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Long-read Ribo-STAMP simultaneously measures transcription and translation with isoform resolution.

Pratibha Jagannatha, Alexandra T Tankka, Daniel A Lorenz, Tao Yu, Brian A Yee, Kristopher W Brannan, Cathy J Zhou, Jason G Underwood, Gene W Yeo
Genome research · 2024 · Vol. 34 (11) · pp. 2012-2024
PubMed DOI PMC Full Text

Abstract

Transcription and translation are intertwined processes in which mRNA isoforms are crucial intermediaries. However, methodological limitations in analyzing translation at the mRNA isoform level have left gaps in our understanding of critical biological processes. To address these gaps, we developed an integrated computational and experimental framework called long-read Ribo-STAMP (LR-Ribo-STAMP) that capitalizes on advancements in long-read sequencing and RNA-base editing-mediated technologies to simultaneously profile translation and transcription at both the gene and mRNA isoform levels. We also developed the EditsC metric to quantify editing and leverage the single-molecule, full-length transcript information provided by long-read sequencing. Here, we report concordance between gene-level translation profiles obtained with long-read and short-read Ribo-STAMP. We show that LR-Ribo-STAMP successfully profiles translation of mRNA isoforms and links regulatory features, such as upstream open reading frames (uORFs), to translation measurements. We apply LR-Ribo-STAMP to discovering translational differences at both the gene and isoform levels in a triple-negative breast cancer cell line under normoxia and hypoxia and find that LR-Ribo-STAMP effectively delineates orthogonal transcriptional and translation shifts between conditions. We also discover regulatory elements that distinguish translational differences at the isoform level. We highlight <i>GRK6</i>, in which hypoxia is observed to increase expression and translation of a shorter mRNA isoform, giving rise to a truncated protein without the AGC Kinase domain. Overall, LR-Ribo-STAMP is an important advance in our repertoire of methods that measures mRNA translation with isoform sensitivity.

Publication Types

["Journal Article"]

Keywords

[]

MeSH Terms

["Humans", "Protein Biosynthesis", "Transcription, Genetic", "RNA, Messenger", "Protein Isoforms", "Open Reading Frames", "RNA Isoforms", "Cell Line, Tumor", "Sequence Analysis, RNA"]

Funding

U24 HG009889 NHGRI NIH HHS (United States)
R01 HG004659 NHGRI NIH HHS (United States)
RF1 MH126719 NIMH NIH HHS (United States)
R01 HG011864 NHGRI NIH HHS (United States)
T32 CA067754 NCI NIH HHS (United States)
U41 HG009889 NHGRI NIH HHS (United States)
R01 CA284315 NCI NIH HHS (United States)
T32 GM145427 NIGMS NIH HHS (United States)
K22 NS112678 NINDS NIH HHS (United States)

Linked Datasets (8)

GSE155726 GSE via ncbi_elink
GEO

Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [scRNA-seq]

Homo sapiens
5 data files
FileTypeSize
control_STAMP_possorted_genome_bam.bam RNA-Seq 12.0 GB
NPC_HEK_control_STAMP_possorted_genome_bam.bam RNA-Seq 8.6 GB
NPC_HEK_RBFOX2_STAMP_possorted_genome_bam.bam RNA-Seq 8.6 GB
RBP_STAMP_possorted_genome_bam.bam RNA-Seq 18.6 GB
RPS2_STAMP_possorted_genome_bam.bam RNA-Seq 12.3 GB
GSE155728 GSE via ncbi_elink
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Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [tia1_eclip]

Homo sapiens
3 data files
FileTypeSize
TIA_293T.rep1_clip.r1.reformatted.fq.gz RIP-Seq 291.5 MB
TIA_293T.rep1_input.NIL.r1.fq.gz RIP-Seq 460.5 MB
TIA_293T.rep2_clip.r1.reformatted.fq.gz RIP-Seq 178.8 MB
GSE166402 GSE via ncbi_elink
GEO

Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [STAMP_new_eclip]

Homo sapiens
9 data files
FileTypeSize
NRBF_1_S3_L001_R1_001.fastq.gz RIP-Seq 567.9 MB
NRBF_2_S4_L001_R1_001.fastq.gz RIP-Seq 583.6 MB
NTIA_1_S1_L001_R1_001.fastq.gz RIP-Seq 636.0 MB
NTIA_2_S2_L001_R1_001.fastq.gz RIP-Seq 333.4 MB
PRBF_1_S9_L001_R1_001.fastq.gz RIP-Seq 1.0 GB
PRBF_2_S10_L001_R1_001.fastq.gz RIP-Seq 888.1 MB
PTIA_1_S7_L001_R1_001.fastq.gz RIP-Seq 597.6 MB
PTIA_2_S8_L001_R1_001.fastq.gz RIP-Seq 878.5 MB
SRR13663712.lite RIP-Seq 333.4 MB
GSE155649 GSE via ncbi_elink
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Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [RNA-seq]

Homo sapiens
77 data files
FileTypeSize
Apobec_plusT_1_S30_L003_R1_001.fastq.gz OTHER 564.3 MB
Apobec_plusT_2_S31_L003_R1_001.fastq.gz OTHER 631.7 MB
Apobec-T_1_S28_L003_R1_001.fastq.gz OTHER 614.7 MB
Apobec-T_2_S29_L003_R1_001.fastq.gz OTHER 814.8 MB
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Sample13_S31_merged.fastq.gz OTHER 148.9 MB
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Sample31_S40_merged.fastq.gz OTHER 139.6 MB
Sample32_S22_merged.fastq.gz OTHER 237.7 MB
Sample_33_S61_L006_R1_001.fastq.gz OTHER 440.1 MB
Sample_34_S62_L006_R1_001.fastq.gz OTHER 556.8 MB
Sample_35_S63_L006_R1_001.fastq.gz OTHER 882.0 MB
Sample_36_S64_L006_R1_001.fastq.gz OTHER 538.0 MB
Sample_37_S65_L006_R1_001.fastq.gz OTHER 664.4 MB
Sample_38_S66_L006_R1_001.fastq.gz OTHER 963.6 MB
Sample_39_S67_L006_R1_001.fastq.gz OTHER 715.0 MB
Sample_3_S57_L006_R1_001.fastq.gz OTHER 980.1 MB
Sample_40_S68_L006_R1_001.fastq.gz OTHER 730.7 MB
Sample41_S41_merged.fastq.gz OTHER 63.0 MB
Sample42_S42_merged.fastq.gz OTHER 124.1 MB
Sample4_S25_merged.fastq.gz OTHER 211.6 MB
Sample5_S26_merged.fastq.gz OTHER 205.2 MB
Sample_6_S58_L006_R1_001.fastq.gz OTHER 587.0 MB
Sample7_S27_merged.fastq.gz OTHER 209.6 MB
Sample8_S28_merged.fastq.gz OTHER 226.2 MB
Sample_9_S59_L006_R1_001.fastq.gz OTHER 720.2 MB
SLBP_0_48hr_A_merged_R1.fastq.gz OTHER 1.2 GB
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SLBP_0_48hr_C_merged_R1.fastq.gz OTHER 1.7 GB
SLBP_0_72hr_A_merged_R1.fastq.gz OTHER 1.2 GB
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TI_55_S13_L001_R1_001.fastq.gz OTHER 883.8 MB
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TI_58_S15_L001_R1_001.fastq.gz OTHER 262.8 MB
TI_59_S16_L001_R1_001.fastq.gz OTHER 718.1 MB
TI_8_S6_L001_R1_001.fastq.gz RNA-Seq 958.8 MB
Tia1_0ng_1_S40_L003_R1_001.fastq.gz OTHER 951.1 MB
Tia1_0ng_2_S41_L003_R1_001.fastq.gz OTHER 708.4 MB
Tia1_1000ng_1_S14_L001_R1_001.fastq.gz OTHER 486.1 MB
Tia1_1000ng_2_S27_L002_R1_001.fastq.gz OTHER 581.7 MB
Tia1_50ng_1_S42_L003_R1_001.fastq.gz OTHER 722.9 MB
Tia1_50ng_2_S13_L001_R1_001.fastq.gz OTHER 792.6 MB
GSE155729 GSE via ncbi_elink
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GSE255844 GSE via ncbi_elink
GEO

Long-read Ribo-STAMP simultaneously measures transcription and translation at full length isoform resolution

Homo sapiens
12 data files
FileTypeSize
APOBEC1_only_HEK293T_rep1_ccs.bam RNA-Seq 493.9 MB
APOBEC1_only_HEK293T_rep2_ccs.bam RNA-Seq 474.4 MB
APOBEC1_only_HEK293T_rep3_ccs.bam RNA-Seq 361.6 MB
Ribo_STAMP_HEK293T_rep1_ccs.bam RNA-Seq 763.2 MB
Ribo_STAMP_HEK293T_rep2_ccs.bam RNA-Seq 498.9 MB
Ribo_STAMP_HEK293T_rep3_ccs.bam RNA-Seq 477.0 MB
Ribo_STAMP_MDA_MB_231_CoCl2_rep1_ccs.bam RNA-Seq 1.7 GB
Ribo_STAMP_MDA_MB_231_CoCl2_rep2_ccs.bam RNA-Seq 1.6 GB
Ribo_STAMP_MDA_MB_231_NT_rep1_ccs.bam RNA-Seq 2.2 GB
Ribo_STAMP_MDA_MB_231_NT_rep2_ccs.bam RNA-Seq 1.9 GB
SRR27982883 RNA-Seq 361.6 MB
SRR27982884 RNA-Seq 474.4 MB
GSE166404 GSE via ncbi_elink
GEO

Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [STAMP_pacbio]

Homo sapiens
30 data files
FileTypeSize
CCS_Demux_5759_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1001_B… OTHER 32.0 MB
CCS_Demux_5759_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1015_B… OTHER 29.7 MB
CCS_Demux_5759_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1016_B… OTHER 20.7 MB
CCS_Demux_5759_RBFOX_1_demultiplex.bc1020_BAK8B_OA__bc1020_… OTHER 12.6 MB
CCS_Demux_5759_RBFOX_2_demultiplex.bc1021_BAK8B_OA__bc1021_… OTHER 19.5 MB
CCS_Demux_5759_RBFOX_3_demultiplex.bc1022_BAK8B_OA__bc1022_… OTHER 28.2 MB
CCS_Demux_5779_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1001_B… OTHER 327.6 MB
CCS_Demux_5779_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1015_B… OTHER 291.7 MB
CCS_Demux_5779_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1016_B… OTHER 228.5 MB
CCS_Demux_5779_RBFOX_1_demultiplex.bc1020_BAK8B_OA__bc1020_… OTHER 141.2 MB
CCS_Demux_5779_RBFOX_2_demultiplex.bc1021_BAK8B_OA__bc1021_… OTHER 208.6 MB
CCS_Demux_5779_RBFOX_3_demultiplex.bc1022_BAK8B_OA__bc1022_… OTHER 293.3 MB
CCS_Demux_5896_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1001_B… OTHER 52.7 MB
CCS_Demux_5896_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1015_B… OTHER 63.0 MB
CCS_Demux_5896_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1016_B… OTHER 46.5 MB
CCS_Demux_5896_RBFOX_1_demultiplex.bc1020_BAK8B_OA__bc1020_… OTHER 39.6 MB
CCS_Demux_5896_RBFOX_2_demultiplex.bc1021_BAK8B_OA__bc1021_… OTHER 55.5 MB
CCS_Demux_5896_RBFOX_3_demultiplex.bc1022_BAK8B_OA__bc1022_… OTHER 60.4 MB
CCS_Demux_6465_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1001_B… OTHER 37.6 MB
CCS_Demux_6465_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1015_B… OTHER 55.7 MB
CCS_Demux_6465_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1016_B… OTHER 37.0 MB
CCS_Demux_6465_RBFOX_1_demultiplex.bc1020_BAK8B_OA__bc1020_… OTHER 38.8 MB
CCS_Demux_6465_RBFOX_2_demultiplex.bc1021_BAK8B_OA__bc1021_… OTHER 49.2 MB
CCS_Demux_6465_RBFOX_3_demultiplex.bc1022_BAK8B_OA__bc1022_… OTHER 52.9 MB
CCS_Demux_6499_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1001_B… OTHER 43.8 MB
CCS_Demux_6499_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1015_B… OTHER 34.3 MB
CCS_Demux_6499_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1016_B… OTHER 28.8 MB
CCS_Demux_6499_RBFOX_1_demultiplex.bc1020_BAK8B_OA__bc1020_… OTHER 17.3 MB
CCS_Demux_6499_RBFOX_2_demultiplex.bc1021_BAK8B_OA__bc1021_… OTHER 27.2 MB
CCS_Demux_6499_RBFOX_3_demultiplex.bc1022_BAK8B_OA__bc1022_… OTHER 39.1 MB
GSE155708 GSE via ncbi_elink
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Homo sapiens
6 data files
FileTypeSize
APOBEC_cDNA_all_pass.fastq.gz RNA-Seq 7.7 GB
APOBEC_dRNA.fastq.gz RNA-Seq 545.2 MB
RBFOX2_cDNA_all_pass.fastq.gz RNA-Seq 21.1 GB
RBFOX2_dRNA.fastq.gz RNA-Seq 1.2 GB
SRR12389274 RNA-Seq 7.7 GB
SRR12389276.lite RNA-Seq 21.1 GB

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