S - Sci Rep (2015)

Bottom Line: At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E.At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues.In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

View Article: PubMed Central - PubMed

Affiliation: 1] Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding/Guangdong Provincial Sericulture and Mulberry Engineering Research Center, College of Animal Sciences, South China Agricultural University, Guangzhou, 510642, China [2] Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

ABSTRACT
Resulted from alternative splicing of the 5' exons, the nuclear receptor gene E75 in the silkworm, Bombyx mori, processes three mRNA isoforms, BmE75A, BmE75B and BmE75C. From the early 5(th) larval instar to the prepupal stages, BmE75A mRNA and protein levels in the prothoracic glands display developmental profiles similar to ecdysteroid titer. In the fat body, mRNA levels but not protein levels of all three BmE75 isoforms correlate with ecdysteroid titer; moreover, proteins of all three BmE75 isoforms disappear at the prepupal stages, and a modified BmE75 protein with smaller molecular weight and cytoplasm localization occurs. At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E. At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues. In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

No MeSH data available.


Attenuation of 20E signaling decreases all BmE75 isoforms in both prothoracic glands and fat body during larval-pupal metamorphosis.(A-B”) dsRNA of BmUSP was injected into Bombyx lava at the initiation of the wandering stage (EW), and the prothoracic gland was isolated 24 hours after RNAi. The mRNA and protein levels of BmUSP/phosphorylated BmUSP (P- BmUSP) were detected by qPCR (A) and Western blotting (A’, A”), and the mRNA and protein levels of BmE75A, BmE75B and BmE75 C were also detected by qPCR (B) and Western blotting (B’, B”). (C-D”) The same as (A-B”) except the fat body was used.
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f7: Attenuation of 20E signaling decreases all BmE75 isoforms in both prothoracic glands and fat body during larval-pupal metamorphosis.(A-B”) dsRNA of BmUSP was injected into Bombyx lava at the initiation of the wandering stage (EW), and the prothoracic gland was isolated 24 hours after RNAi. The mRNA and protein levels of BmUSP/phosphorylated BmUSP (P- BmUSP) were detected by qPCR (A) and Western blotting (A’, A”), and the mRNA and protein levels of BmE75A, BmE75B and BmE75 C were also detected by qPCR (B) and Western blotting (B’, B”). (C-D”) The same as (A-B”) except the fat body was used.

Mentions: Finally, we examined whether 20E signaling is absolutely required for expression of BmE75 isoforms at both mRNA and protein levels during larval-pupal metamorphosis in Bombyx. In a previous study, we documented that RNAi knockdown of BmEcR-BmUSP at the initiation of the wandering stage resulted in significant prepupal or pupal lethality and a decrease of 20E signaling, with BmUSP RNAi exhibiting stronger inhibitory effects than BmEcR RNAi2829. RNAi-mediated reduction of BmUSP expression was performed at the initiation of the wandering stage, and the effects were determined 24 hours after RNAi. In the prothoracic glands, BmUSP RNAi not only decreased BmUSP mRNA (Fig. 7A) and protein (Fig. 7A’) levels, but also decreased mRNA (Fig. 7B) and protein (Fig. 7B’) levels of all three BmE75 isoforms. Similar results were also observed in the fat body (Fig. 7C-7D”). The BmUSP RNAi experimental data demonstrated that attenuation of 20E signaling decreases mRNA and protein levels of all BmE75 isoforms in prothoracic glands and fat body during larval-pupal metamorphosis.


Bombyx E75 isoforms display stage- and tissue-specific responses to 20-hydroxyecdysone.

Li K, Guo E, Hossain MS, Li Q, Cao Y, Tian L, Deng X, Li S - Sci Rep (2015)

Attenuation of 20E signaling decreases all BmE75 isoforms in both prothoracic glands and fat body during larval-pupal metamorphosis.(A-B”) dsRNA of BmUSP was injected into Bombyx lava at the initiation of the wandering stage (EW), and the prothoracic gland was isolated 24 hours after RNAi. The mRNA and protein levels of BmUSP/phosphorylated BmUSP (P- BmUSP) were detected by qPCR (A) and Western blotting (A’, A”), and the mRNA and protein levels of BmE75A, BmE75B and BmE75 C were also detected by qPCR (B) and Western blotting (B’, B”). (C-D”) The same as (A-B”) except the fat body was used.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499807&req=5

f7: Attenuation of 20E signaling decreases all BmE75 isoforms in both prothoracic glands and fat body during larval-pupal metamorphosis.(A-B”) dsRNA of BmUSP was injected into Bombyx lava at the initiation of the wandering stage (EW), and the prothoracic gland was isolated 24 hours after RNAi. The mRNA and protein levels of BmUSP/phosphorylated BmUSP (P- BmUSP) were detected by qPCR (A) and Western blotting (A’, A”), and the mRNA and protein levels of BmE75A, BmE75B and BmE75 C were also detected by qPCR (B) and Western blotting (B’, B”). (C-D”) The same as (A-B”) except the fat body was used.
Mentions: Finally, we examined whether 20E signaling is absolutely required for expression of BmE75 isoforms at both mRNA and protein levels during larval-pupal metamorphosis in Bombyx. In a previous study, we documented that RNAi knockdown of BmEcR-BmUSP at the initiation of the wandering stage resulted in significant prepupal or pupal lethality and a decrease of 20E signaling, with BmUSP RNAi exhibiting stronger inhibitory effects than BmEcR RNAi2829. RNAi-mediated reduction of BmUSP expression was performed at the initiation of the wandering stage, and the effects were determined 24 hours after RNAi. In the prothoracic glands, BmUSP RNAi not only decreased BmUSP mRNA (Fig. 7A) and protein (Fig. 7A’) levels, but also decreased mRNA (Fig. 7B) and protein (Fig. 7B’) levels of all three BmE75 isoforms. Similar results were also observed in the fat body (Fig. 7C-7D”). The BmUSP RNAi experimental data demonstrated that attenuation of 20E signaling decreases mRNA and protein levels of all BmE75 isoforms in prothoracic glands and fat body during larval-pupal metamorphosis.

Bottom Line: At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E.At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues.In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

View Article: PubMed Central - PubMed

Affiliation: 1] Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding/Guangdong Provincial Sericulture and Mulberry Engineering Research Center, College of Animal Sciences, South China Agricultural University, Guangzhou, 510642, China [2] Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

ABSTRACT
Resulted from alternative splicing of the 5' exons, the nuclear receptor gene E75 in the silkworm, Bombyx mori, processes three mRNA isoforms, BmE75A, BmE75B and BmE75C. From the early 5(th) larval instar to the prepupal stages, BmE75A mRNA and protein levels in the prothoracic glands display developmental profiles similar to ecdysteroid titer. In the fat body, mRNA levels but not protein levels of all three BmE75 isoforms correlate with ecdysteroid titer; moreover, proteins of all three BmE75 isoforms disappear at the prepupal stages, and a modified BmE75 protein with smaller molecular weight and cytoplasm localization occurs. At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E. At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues. In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

No MeSH data available.