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. Author manuscript; available in PMC: 2016 Jun 4.
Published in final edited form as: Cell. 2015 Jun 4;161(6):1400–1412. doi: 10.1016/j.cell.2015.05.008

Figure 2. Ribosome protection shapes the abundance of 5’P degradation intermediates.

Figure 2

A) Metagene analysis displaying the abundance of 5’P reads relative to ORF stop codons for cells in rich media (5PSeq YPD, in blue), in a mutant for the 5’-3’ RNA exonuclease (xrn1Δ, in pink), or after random fragmentation (5PSeq control, dotted light blue line). The blue peak at −17 nt corresponds to the protection of a putative termination-paused ribosome. Reads are represented in rpm (reads per million) and biological replicates are merged.

B) Three nucleotide periodicity pattern, displayed by histograms with the total number of reads overlapping each of the three frames along coding regions for 5PSeq in YPD (blue), random fragmentation (light blue), xrn1Δ strain (pink), and after cycloheximide treatment for isolated monosomes (yellow), polyribosome fractions (green) or total RNA (in purple).

C) Genome tracks of 5’ ends of 5’P molecules after cycloheximide treatment (in red), standard growth in YPD for a wild-type strain (in blue) and for xrn1Δ (in purple). For comparison a track displaying 5’ cap molecules (in grey) and random fragmented 5’ ends (in black) are shown. Coverage is expressed in reads per million (rpm). Horizontal bars represent regions significantly (FDR<0.1) enriched (in red) for 5PSeq coverage, when comparing CHX treatment with standard YPD conditions.

D) Metagene analysis displaying the abundance of 5’P reads relative to ORF start and stop codons as in panel A. 5’P reads after cycloheximide treatment is shown in purple (5PSeq CHX).

E) Proportion of 5PSeq reads in the ribosome-protected frame (Frame 1 in panel B), shown as smoothed lines (smooth splines) for genes without introns and longer than 600bp. 5PSeq samples from cells grown YPD (blue), after 5 min oxidative stress (brown), 3-AT treatment (orange), cycloheximide treatment (purple) or random fragmentation (light blue dashed line) are shown. The dotted black line shows the random expected frequency of 0.33.

F) Model for differential mRNA footprinting in 5PSeq compared to ribosome profiling.