Sucrose gradient centrifugation of Nab2-precipitated mRNPs. A, affinity-purified Nab2-TAP under conditions of mRNA protection was separated on a 10–30% sucrose gradient. Fractions 1 (top) to 15 (bottom) were analyzed by SDS-PAGE and Coomassie staining. Nab2 and ribosomal proteins (rps) were indicated. B, shown is Western blot analysis of the sucrose gradient fractions shown in A. Antibodies against the indicated proteins were used to show their distribution on the sucrose gradient. C, shown is Western blot analysis of sucrose gradient fractions using the indicated antibodies, but the Nab2-TAP eluate was treated with RNase before sucrose gradient centrifugation. D, Northern analysis of the gradient fractions (10–30% sucrose) of the Nab2-TAP eluate using an oligo(dT) probe is shown.

Electron microscopic analysis of mRNPs isolated via Nab2-TAP. Shown is an electron micrograph overview (A) and a gallery (B) of single particles showing mRNPs affinity-purified by Nab2-TAP (fraction 8 from the GraFix sucrose gradient). Scale bars, 200 nm (A) and 30 nm (B). C, shown is an increase in particle size of Nab2-purified mRNPs with increasing gradient fraction number. Electron micrographs of mRNPs present in the GraFix sucrose gradient fractions 2, 5, 7, 8, and 9 (Overview: scale bar, 100 nm; inset: characteristic single mRNP enlarged; scale bar, 35 nm). Below the gallery is a graph with measured mRNP particle size (in nm) plotted against GraFix sucrose gradient fraction number. The upper line indicates the average length, and the lower line indicates the average width of the mRNP particles. 250–300 single particles were selected to measure length and width of the mRNPs, and the S.D. is given. Note that in the GraFix sucrose gradient fractions are shifted by ∼1–2 fractions to higher density when compared with the sucrose gradient without glutaraldehyde (see Fig. 2A).

Affinity purification of Nab2-TAP under different mRNA protection conditions. A and B, shown is bead lysis (lanes 1–4) versus N₂ grinding (lanes 5–8) with or without RVC. TEV and EGTA eluates of Nab2-TAP were analyzed by SDS-PAGE/Coomassie staining (A) and Northern to reveal poly(A)⁺ RNA (B). The indicated bands were identified by mass spectrometry. rps, ribosomal proteins; *, fatty acid synthetase; #, RNase inhibitor. S, protein standard. C, shown is a comparison of Nab2-TAP (lane 1) and Rix1-TAP purifications (lane 2) using mRNPs protection conditions by SDS-PAGE and Coomassie staining. The labeled bands 1–11 were identified by mass spectrometry. S, protein standard. 1, Tho2; 2, Nab2; 3, Yra1; 4, Rea1; 5, Rix1-CBP; 6, Nog1; 7, Ipi3; 8, Nug2; 9, Rpl3; 10, Rpl4; 11, Ipi1. Lanes 3–6, shown is Western blot analysis of the Rix1-TAP EGTA eluate (lane 3) and Nab2-TAP TEV (lane 5) and EGTA (lane 6) eluates and RS453 whole cell lysate (lane 4). D, Nab2-TAP/Tho2-FLAG split affinity purification is shown. Nab2-TAP TEV eluate and second eluate after Tho2-FLAG immunoprecipitation (anti-FLAG) is shown. 1, Tho2-FLAG; 2, Hpr1; 3, Nab2-CBP; 4, Yra1; §, flag peptide.

Analysis of Nab2-TAP associated transcripts. A, shown is a comparison of total yeast transcript distribution (black; GO annotation criteria function; for component and process, see supplemental Fig. S3) with Nab2-associated mRNAs identified by high-throughput sequencing (gray). B, shown is a comparison of total yeast transcript distribution (left) according to abundance versus Nab2-associated mRNAs (right). Transcripts were sorted according to abundance and divided in 5 classes: <0.5 t/c, 0.5–1 t/c, 1–10 t/c, >10 t/c, and unknown. C, shown is RT-PCR analysis of the indicated transcripts associated with Nab2-TAP (upper panel) or Rix1-TAP (lower panel).