A Secreted RNA Binding Protein Forms RNA- Stabilizing Granules in the Honeybee Royal Jelly (original) (raw)

Abstract

Graphical Abstract Highlights d Worker and royal jellies harbor robust RNA-binding activity d Major royal jelly protein 3 (MRJP-3) is the dietary RNA-binding factor d RNA stimulates higher-order assembly of MRJP-3 into extracellular RNP granules d MRJP-3 granules concentrate, stabilize, and enhance environmental RNA bioavailability

Figures (6)

Highlights Graphical Abstract

Highlights Graphical Abstract

0 ee Se ee ee ee ee ee ee ee ee ee ee ee ee ee ee ee ee (A) Experimental design for RNA detection in RJ. Hives were fed with a 10% sucrose solution with or without the addition of Alexa Fluor-488-labeled dsRNA (dsRNA’). (B) Immunohistochemistry-based detection of dsRNA* in RJ samples, which were reacted with Alexa Fluor-488 antibody. Scale bar represents 25 jum. (C) RJ proteins bind dsRNA. dsRNA-binding activity was tested using EMSA. Treatments included dsRNA mixed in Ru buffer, 10% RJ mixed with dsRNA, 10% RJ digested by Proteinase K (PK) and then mixed with dsRNA, 10% RJ mixed with dsRNA and then digested by PK, 10% RJ mixed with dsRNA and PK buffer, 27.3 uM purified BSA mixed with dsRNA, 10% Ru only, and 10% Ru only digested by PK. dsRNA (0.05 1M) was applied in all dsRNA-containing treatments. (D) Precipitation dynamics of dsRNA-protein complexes in RJ. Two percent RJ was mixed with increasing dsRNA concentrations. (E) MRUP-3 and its prion-like TRR. Amino acid sequence in bold: secretion signal peptide. Amino acid sequence highlighted in color: tandem repeats. Alignment of the tandem repeats, QN (in gray) and positively charged amino acids (in red). See also Figure S1 and Table S1.

0 ee Se ee ee ee ee ee ee ee ee ee ee ee ee ee ee ee ee (A) Experimental design for RNA detection in RJ. Hives were fed with a 10% sucrose solution with or without the addition of Alexa Fluor-488-labeled dsRNA (dsRNA’). (B) Immunohistochemistry-based detection of dsRNA* in RJ samples, which were reacted with Alexa Fluor-488 antibody. Scale bar represents 25 jum. (C) RJ proteins bind dsRNA. dsRNA-binding activity was tested using EMSA. Treatments included dsRNA mixed in Ru buffer, 10% RJ mixed with dsRNA, 10% RJ digested by Proteinase K (PK) and then mixed with dsRNA, 10% RJ mixed with dsRNA and then digested by PK, 10% RJ mixed with dsRNA and PK buffer, 27.3 uM purified BSA mixed with dsRNA, 10% Ru only, and 10% Ru only digested by PK. dsRNA (0.05 1M) was applied in all dsRNA-containing treatments. (D) Precipitation dynamics of dsRNA-protein complexes in RJ. Two percent RJ was mixed with increasing dsRNA concentrations. (E) MRUP-3 and its prion-like TRR. Amino acid sequence in bold: secretion signal peptide. Amino acid sequence highlighted in color: tandem repeats. Alignment of the tandem repeats, QN (in gray) and positively charged amino acids (in red). See also Figure S1 and Table S1.

Please cite this article in press as: Maori et al., A Secreted RNA Binding Protein Forms RNA-Stabilizing Granules in the Honeybee Royal Jelly, Molecul Cell (2019), https://doi.org/10.1016/j.molcel.2019.03.010

Please cite this article in press as: Maori et al., A Secreted RNA Binding Protein Forms RNA-Stabilizing Granules in the Honeybee Royal Jelly, Molecul Cell (2019), https://doi.org/10.1016/j.molcel.2019.03.010

Figure 4. MRJP-3 RNP Granules Protect RNA From Degradation and Enhance RNA Bioavailability (A) MRJP-3-bound RNA is protected from RNase-A digestion. Treatments included ssRNA mixed with MRJP-3, ssRNA mixed with MRJP-3 followed by incu bation with PK, ssRNA mixed with MRUP-3 and RNase-A, ssRNA mixed with MRJP-3 and RNase-A followed by incubation with PK, ssRNA mixed with MRJP-1 and ssRNA mixed with MRJP-1 and RNase-A. ssRNA (0.3 uM) and MRJP-3 or MRJP-1 (42.8 1M) were used in all ssRNA- and protein-containing treatments RNase challenge was performed by introducing 5 pg RNase-A followed by 1 h incubation at room temperature. (B) RNase-A presence does not affect MRJP-3 RNPs. Images of RNPs formed with ssRNA* with or without RNase-A. ssRNA* (0.3 uM) and MRJP-3 or MRJP-" (42.8 uM) were used in all ssRNA*- and protein-containing treatments. RNase challenge was performed by introducing 5 1g RNase-A followed by 1-3 h incubatior at room temperature. Scale bar represents 2 «um. (C) dsRNA-MRUP-3 RNPs enhance unc-22 RNAi phenotype in C. elegans. Each treatment contained three biological repeats (n = 150 animals per treatment). Please cite this article in press as: Maori et al., A Secreted RNA Binding Protein Forms RNA-Stabilizing Granules in the Honeybee Royal Jelly, Molecu Cell (2019), https://doi.org/10.1016/j.molcel.2019.03.010

Figure 4. MRJP-3 RNP Granules Protect RNA From Degradation and Enhance RNA Bioavailability (A) MRJP-3-bound RNA is protected from RNase-A digestion. Treatments included ssRNA mixed with MRJP-3, ssRNA mixed with MRJP-3 followed by incu bation with PK, ssRNA mixed with MRUP-3 and RNase-A, ssRNA mixed with MRJP-3 and RNase-A followed by incubation with PK, ssRNA mixed with MRJP-1 and ssRNA mixed with MRJP-1 and RNase-A. ssRNA (0.3 uM) and MRJP-3 or MRJP-1 (42.8 1M) were used in all ssRNA- and protein-containing treatments RNase challenge was performed by introducing 5 pg RNase-A followed by 1 h incubation at room temperature. (B) RNase-A presence does not affect MRJP-3 RNPs. Images of RNPs formed with ssRNA* with or without RNase-A. ssRNA* (0.3 uM) and MRJP-3 or MRJP-" (42.8 uM) were used in all ssRNA*- and protein-containing treatments. RNase challenge was performed by introducing 5 1g RNase-A followed by 1-3 h incubatior at room temperature. Scale bar represents 2 «um. (C) dsRNA-MRUP-3 RNPs enhance unc-22 RNAi phenotype in C. elegans. Each treatment contained three biological repeats (n = 150 animals per treatment). Please cite this article in press as: Maori et al., A Secreted RNA Binding Protein Forms RNA-Stabilizing Granules in the Honeybee Royal Jelly, Molecu Cell (2019), https://doi.org/10.1016/j.molcel.2019.03.010

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