Figure 5.
Rie1 forms a functional complex with the RBP Sgn1. (A) Strains harboring RIE1-3V5 (B3114) or wild type RIE1 (no tag, B47) were induced to sporulate at 30°C and cells were collected at the indicated times. Lysates were prepared and Rie1 IP was conducted under non-denaturing conditions using anti-V5 agarose beads. Precipitated proteins were tandem mass tag(TMT)–labeled and analyzed by mass spectrometry. Shown are the Log2 ratios of enrichment over no-tag control for each identified protein (minimum 2 unique peptides). (B) Diagram of Sgn1. RRM domain is shown in blue, RNP motifs in green, and eIF4B-related region is outlined in orange. (C) Strains harboring RIE1-3V5 and either SGN1-FLAG (B2668) or wild type SGN1 (B3114) were induced to sporulate at 30°C. Sgn1-FLAG was IPed from meiotic lysate using anti-FLAG agarose beads at the indicated time points. Shown are Sgn1 and Rie1 protein levels by immunoblot (IB) in lysate, unbound, and IP samples. Biological replicates = 2. (D) Strains harboring SGN1-FLAG (B2668) were grown in either YPD (log phase), BYTA (stationary phase), or induced to sporulate at 30°C. Samples were collected at the indicated times and Sgn1 and Pgk1 (loading) protein levels were determined by immunoblot. Quantifications of Sgn1/Pgk1 compared to YPD (set at 1) are indicated below. (E and F) Strains harboring N-terminally tagged sfGFP-IME1 and rie1∆ (B2430, red), wild type RIE1 and SGN1 (B2459, blue), sgn1∆ (B3375, orange), or rie1∆ sgn1∆ (B3378, gray) were induced to sporulate at 30°C. Protein levels of Ime1 and Pgk1 (loading) protein levels were determined by immunoblot and mRNA levels of IME1 and rRNA (loading) were determined by Northern blot. (F) Quantification of Ime1 protein levels corrected for IME1 mRNA levels are shown. Biological replicates = 3. (G) AlphaFold2 output of individual Rie1–Sgn1 structures and AlphaFold2 Multimer output of predicted Rie1–Sgn1 complex. Rie1 is shown in blue and Sgn1 is shown in orange. A ribbon diagram zoomed in on the predicted Rie1–Sgn1 interface is shown on right with key interacting residues on Rie1 highlighted. (H and I) Strains harboring SGN1-FLAG and 3V5 tagged RIE1 (B2668) or rie1 D429A, S433A, and S435A (B3551) were induced to sporulate at 30°C. RIE1-3V5 with untagged wild type SGN1 (no tag control, B3114) was also included. Rie1-3V5 was IPed from meiotic lysate using anti-V5 agarose at the indicated time points. (H) Shown are Sgn1 and Rie1 protein levels by immunoblot in lysate, unbound, and 1% IP and 99% IP samples. (I) Protein levels of Ime1 and Pgk1 (loading) were determined by immunoblot with quantifications shown below. Source data are available for this figure: SourceData F5.

Rie1 forms a functional complex with the RBP Sgn1. (A) Strains harboring RIE1-3V5 (B3114) or wild type RIE1 (no tag, B47) were induced to sporulate at 30°C and cells were collected at the indicated times. Lysates were prepared and Rie1 IP was conducted under non-denaturing conditions using anti-V5 agarose beads. Precipitated proteins were tandem mass tag(TMT)–labeled and analyzed by mass spectrometry. Shown are the Log2 ratios of enrichment over no-tag control for each identified protein (minimum 2 unique peptides). (B) Diagram of Sgn1. RRM domain is shown in blue, RNP motifs in green, and eIF4B-related region is outlined in orange. (C) Strains harboring RIE1-3V5 and either SGN1-FLAG (B2668) or wild type SGN1 (B3114) were induced to sporulate at 30°C. Sgn1-FLAG was IPed from meiotic lysate using anti-FLAG agarose beads at the indicated time points. Shown are Sgn1 and Rie1 protein levels by immunoblot (IB) in lysate, unbound, and IP samples. Biological replicates = 2. (D) Strains harboring SGN1-FLAG (B2668) were grown in either YPD (log phase), BYTA (stationary phase), or induced to sporulate at 30°C. Samples were collected at the indicated times and Sgn1 and Pgk1 (loading) protein levels were determined by immunoblot. Quantifications of Sgn1/Pgk1 compared to YPD (set at 1) are indicated below. (E and F) Strains harboring N-terminally tagged sfGFP-IME1 and rie1∆ (B2430, red), wild type RIE1 and SGN1 (B2459, blue), sgn1∆ (B3375, orange), or rie1∆ sgn1∆ (B3378, gray) were induced to sporulate at 30°C. Protein levels of Ime1 and Pgk1 (loading) protein levels were determined by immunoblot and mRNA levels of IME1 and rRNA (loading) were determined by Northern blot. (F) Quantification of Ime1 protein levels corrected for IME1 mRNA levels are shown. Biological replicates = 3. (G) AlphaFold2 output of individual Rie1–Sgn1 structures and AlphaFold2 Multimer output of predicted Rie1–Sgn1 complex. Rie1 is shown in blue and Sgn1 is shown in orange. A ribbon diagram zoomed in on the predicted Rie1–Sgn1 interface is shown on right with key interacting residues on Rie1 highlighted. (H and I) Strains harboring SGN1-FLAG and 3V5 tagged RIE1 (B2668) or rie1 D429A, S433A, and S435A (B3551) were induced to sporulate at 30°C. RIE1-3V5 with untagged wild type SGN1 (no tag control, B3114) was also included. Rie1-3V5 was IPed from meiotic lysate using anti-V5 agarose at the indicated time points. (H) Shown are Sgn1 and Rie1 protein levels by immunoblot in lysate, unbound, and 1% IP and 99% IP samples. (I) Protein levels of Ime1 and Pgk1 (loading) were determined by immunoblot with quantifications shown below. Source data are available for this figure: SourceData F5.

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