Fig. 6. Two populations of ribosomes at uORFs and UDRs.

a, A₂₅₄ absorbance profile of 10% to 50% sucrose density gradients of testis lysates from adult mouse that was lysed in conventional lysis buffer disrupted mitochondria. Compared to the amended lysis condition that preserve mitochondria (Fig. 1a, Extended Data Fig. 2a), less prominent polysome peaks were observed here. From top to bottom, the relative abundance of 28S rRNA, β-Actin mRNA, and two pachytene piRNA precursors (17-qA3.3-26735 and 7-qD2-11976). The long transcripts (>200nt) were quantified using RT-qPCR. They were normalized to spike-in controls. b, Aggregated data for ribosome occupancy on pachytene piRNA precursors (10% trimmed mean) using Ribo-seq data from polysome fraction (left) and monosome fraction (right). c, (upper) A₂₅₄ absorbance profile of testis lysates lysed in mitochondria preserved lysis condition from adult mice following separation in 15% to 60% sucrose density gradients with (red) and without (blue) RNase treatment. (lower) Western blot analysis of VDAC, TDRKH, PLD6 and HA-tagged RPL22. Experiments have been repeated for at least three times independently with similar results. d, Aggregated data for ribosome occupancy on pachytene piRNA precursors (10% trimmed mean) using Ribo-seq data from 80S fraction (left) and mitochondria fraction (right). See also Extended Data Fig. 9a,b. Statistical source data are provided in Source Data Fig.6. Unprocessed blots are provided in Source Data Fig 6.