Abstract
Ribosome profiling is widely used to study translation in vivo, but not all sequence reads correspond to ribosome-protected RNA. Here we describe Rfoot, a computational pipeline that analyzes ribosomal profiling data and identifies native, nonribosomal RNA-protein complexes. We use Rfoot to precisely map RNase-protected regions within small nucleolar RNAs, spliceosomal RNAs, microRNAs, tRNAs, long noncoding (lnc)RNAs and 3′ untranslated regions of mRNAs in human cells. We show that RNAs of the same class can show differential complex association. Although only a subset of lncRNAs show RNase footprints, many of these have multiple footprints, and the protected regions are evolutionarily conserved, suggestive of biological functions.
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Acknowledgements
This work was supported by grants to K.S. from the National Institutes of Health (CA 107486). A.R. is a Howard Hughes Investigator.
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Z.J., R.S., A.R. and K.S. conceived of and designed experiments, R.S. performed experiments, Z.J. and H.H. performed the data analysis, and Z.J., R.S., A.R. and K.S. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 tRNA-protein complexes.
(a) Structure of Chr1 tRNA9–ArgUCU with the protected region highlighted. (b) Structure of Chr12 tRNA2–SerCGA with the protected region highlighted.
Supplementary Figure 2 microRNA-protein complexes.
(a) Read distribution in MIR3609. The mature microRNA region was highlighted. (b) Read distribution in MIR4497. (c) Read distribution in MIR21. MIR21 transcript generates two mature microRNAs. The mature microRNA regions were highlighted.
Supplementary Figure 3 mRNA-protein complexes.
Read distribution in 3’ UTRs of protein-coding gene AMD1 with protected regions highlighted with respect to the putative secondary structure of the AMD1 3’ UTR.
Supplementary Figure 4 lncRNA-protein complexes in TERC lncRNA.
(a) Read distribution in the TERC lncRNA with protected regions highlighted along with PhastCon scores based on 44-vertebrate Multiz alignment. (b) RNA structure of TERC with protected regions highlighted.
Supplementary Figure 5 lncRNA-protein complexes in RNA45S5 lncRNA.
(a) Read distribution in the RNA45S5 lncRNA in two cell types with read peaks representing non-ribosomal protein–RNA complexes. (b) Fragment length distribution in two protected regions. Two fragment length peaks were detected for the protected region on the left, suggesting different RNA–protein complexes or alternative conformations of the same complex.
Supplementary Figure 6 Identify non-ribosomal RNA-protein complexes using published mouse ribosome profiling data.
(a) Distribution of PME values across transcripts (60 nt window). The pattern is comparable to the PME profile in Fig. 1b. (b) Non-ribosomall RNA–protein complexes identified using 65 million randomly selected ribosome profiling reads in mouse cells. The low number of spliceosomal RNA–protein complexes reflects inadequate database annotation.
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Ji, Z., Song, R., Huang, H. et al. Transcriptome-scale RNase-footprinting of RNA-protein complexes. Nat Biotechnol 34, 410–413 (2016). https://doi.org/10.1038/nbt.3441
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DOI: https://doi.org/10.1038/nbt.3441
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