Selective profiling of ribosomes associated with yeast Upf proteins

Abstract

Ribosomes associated with nonsense-mediated decay factors Upf1, Upf2, or Upf3 were purified by immunoprecipitation, and enrichment and stoichiometry of Upf factors and ribosomal proteins were analyzed by western blot and mass spectrometry. Using a small RNA library preparation protocol that eliminates in-gel RNA and cDNA size selection and incorporates four random nucleotides on each side of the ribosome-protected RNA fragment allowed recovery, detection, and analysis of all size classes of protected fragments from a sample simultaneously.

Keywords: NMD; Selective ribosome profiling; Upf proteins.

Figure 1.

Diagram of protocol workflow.

Figure 2.. Fragment analyzer examples.

Starting material lanes 1–4, yeast lysate, untreated with RNase I; lanes 5–6, immunopurified ribosomes; lanes 7–10, total ribosomes. Numbers to the left of each figure indicate marker sizes in nt. A. RNA before removal of ribosomal RNA. Samples in lanes 2 and 5 are degraded and should be re-prepared; if the problem persists, it is necessary to make another preparation of lysate and ribosomes. Samples in lanes 5–10 have fragmented ribosomal RNA due to incubation of the lysate from which they were prepared with RNase I; this is expected. B. RNA after removal of ribosomal RNA. Samples in lanes 5 and 9 have incomplete removal of ribosomal RNA; these RNAs should be subjected to another round of ribosomal RNA removal. C. Finished libraries after PCR and cleanup. Sample in lane 5 is a library with a broad size range. This library should be prepared again if sequencing results indicate poor quality.

Figure 3.. Overexpressed N-terminally FLAG-tagged Upf proteins complement nonsense-mediated mRNA decay phenotypes.

Northern blot of total RNA from yeast strains expressing: empty vector (WT); high-copy FLAG-tagged Upf1, Upf2, or Upf3 in the respective deletion strains; or bearing a deletion of the UPF1, UPF2 or UPF3 coding region (designated upf1Δ, upf2Δ, or upf3Δ). NMD phenotype was determined by hybridization with a random-primed labeled probe for CYH2. SCR1 was used as a loading control.

Figure 4.. Growth curve comparisons.

Left panel, between WT and FLAG-tagged Upf strains; center panel, between WT and deletion strains; right panel, between FLAG-tagged Upf strains and corresponding deletion strains. WT, ●; FLAG-Upf1, ■; FLAG-Upf2, ▲; FLAG-Upf3, ▼; upf1Δ, ♦; upf2Δ, ○; upf3Δ, □. Timepoints as mean A₆₀₀ from cultures of two independent isolates. Error bars=range of values.

Figure 5.. Overexpressed N-terminally FLAG-tagged Upf proteins allow efficient recovery of Upf-associated ribosomes.

A. Western blot of input (I), flowthrough (F), final wash (W), or pelleted ribosomes (P) from anti-FLAG immunoprecipitation reactions. Cultures were cycloheximide treated (+CHX) or untreated (-CHX) prior to cell collection. Western blots were probed with anti-FLAG (top panel per set) or anti-TCM1/RPL3 antibodies (bottom panel per set). B. Relative abundance of Upf protein per average relative abundance of core ribosomal proteins present in a sample (Upf:RP) in pellet as determined by mass spectrometry; mean and range of two biological replicates per strain and condition. C. Fold enrichment of Upf protein in pellet vs input; mean and range of two biological replicates per strain and condition. FLAG-tagged proteins are indicated, all panels.