Abstract
Ribosome profiling has unveiled diverse regulation and perturbations of translation through a transcriptome-wide survey of ribosome occupancy, read out by sequencing of ribosome-protected messenger RNA fragments. Generation of ribosome footprints and their conversion into sequencing libraries is technically demanding and sensitive to biases that distort the representation of physiological ribosome occupancy. We address these challenges by producing ribosome footprints with P1 nuclease rather than RNase I and replacing RNA ligation with ordered two-template relay, a single-tube protocol for sequencing library preparation that incorporates adaptors by reverse transcription. Our streamlined approach reduced sequence bias and enhanced enrichment of ribosome footprints relative to ribosomal RNA. Furthermore, P1 nuclease preserved distinct juxtaposed ribosome complexes informative about yeast and human ribosome fates during translation initiation, stalling and termination. Our optimized methods for mRNA footprint generation and capture provide a richer translatome profile with low input and fewer technical challenges.
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Data availability
S. cerevisiae genome annotations from SGD are at http://sgd-archive.yeastgenome.org/sequence/S288C_reference/genome_releases/S288C_reference_genome_R64-2-1_20150113.tgz). H. sapiens genomic annotations were from GRCh38 and transcript annotations from NCBI RefSeq MANE v.0.95. Transcriptomic references were also included in Zenodo at https://zenodo.org/record/7574339#.Y9MrtHbMKrQ. High-throughput sequencing data has been deposited with the NCBI Short Read Archive under accession no. SRP419250 (https://trace.ncbi.nlm.nih.gov/Traces/?view=study&acc=SRP419250). The short read sequencing data generated are as described below (accession code; library name; and title):
SAMN32928056; McGlincy; ligation-based ribosome profiling from a pool of yeast RNase I digested RPFs (technical replicate 1 with linker CGTAA, technical replicate 2 with linker GCATA was not successful).
SAMN32928057; OTTR1; OTTR ribosome profiling from a pool of yeast RNase I digested RPFs (technical replicate 1, paired with McGlincy linker CGTAA).
SAMN32928058; OTTR2; OTTR ribosome profiling from a pool of yeast RNase I digested RPFs (technical replicate 2, paired with McGlincy linker GCATA).
SAMN32928059; yP1; nuclease digestion replicate 1 of yeast lysate with P1 nuclease.
SAMN32928060; yP2; nuclease digestion replicate 2 of yeast lysate with P1 nuclease.
SAMN32928061; yR1; nuclease digestion replicate 1 of yeast lysate with RNase I.
SAMN32928062; yR2; nuclease digestion replicate 2 of yeast lysate with RNase I.
SAMN32928063; M2; monosome-sized RPFs from monosome fraction of a polysome profile after P1 nuclease digestion, biological replicate 1 of untreated cells (no HTS1 knockdown).
SAMN32928064; M3; monosome-sized RPFs from monosome fraction of a polysome profile after P1 nuclease digestion, biological replicate 2 of untreated cells (no HTS1 knockdown).
SAMN32928065; M4; monosome-sized RPFs from monosome fraction of a polysome profile after P1 nuclease digestion, biological replicate 1 of anhydrotetracycline-treated cells (HTS1 knockdown).
SAMN32928066; M5; monosome-sized RPFs from monosome fraction of a polysome profile after P1 nuclease digestion, biological replicate 2 of anhydrotetracycline-treated cells (HTS1 knockdown).
SAMN32928067; DM2; monosome-sized RPFs from disome fraction of a polysome profile after P1 nuclease digestion, biological replicate 1 of untreated cells (no HTS1 knockdown).
SAMN32928068; DM3; monosome-sized RPFs from disome fraction of a polysome profile after P1 nuclease digestion, biological replicate 2 of untreated cells (no HTS1 knockdown).
SAMN32928069; DM4; monosome-sized RPFs from disome fraction of a polysome profile after P1 nuclease digestion, biological replicate 1 of anhydrotetracycline-treated cells (HTS1 knockdown).
SAMN32928070; DM5; monosome-sized RPFs from disome fraction of a polysome profile after P1 nuclease digestion, biological replicate 2 of anhydrotetracycline-treated cells (HTS1 knockdown).
SAMN32928071; D2; disome-sized RPFs from disome fraction of a polysome profile after P1 nuclease digestion, biological replicate 1 of untreated cells (no HTS1 knockdown).
SAMN32928072; D3; disome-sized RPFs from disome fraction of a polysome profile after P1 nuclease digestion, biological replicate 2 of untreated cells (no HTS1 knockdown).
SAMN32928073; D4; disome-sized RPFs from disome fraction of a polysome profile after P1 nuclease digestion, biological replicate 1 of anhydrotetracycline-treated cells (HTS1 knockdown).
SAMN32928074; D5; disome-sized RPFs from disome fraction of a polysome profile after P1 nuclease digestion, biological replicate 2 of anhydrotetracycline-treated cells (HTS1 knockdown).
SAMN32928075; TetMMRMonoR1; mirRICH small RNA enrichment and cDNA size selection of P1 nuclease-digested RPFs from a sucrose cushion pellet, biological replicate 1 of untreated cells (no HTS1 knockdown).
SAMN32928076; TetMMRMonoR2; mirRICH small RNA enrichment and cDNA size selection of P1 nuclease-digested RPFs from a sucrose cushion pellet, biological replicate 2 of untreated cells (no HTS1 knockdown).
SAMN32928077; TetMDZMonoR1; Direct-Zol RNA purification and cDNA size selection of P1 nuclease-digested RPFs from a sucrose cushion pellet, biological replicate 1 of untreated cells (no HTS1 knockdown).
SAMN32928078; TetMDZMonoR2; Direct-Zol RNA purification and cDNA size selection of P1 nuclease-digested RPFs from a sucrose cushion pellet, biological replicate 2 of untreated cells (no HTS1 knockdown).
SAMN32928079; TetPMRMonoR1; mirRICH small RNA enrichment and cDNA size selection of P1 nuclease-digested RPFs from a sucrose cushion pellet, biological replicate 1 of anhydrotetracycline-treated cells (HTS1 knockdown).
SAMN32928080; TetPMRMonoR2; mirRICH small RNA enrichment and cDNA size selection of P1 nuclease-digested RPFs from a sucrose cushion pellet, biological replicate 2 of anhydrotetracycline-treated cells (HTS1 knockdown).
SAMN32928081; TetPMRDiR1; mirRICH small RNA enrichment and cDNA size selection of P1 nuclease-digested disome-sized RPFs from a sucrose cushion pellet, biological replicate 1 of anhydrotetracycline-treated cells (HTS1 knockdown).
SAMN32928082; TetPMRDiR2; mirRICH small RNA enrichment and cDNA size selection of P1 nuclease-digested disome-sized RPFs from a sucrose cushion pellet, biological replicate 2 of anhydrotetracycline-treated cells (HTS1 knockdown).
SAMN32928153; hP1; Nuclease digestion of biological replicate 1 of 293T cell lysate with P1 nuclease.
SAMN32928154; hP2; nuclease digestion of biological replicate 2 of 293T cell lysate with P1 nuclease.
SAMN32928155; hR1; nuclease digestion of biological replicate 1 of 293T cell lysate with RNase I.
SAMN32928156; hR2; nuclease digestion of biological replicate 2 of 293T cell lysate with RNase I.
SAMN32928157; T293MRMonoR1; mirRICH small RNA enrichment and cDNA size selection of P1 nuclease-digested RPFs from a sucrose cushion pellet, biological replicate 1 of 293T cell lysate (different batch of lysates separate from hP1, hP2, hR1 and hR2).
SAMN32928158; T293MRMonoR2; mirRICH small RNA enrichment and cDNA size selection of P1 nuclease-digested RPFs from a sucrose cushion pellet, biological replicate 2 of 293T cell lysate (different batch of lysates separate from hP1, hP2, hR1 and hR2).
SAMN32928159; T293MRDiR1; mirRICH small RNA enrichment and cDNA size selection of P1 nuclease-digested disome-sized RPFs from a sucrose cushion pellet, biological replicate 1 of 293T cell lysate (different batch of lysates separate from hP1, hP2, hR1 and hR2).
SAMN32928160; T293MRDiR2; mirRICH small RNA enrichment and cDNA size selection of P1 nuclease-digested disome-sized RPFs from a sucrose cushion pellet, biological replicate 2 of 293T cell lysate (different batch of lysates separate from hP1, hP2, hR1 and hR2).
SAMN32928161; T293SSMonoR1; RNA size selection and cDNA size selection of P1 nuclease-digested RPFs from a sucrose cushion pellet, biological replicate 1 of 293T cell lysate (different batch of lysates separate from hP1, hP2, hR1 and hR2).
SAMN32928162; T293SSMonoR2; RNA size selection and cDNA size selection of P1 nuclease-digested RPFs from a sucrose cushion pellet, biological replicate 1 of 293T cell lysate (different batch of lysates separate from hP1, hP2, hR1 and hR2). Source data are provided with this paper.
Code availability
Custom software, a workflow used to analyze data and prepared human and yeast genomic and transcriptomic references used in this study are provided in Zenodo at https://zenodo.org/record/7574339#.Y9MrtHbMKrQ.
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Acknowledgements
L.F., H.E.U., S.C.P. and K.C. were supported by a Bakar Fellows Program Award and National Institutes of Health (NIH) DP1 HL156819 (to K.C.). L.F. was also supported by NIH T32 GM007232. N.T.I. was supported by NIH grant R01 GM130996 (to N.T.I.). A.M. and L.F.L. were supported by NSF award 1936069 (to L.F.L.), NIH award R01GM132104 (to L.F.L.) and a Rose Hills Innovator Award (to L.F.L.). We acknowledge the Vincent J. Coates Genomics Sequencing Laboratory QB3 Genomics, UC Berkeley, RRID:SCR_022170, for sequencing support. We thank the staff at the UC Berkeley Electron Microscope Laboratory for advice and assistance in electron microscopy sample preparation and data collection. We also thank R. Muller, S. Fernandez, R. Flynn and past and present members of the Collins, Ingolia and Lareau laboratories of UC Berkeley for their various levels of support in the development of this manuscript.
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Authors and Affiliations
Contributions
All experiments and analysis were entirely completed by L.F. The manuscript was written and edited by L.F., K.C., N.T.I. and L.F.L. Planning of the OTTR-seq library generation strategy employed was carried out by L.F., H.E.U., S.C.P. and K.C. Ribosome profiling strategy employed was developed by L.F., K.C. and N.T.I. The ribosome profiling and associated analysis related specifically to library generation bias was planned by L.F., A.M., L.F.L. and N.T.I. and executed and analyzed by L.F.
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Competing interests
L.F., H.E.U., S.C.P. and K.C. are named inventors on patent applications filed by the University of California describing biochemical activities of RTs used for OTTR. H.E.U. and K.C. have equity in Karnateq, which licensed the technology and is producing kits for OTTR cDNA library preparation. N.T.I. declares equity in Tevard Biosciences and Velia Therapeutics. A.M. and L.F.L. declare no competing interests.
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Nature Methods thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.
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Extended data
Extended Data Fig. 1 Additional comparison of RNase I RPF libraries by ligation-based or OTTR protocol.
a, Fraction of RNase I RPF sequencing library reads that mapped to each transcript class. Library generation artifacts included sequences that were adapter-only, shorter than 15 bases, or unmapped. b, CDS RPF length distribution from OTTR (blue) and ligation-based (red) RNase I ribosome profiling libraries. RPFs aligned to the first 15 or final 10 codons were excluded. Counts were represented in reads per million mapped reads (RPM) and averaged across replicates. c, For each read length from 26 to 29 nt, the fraction of RPF alignments with mismatches at the 5′-most base of the alignment. For this analysis, alignments were permitted to only have a single mismatch to the reference. d, For each read length from 26 to 29 nt, the fraction of RPF alignments with an adenosine (A) at the 3′-most base of the alignment. For this analysis, alignments were permitted to only have a single mismatch to the reference. e, For each read length from 26 to 29 nt, the fraction of RPF alignments with a thymine (T) at the 3′-most base of the alignment. For this analysis, alignments were permitted to only have a single mismatch to the reference.
Extended Data Fig. 2 Optimization of P1 nuclease digestion and cell lysis conditions.
a, Polysome collapse efficiency analysis comparing lysis with either pH 6.5 or pH 7.5 polysome lysis buffer and addition of a range of P1 nuclease U/µg. All assays were carried out with lysate measuring 30 µg of total RNA diluted to 200 µL with either pH 6.5 or pH 7.5 polysome buffer. At the point of nuclease digestion, pH 7.5 lysate was adjusted to ~pH 6.5 with 14 µl 300 mM Bis-Tris (pH 6.0) and pH 6.5 lysate was supplemented with an additional 14 µL of pH 6.5 polysome buffer. Collapse efficiency was calculated as the ratio of the integrated monosome peak absorbance relative to the integrated polysome region absorbance, normalized by the undigested control (n = 1 for each condition, except n = 2 for pH 6.5 with 3.33 U/µg or 20 U/µg and pH 7.5 with 0 U/µg or 10 U/µg). b, Comparison of polysome collapse efficiency by P1 nuclease digestion at either 30 °C or 37 °C using Calu-3 human cell lysate. Briefly, lysate measuring 15 µg of total RNA was diluted to 200 µL in pH 7.5 polysome buffer and pH adjusted with 14 µL of 300 mM Bis-Tris (pH 6.0) before supplemented with P1 nuclease and digestion at 30 °C or 37 °C. Undigested control incubated at 4 °C for an hour without nuclease (n = 2 for 4 °C no-nuclease controls, n = 2 or 3 for 15 U/µg as shown, and n = 1 for 20 U/µg). c, Representative polysome profile from P1 nuclease digestion of Calu-3 human cell lysate at 30 °C or 37 °C with nuclease at 15 U/µg total RNA. Undigested control incubated at 4 °C for an hour without nuclease.
Extended Data Fig. 3 OTTR library production and ribosome profiles.
a, A 0.6X TBE 8% denaturing urea–PAGE gel of OTTR library cDNA generated from human 293 T and yeast cell P1 nuclease RPFs purified by sucrose cushion. The cDNA was directly imaged by the Cy5 dye covalently linked to the ORRT adapter duplex (see Fig. 1a). A parallel OTTR library was synthesized from size marker oligonucleotides (Table 1). Xylene cyanol co-migrates with OTTR adapter dimer cDNA. Horizontal lines indicate excision. b-c, CDS RPF length distribution from yeast (b) and human 293 T (c) RNase I (blue) and P1 nuclease (red) ribosome profiling libraries, as in Extended Data Fig. 1b. d-e, Fraction of sequencing reads mapped to each transcript class yeast (d) and human 293 T (e) ribosome profiling libraries, as in Extended Data Fig. 1a. f, Mean per-base read coverage of cytosolic 18 S and 25 S (or 28 S) rRNA from yeast (left) or human (right) ribosome profiles made from P1 nuclease (red) or RNase I (blue) digestion. Coverage was represented in reads per million total mapped reads (that is, rRNA, tRNA, ncRNA, mRNA, and other genomic loci). g, As in (f) but for 5.8 S and 5 S rRNA coverage. h, As in (f) for mitochondrial rRNA coverage.
Extended Data Fig. 4 Abundance and properties of monosome, sub-disome, and true disome RPFs from yeast cells with or without histidine starvation.
a, Ratio of sucrose density gradient disome to monosome peak area from polysome profiles after P1 nuclease digestion of yeast lysates from cells with or without HTS1 knockdown. b, Fraction of sequencing reads mapped to each transcript class for sucrose density gradient light monosome, heavy monosome, and disome profiling from P1 nuclease digestion and OTTR library cDNA synthesis, as in Extended Data Fig. 1a. c, Average profile of sucrose density gradient light monosome RPFs around isolated histidine codons after HTS1 depletion, as in Fig. 4g. d, Average profile of sucrose density gradient heavy monosome RPFs around isolated histidine codons after HTS1 depletion, as in Fig. 4g. e, Average profile of sucrose density gradient light monosome RPFs around start codons after HTS1 depletion, as in Fig. 3e. f, Average profile of sucrose density gradient heavy monosome RPFs around start codons after HTS1 depletion, as in Fig. 3e.
Extended Data Fig. 5 Complete GCN4 uORF1 and uORF2 and CPA1 uORF profiles for monosome, sub-disome, and true disome footprints with and without HTS1 knockdown.
a, Extended representation from Fig. 5c. Rescaled counts of 5′ and 3′ ends of aligned true disome (top), sub-disome (middle), and monosome (bottom) footprints for GCN4 uORF1 without HTS1 knockdown. Results from replicates were summed together from sucrose density gradient purified material. b, Footprint length 5′ and 3′ profile of (a). c, Rescaled counts of 5′ and 3′ ends of aligned true disome (top), sub-disome (middle), and monosome (bottom) footprints for GCN4 uORF1 after HTS1 knockdown. Results from replicates were summed together. d, Footprint length 5′ and 3′ profile of (c). e, Extended representation from Fig. 5d. Rescaled counts of 5′ and 3′ ends of aligned true disome (top), sub-disome (middle), and monosome (bottom) footprints for GCN4 uORF2 without HTS1 knockdown. Results from replicates were summed together. f, Footprint length 5′ and 3′ profile of (e). g, Rescaled counts of 5′ and 3′ ends of aligned true disome (top), sub-disome (middle), and monosome (bottom) footprints for GCN4 uORF2 after HTS1 knockdown. Results from replicates were summed together. h, Footprint length 5′ and 3′ profile of (g). i, Rescaled counts of 5′ and 3′ ends of aligned true disome (top), sub-disome (middle), and monosome (bottom) footprints for CPA1 uORF without HTS1 knockdown. Results from replicates were summed together. Both a terminating ribosome (leading) and elongating ribosome (trailing) at various codon positions are schematized. Results from replicates were summed together. j, Footprint length 5′ and 3′ profile of (i). k, Rescaled counts of 5′ and 3′ ends of aligned true disome (top), sub-disome (middle), and monosome (bottom) footprints for CPA1 uORF with HTS1 knockdown. Both an elongating ribosome stalled at the penultimate histidine codon (leading) and an elongating ribosome (trailing) are schematized. Results from replicates were summed together. l, Footprint length 5′ and 3′ profile of (k).
Extended Data Fig. 6 Additional comparisons of libraries made from mirRICH, total RNA, or gel-base size-selected RPFs.
a, A SYBR Gold stained 0.6X TBE 12% denaturing urea–PAGE gel of resolved P1 nuclease digested yeast RNA purified from either Direct-Zol Total RNA extraction or mirRICH small RNA enrichment following purification by sucrose cushion. Roughly 5% of the Direct-Zol extract and 25% of the mirRICH extract was analyzed in this gel. The migration of the 30 nt (light blue) and 40 nt (dark blue) size selection RNA oligos are demarcated (left). Both lanes are from the same gel, and both purification methods were replicated twice. b, As in Extended Data Fig. 3a, cDNA size selection from a OTTR library generated from 40 ng of mirRICH small RNA from (a). Parallel positive control OTTR libraries were synthesized from either 30 or 40 nt size selection RNA oligos to aid in cDNA size selection. The single light blue and dark-blue dots (left) demarcate the cDNA with inserts derived from the 30 nt and 40 nt RNA oligos, respectively. cDNA concatemers from deliberately using more templates in OTTR, for example ~80 nt insert, are demarcated by two dark blue dots. monosome cDNA size selection area was demarcated by the bottom two black lines (right), and the disome cDNA size selection by the top two black lines. Both lanes are from the same gel. Four yeast and two human libraries from mirRICH purified RNA were constructed. c, Library length distribution of bifurcated monosome (top) and disome (bottom) libraries by Agilent 2200 TapeStation. Libraries were bifurcated at the cDNA size selection step as shown in Extended Data Fig. 6b. d, Gene-level estimates for CDS occupancy, as in Fig. 1a, but here for mirRICH or Direct-Zol P1 nuclease RPF from the yeast lysate without HTS1 knockdown, purified by sucrose cushion. Both libraries relied on cDNA size selection rather than RNA size selection. The counts were deduplicated before analysis. e, Distribution of the log2 ratio of PCR deduplicated counts (corrected) versus uncorrected counts for each CDS from the libraries generated from the no HTS1 knockdown lysate, purified by sucrose cushion. The number of necessary PCR cycles used for library multiplexing is defined above. Two technical replicates per library condition were analyzed. f, Read length distribution represented as a fraction of mRNA mapping reads for the sucrose cushion purified mirRICH monosome and disome reads bifurcated by cDNA size-selected libraries from the HTS1 knockdown lysate. Two technical replicates per library condition were analyzed. Monosome replicates are in light blue and purple; disome replicates are in red and green. g, Read length distribution represented as a fraction of mRNA mapping reads for the sucrose cushion purified mirRICH monosome and disome reads bifurcated by cDNA size-selected libraries from the 293 T lysate. Two technical replicates per library condition were analyzed. Monosome replicates are in light blue and purple; disome replicates are in red and green. h, 5′ aligned ends and footprint length profile at initiating codons for P1 sub-disome (red line) and true disome (blue line) RPFs captured by mirRICH from human 293 T cell lysates. Material was purified by a sucrose cushion. The summed 5′ end profile is depicted at top and the contributions from each footprint length at each 5′ end are shown at bottom. Sub-disome and true disome-sized reads were analyzed separately.
Supplementary information
Supplementary Information
Supplementary Protocols 1–3.
Source data
Source Data Fig. 2e
Unprocessed SYBR Gold stained denaturing 7 M urea 12% PAGE gel (0.6× TBE) of RNA.
Source Data Fig. 4d
Unprocessed negative-stain electron micrograph of material concentrated from the disome fraction of a sucrose density gradient.
Source Data Extended Data Fig. 3a
Unprocessed Cy5 imaged denaturing 7 M urea 8% PAGE gel (0.6× TBE) of cDNA.
Source Data Extended Data Fig. 6a
Unprocessed SYBR Gold stained denaturing 7 M urea 12% PAGE gel (0.6× TBE) of RNA. b, Unprocessed Cy5 imaged denaturing 7 M urea 8% PAGE gel (0.6× TBE) of cDNA.
Source Data Extended Data Fig. 6b
Unprocessed Cy5 imaged denaturing 7 M urea 8% PAGE gel (0.6× TBE) of cDNA.
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Ferguson, L., Upton, H.E., Pimentel, S.C. et al. Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells. Nat Methods 20, 1704–1715 (2023). https://doi.org/10.1038/s41592-023-02028-1
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DOI: https://doi.org/10.1038/s41592-023-02028-1
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