Experiment / E1DRLR3QF5' UTR / Translation Efficiency MPRA (MPTA)

In vivo zebrafish 5′ UTR MPRA with polysome profiling

The regulatory landscape of 5′ UTRs in translational control during zebrafish embryogenesis

A capped mRNA library of 18,154 endogenous Danio rerio 5′ UTR full-length and split-segment sequences, each upstream of a common sfGFP reporter, was injected into one-cell TLAB embryos. Total RNA and 80S, low-molecular-weight (LMW), and high-molecular-weight (HMW) polysome fractions were sequenced at 2, 4, 6, and 10 hpf in three independent replicates to calculate ribosome recruitment scores (RRS).

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Organism: Zebrafish
Taxonomy ID: NCBITaxon:7955
Biosample: UBERON:0000922
Reference genome: GRCz11
Design focus: Region-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated (5′ UTR mRNA library microinjected at the one-cell stage)

In vivo mRNA MPRA using a common 5′ adaptor–5′ UTR–superfolder GFP–BG1 3′ UTR–36-nt poly(A) reporter. Polysome profiling pooled fractions 4+5 as 80S, 6+7 as LMW, and 8+9 as HMW; UMI-based paired-end sequencing quantified total-input and fractionated reporter abundance at 2, 4, 6, and 10 hpf for three independent replicates. RRS was calculated as fraction TPM divided by the matched total-input TPM, and the processed scores are the authors’ arithmetic means of replicate log2 RRS values.

Processed data

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Filters apply to this table only. The CSV download contains the complete processed table; filtered rows are available through the API.

Column dictionary · 31 definitions

element_id: Unique MPRA insert identifier from the authors’ 5′ UTR library.
chromosome: GRCz11 chromosome containing the source 5′ UTR.
strand: Genomic strand of the source transcript.
ensembl_gene_id: Ensembl gene identifier for the source transcript.
gene_name: Author-provided external gene name.
ensembl_transcript_id: Ensembl transcript identifier for the source 5′ UTR.
consensus_cluster: CAGE-derived consensus transcription-start-site cluster identifier.
gene_transcript_cluster_id: Combined gene, transcript, and CAGE consensus-cluster identifier.
switching_status: Author library annotation indicating switching or non-switching 5′ UTR status.
sequence_type: Whether the assayed insert is a full-length UTR or a split segment of a longer UTR.
original_utr_length_nt: Length in nucleotides of the original recovered 5′ UTR.
assayed_utr_length_nt: Length in nucleotides of the sequence actually assayed in this insert.
split_segment_length_nt: Length in nucleotides of the split segment; blank for full-length inserts.
full_utr_sequence: Full recovered 5′ UTR sequence; for split inserts this preserves the parent sequence.
assayed_sequence: 5′ UTR sequence present in the MPRA reporter insert.
n_uORFs: Number of predicted AUG-initiated upstream open reading frames in the assayed sequence.
gc_content_percent: GC content of the assayed sequence, in percent.
mxfold2_score: Authors’ MXfold2 sequence-based RNA folding score.
mean_log2_RRS_80S_2hpf: Mean across three replicates of log2(80S-fraction TPM / total-input TPM) at 2 hpf.
mean_log2_RRS_80S_4hpf: Mean across three replicates of log2(80S-fraction TPM / total-input TPM) at 4 hpf.
mean_log2_RRS_80S_6hpf: Mean across three replicates of log2(80S-fraction TPM / total-input TPM) at 6 hpf.
mean_log2_RRS_80S_10hpf: Mean across three replicates of log2(80S-fraction TPM / total-input TPM) at 10 hpf.
mean_log2_RRS_LMW_2hpf: Mean across three replicates of log2(LMW-fraction TPM / total-input TPM) at 2 hpf.
mean_log2_RRS_LMW_4hpf: Mean across three replicates of log2(LMW-fraction TPM / total-input TPM) at 4 hpf.
mean_log2_RRS_LMW_6hpf: Mean across three replicates of log2(LMW-fraction TPM / total-input TPM) at 6 hpf.
mean_log2_RRS_LMW_10hpf: Mean across three replicates of log2(LMW-fraction TPM / total-input TPM) at 10 hpf.
mean_log2_RRS_HMW_2hpf: Mean across three replicates of log2(HMW-fraction TPM / total-input TPM) at 2 hpf.
mean_log2_RRS_HMW_4hpf: Mean across three replicates of log2(HMW-fraction TPM / total-input TPM) at 4 hpf.
mean_log2_RRS_HMW_6hpf: Mean across three replicates of log2(HMW-fraction TPM / total-input TPM) at 6 hpf.
mean_log2_RRS_HMW_10hpf: Mean across three replicates of log2(HMW-fraction TPM / total-input TPM) at 10 hpf.
geometric_mean_RRS_HMW_10hpf: 2 raised to mean_log2_RRS_HMW_10hpf, the geometric mean fraction/input TPM ratio at 10 hpf.

Quality control

The authors retained reads with a UMI immediately followed by the common adaptor GAAGAGTAGCCTGCAGATAGAC, partially trimmed the common adaptor and sfGFP flanks, allowed up to 2 adaptor and 3 sfGFP mismatches, aligned end-to-end with Bowtie2 very-sensitive settings to 18,154 library references, removed multimapped reads using MAPQ ≥ 2 and SAMtools flag 256 filtering, and deduplicated with UMI-tools. TPM values included a one-count pseudocount; RRS calculations required TPM ≥ 2. The processed table retains the authors’ 17,879 inserts with complete mean log2 RRS values for all 12 fraction-by-stage conditions across the three replicates. It excludes 142 of 18,021 RRS-result rows containing one or more NA values and does not include the 133 library inserts absent from the RRS-result sheet.

Curation notes

This is a natural-sequence 5′ UTR activity library rather than an allele-contrast or variant-effect library. It includes full-length UTRs plus split segments for UTRs longer than the oligo limit and includes maternal/zygotic isoform pairs. Embryos were TLAB strain and received no drug or trans-factor perturbation. The processed table uses the authors’ mean_log2_RRS_data results joined to their 5'UTR_library annotations; raw UMI-deduplicated mapped counts, TPMs, sequencing QC, and supplementary analyses are retained in raw_data. Source annotation used GRCz11 with Ensembl release 100.

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