PMC

MFS2 is highly expressed in the Malpighian tubules of y w flies, induced by dietary Pi and ablation of MFS2 increases fly blood Pi and prolongs life span. (A) qRT-PCR for MFS2 mRNA in fly tissues after culture of y w adults on control medium (mean ± SEM, n = 7–40, *p = 0.01 vs. Whole Body). (B) qRT-PCR for MFS2 mRNA in Malpighian tubules dissected from y w adults after culture on control and P30 medium for 5 days (mean ± SEM, n = 5, *p = 0.04 vs. control). (C) when MFS2 is ablated universally using da-Gal4^ts driving expression of four different transgenic RNAis targeting MFS2 (TRIP or VDRC ID indicated on the x-axis) blood Pi levels are increased after culture at 29 °C for 5 days (mean ± SEM, n = 4–20 pooled collections of 15 female flies, ^#p = 0.002, **p = 0.002, *p = 0.04 vs. white-RNAi). (D) Male siblings were used to determine effect of MFS2 ablation on life span (mean ± SEM, n = 51–381 flies, **p = 0.002, *p = 0.03 vs. white-RNAi) (E) and to confirm MFS2 knockdown (F) (mean ± SEM, n = 4 flies, *p = 0.03 vs. white-RNAi).

A stem-loop structure in the mfs2 nascent transcript is a Pac1 substrate. (A) Predicted RNA secondary structure at the beginning of mfs2 and SPBC530.02 open reading frame. The nucleotides mutated in the mfs2 stemdead mutant are shown in red and indicated by arrows. Colored blocks highlight regions of high structural and sequence similarity between the two paralog genes. The nucleotide positions are numbered relative to the beginning of the genes. (B) Top, ChIP-qPCR analysis of RNAPII relative occupancy (8WG16 antibody) across the mfs2 gene in wild-type and mfs2 stemdead mutant. n = 3 biological replicates. Bottom, schematic of the mfs2 gene with bars showing the positions of PCR products analyzed. (C) Relative gene expression analysis determined by RT-qPCR in the mfs2 stemdead mutant normalized to the nda2 housekeeping gene and expressed relative to the wild-type control strain. The expression of full-length mfs2 was measured using amplicon #3 (as in figure .B). a.u.: arbitrary units. n = 3 biological replicates. (D) ChIP-qPCR analysis of Pac1-TAP occupancy at the mfs2 gene in wild-type and mfs2 stemdead mutant. The amplicon numbers refer to the same regions as in (B). n = 3 biological replicates. (E, F) Heatmap of the fold-change of the normalized RNA-seq coverage upon thiamine-dependent depletion of the 3′-5′ exonuclease Dis3 (in red), of its cofactor Mtr4 (in red), of the 5′-3′ exonuclease Dhp1 (in green), or of the double Dis3/Dhp1 depletion (in orange) relative to their appropriate wild-type strain on the SPBC530.02 (E) and mfs2 (F) genes. Hairpin RNA secondary structures are represented by arcs connecting base pairs on top of the gene annotation.

Phenotypes of spikelets of the wild-type and mfs2. A1, Complete spikelet of the wild type. A2, Spikelet from A1with lemma (le) removed. A3, Spikelet from A1 with lemma and palea (pa) removed. A4, Surface of a wild-type spikelet. A5 and A6, Surface characters of le, lodicule (lo), bop, and mrp, respectively. A7, Transverse sections of a wild-type spikelet. B1, mfs2 spikelet with degraded palea (dp). B2 and B3, Spikelet with lemma and palea removed. B4 and B5, Surfaces of the degraded palea and extra glume, respectively. B6, Magnification of B5. B7, Surface of the stamen-pistil fusion organ (sp). B8, Transverse sections of a mfs2 spikelet with degraded palea. C1, mfs2 spikelet with a partially lemma-like palea (lp). C2 and C3, Spikelet with lemma and palea removed. C4 and C5, Surfaces of the partially lemma-like palea and extra glume, respectively. C6, Transverse sections of a mfs2 spikelet with a partially lemma-like palea. D1, mfs2 spikelet with a lemma-like palea. D2, Spikelet from D1 with lemma and lemma-like palea removed. D3, Spikelet from D2 with the extra palea/mrp-like organ removed. D4 and D5, Surfaces of the lemma-like palea and extra glume, respectively. D6, Transverse sections of a mfs2 spikelet with lemma-like palea. E, Schematic diagrams of wild-type (WT) and mfs2 spikelet structure in transverse section. ep, Extra palea/mrp-like organ; pi, pistil; sl, sterile lemma; st, stamen. Scale bars = 1 mm (A1–A3, B1–B3, C1–C3, and D1– D3), 200 μm (A4–A6, B4– B7, C4, C5, D4, and D5), and 500 μm (A7, B8, C6, and D6).

Expression pattern of floral-organ identity genes in spikelets of the wild-type and mfs2 mutant. A1 to A3, Expression of OsMADS6 in wild-type (WT) spikelets at the Sp4, Sp5, Sp6, Sp7, and Sp8 developmental stages. B1 to B3, Expression of OsMADS6 in mfs2 spikelets with degraded palea (dp) at the Sp4, Sp5, Sp6, Sp7, and Sp8 developmental stages. C1 to C3, Expression of OsMADS6 in mfs2 spikelets with lemma-like palea (lp) at the Sp4, Sp5, Sp6, Sp7, and Sp8 developmental stages. D1 to D3, Expression of DL in wild-type spikelets at the Sp4, Sp5, Sp6, Sp7, and Sp8 developmental stages. E1 to E3, Expression of DL in mfs2 with degraded spikelets at the Sp4, Sp5, Sp6, Sp7, and Sp8 developmental stages. F1 to F3, Expression of DL in mfs2 spikelets with lemma-like palea at the Sp4, Sp5, Sp6, Sp7, and Sp8 developmental stages. G1 to G3, Expression of OsMADS1 in wild-type spikelets at the Sp4, Sp5, Sp6, Sp7, and Sp8 developmental stages. H1 to H3, Expression of OsMADS1 in mfs2 with degraded spikelets at the Sp4, Sp5, Sp6, Sp7, and Sp8 developmental stages. I1 to I3, Expression of OsMADS1 in mfs2 spikelets with lemma-like palea at the Sp4, Sp5, Sp6, Sp7, and Sp8 developmental stages. ep, Extra palea/mrp-like organ; le, lemma; lo, lodicule; pa, palea; pi, pistil; sl, sterile lemma; sp, stamen-pistil fusion organ. Scale bars = 100 μm.