Journal: Nature biotechnology

Article Title: Human 5′ UTR design and variant effect prediction from a massively parallel translation assay

doi: 10.1038/s41587-019-0164-5

Figure Lengend Snippet: A library of 280,000 random 50-mers as 5′ UTRs for eGFP. (a) A 5′ UTR model capable of predicting translation from sequence is used to evaluate the effect of 5′ UTR SNVs and to engineer new sequences for optimal protein expression. (b) A library of 280,000 members was built by inserting a T7 promoter followed by 25 nt of defined 5′ UTR sequence, a random 50-mer, and the eGFP coding sequence into a plasmid backbone. Library IVT mRNA was produced by in vitro transcription from a linearized DNA template obtained through PCR from the plasmid library. Cells transfected with library IVT mRNA were grown for 12 hours before polysome profiling. Read counts per fraction were used to calculate Mean Ribosome Loads (MRL) for each UTR and the resulting data were used to train a convolutional neural network (CNN). (c) Out-of-frame upstream AUGs (uAUGs) reduce ribosome loading (vertical lines indicate positions that are in-frame with the eGFP CDS). A similar but much weaker periodicity is observed for CUG and GUG. (d) The repressive strength of all out-of-frame variations of NNNATGNN. (e) Nucleotide frequencies were calculated for the 20 most repressive (‘strong’) and least repressive (‘weak’) TIS sequences.

Article Snippet: All designed and human 5′ UTR sequences were synthesized by CustomArray, Inc. Fragments were PCR amplified and cloned into the pET 28 eGFP vector described above.

Techniques: Sequencing, Expressing, Plasmid Preparation, Produced, In Vitro, Transfection

Journal: Nature biotechnology

Article Title: Human 5′ UTR design and variant effect prediction from a massively parallel translation assay

doi: 10.1038/s41587-019-0164-5

Figure Lengend Snippet: (a) Optimus 5-Prime structure: A one-hot encoded 5′ UTR sequence is fed into a CNN composed of three convolution layers and a fully connected layer to produce a linear output predicting MRL. (b) Optimus 5-Prime trained on 260,000 UTRs and tested on 20,000 held-out sequences could explain 93% of the variability in observed MRLs. Blue dots represent sequences with an uAUG while red dots represent sequences without uAUG (n = 20,000). (c) A similar model was trained to predict the polysome profile distribution of an individual 5′ UTR. The observed (blue) and predicted (red) polysome distribution of 5 random picked example UTRs out of 20,000 in the test set spanning MRLs from 4 to 8 (top to bottom) are shown. (d) The performance of the polysome profile model per fraction ranged from an r2 of 0.621 to 0.915 and an average of 0.834 across all fractions (n = 20,000). (e) eGFP expression for ten UTRs selected from the library were evaluated via eGFP fluorescence using IncuCyte live cell imaging (n = 3, centers are the means, error bars are s.e.m.). Predicted MRL and fluorescence are highly correlated (r2: 0.87, n = 10). For details, see Supplementary Table 2. (f) Visualization of four out of 120 filters from the first convolution layer (left) and four out of 120 filters from the second convolution layer. Boxes below show correlation (Pearson r) between filter activation and MRL at each UTR position. Filters learned important regulatory motifs such as start and stop codons, uORFs, and GC-rich regions likely involved in secondary structure formation. (g) IVT mRNA from the eGFP library were generated with pseudouridine (Ψ) or 1-methylpseudouridine (m1 Ψ) in place of uridine (U) and evaluated by polysome profiling and modeling. (h) Model performance trained and tested on different data sets (r-squared). The unmodified RNA (U) models perform best with U data, while the Ψ and m1 Ψ models perform equally well with Ψ and m1 Ψ test data (n = 20,000). (i) Ribosome loading as a function of MFE. U is less dependent on secondary structure than Ψ and m1 Ψ (Pearson r: 0.43, 0.56, and 0.58, respectively. n = 19,976).

Article Snippet: All designed and human 5′ UTR sequences were synthesized by CustomArray, Inc. Fragments were PCR amplified and cloned into the pET 28 eGFP vector described above.

Techniques: Sequencing, Expressing, Fluorescence, Live Cell Imaging, Activation Assay, Generated

Journal: Nature biotechnology

Article Title: Human 5′ UTR design and variant effect prediction from a massively parallel translation assay

doi: 10.1038/s41587-019-0164-5

Figure Lengend Snippet: (a) The first 50 nucleotides preceding the CDS of 35,212 human transcripts and an additional 3,577 UTRs with SNVs (ClinVar) were evaluated using our polysome profiling method with eGFP used as the CDS. The retrained Optimus 5-Prime could explain 81.1% of the observed variation in MRL (n = 25,000). (b) The log2 change in MRL between an SNV and its common sequence was compared to the predicted change between the two (r2: 0.555, n = 1,597). SNV classification labels are from the ClinVar database. (c) In silico saturation mutagenesis and model prediction of MRL change for all 5’ UTR variants of CPOX, TMEM127 and RPL5. The three annotated Clinvar variants, rs867711777 (CPOX, G > A), rs121908813 (TMEM127, C > U), and rs376208311 (RPL5, C > A), are predicted to have the most dramatic effect on ribosome loading. (d) A library of 76,319 random 5’UTRs with varying lengths from 25 to 100 nucleotides was used to train the generalized Optimus 5-Prime. Sequences are one-hot encoded and zero padded to 100 nucleotides long if shorter than 100. (e) 7,600 random (blue dots) and 7,600 human (red dots) sequences are tested using the generalized Optimus 5-Prime. 100 sequences of each length (25–100) are represented. Model accuracy (r2: 0.754 to 0.838) is shown in predicting MRLs on different range of lengths of 5’ UTRs (From left to right: n = 4,000; n = 4,000; n = 4,000; n = 3,200.).

Article Snippet: All designed and human 5′ UTR sequences were synthesized by CustomArray, Inc. Fragments were PCR amplified and cloned into the pET 28 eGFP vector described above.

Techniques: Sequencing, In Silico, Mutagenesis

Journal: Molecular Systems Biology

Article Title: Pervasive isoform‐specific translational regulation via alternative transcription start sites in mammals

doi: 10.15252/msb.20166941

Figure Lengend Snippet: Scatter plot showing the bootstrap means ( x ‐axis) and standard deviations ( y ‐axis) for log2‐transformed TE difference between 13,118 TSS isoform pairs in the 4,153 multi‐TSS genes. Dashed purple lines indicated the Benjamini–Hochberg adjusted P ‐value of 0.01, and dashed orange lines indicated the 1.5‐fold divergence. Genes with significant TE divergence (Benjamini–Hochberg adjusted P ‐value < 0.01, TE divergence > 1.5‐fold) are depicted in blue. See also Table EV2 . Independent validation of TSS isoforms and their associated translational efficiency in genes Ndufb11 , Ube4b , Nedd8 , and Ssu72 , respectively. Left: Under each gene structure, cumulative reads were shown for the alternative TSSs in the “free” fraction and poly9+ fraction. Green arrows above the gene structure indicate the locations of the reverse PCR primer. Red and blue bars represented sequencing reads mapped within distal and proximal TSSs, respectively; gray bars represented reads mapped outside of the identified TSSs. Right: Agarose gel electrophoresis of amplified products of mRNA 5ʹ ends obtained from non‐ribosomal fraction and polysomal fraction. Positions of the distal TSS isoform and the proximal TSS isoforms are indicated with red and blue arrows, respectively. In the case of gene Ndufb11 , the band below the distal TSS (indicated by a yellow arrow) in the gel image was caused by an alternative splicing event, which removed an 88‐nt region for a minor fraction of transcripts initiating at the distal TSS. L, HyperLadder I; N, non‐ribosomal fraction; P, polysomal fraction. The description of these genes can be found in Table EV3 . Alternative 5ʹUTR sequences are able to drive the observed isoform‐specific TE divergence. An in vivo reporter system was used to compare the TE of a Renilla luminescent reporter gene led by the 5ʹUTR sequences derived from eight pairs of alternative TSS isoforms identified in eight genes. TE is calculated by luciferase activity normalized to mRNA abundance. Seven out of eight reporter pairs showed significant differential TE biased toward the same TSS isoforms as observed in our global analysis ( n = 3; mean ± SEM; * P < 0.05, ** P < 0.01; Student's t ‐test). The description of these genes can be found in Table EV3 .

Article Snippet: Both in vitro and in vivo analyses have demonstrated that different 5ʹUTR sequences derived from the same yeast genes can lead to large difference in translational efficiency (TE) (Rojas‐Duran & Gilbert, ).

Techniques: Transformation Assay, Biomarker Discovery, Sequencing, Agarose Gel Electrophoresis, Amplification, Alternative Splicing, In Vivo, Derivative Assay, Luciferase, Activity Assay

Journal: Molecular Systems Biology

Article Title: Pervasive isoform‐specific translational regulation via alternative transcription start sites in mammals

doi: 10.15252/msb.20166941

Figure Lengend Snippet: Barplots showing the fraction of alternative TSS isoform pairs with and without significant differential TE. Isoform pairs with certain 5ʹUTR length difference were grouped together. The larger the length difference between the two isoforms, the higher the fraction associated with significant TE divergence. Scatter plot comparing the number of ribosomes per mRNA between shorter 5ʹUTR isoforms ( x ‐axis) and longer 5ʹUTR isoforms ( y ‐axis) from the same genes. Purple and green dots were isoform pairs with significant differential TE biased toward longer and shorter isoforms, respectively.

Article Snippet: Both in vitro and in vivo analyses have demonstrated that different 5ʹUTR sequences derived from the same yeast genes can lead to large difference in translational efficiency (TE) (Rojas‐Duran & Gilbert, ).

Techniques:

Journal: Molecular Systems Biology

Article Title: Pervasive isoform‐specific translational regulation via alternative transcription start sites in mammals

doi: 10.15252/msb.20166941

Figure Lengend Snippet: Left: Boxplots comparing the log2 TE fold changes between two groups of alternative isoform pairs, one group with at least one uORF present in the isoform‐divergent 5ʹUTR and the other without. Right: The group with uORF was further separated into three subgroups according to the number of uORFs present in the divergent 5ʹUTR. Same as (A)—left, but the sequence feature of interest is the out‐of‐frame uAUGs. Same as (A)—left, but the sequence feature of interest is the in‐frame uAUGs. Same as (A)—left, but the sequence feature of interest is the translated uORFs (i.e. supported by ribosome footprinting) with canonical AUG start codon. Same as (A)—left, but the sequence feature of interest is the translated out‐of‐frame uAUGs (i.e. supported by ribosome footprinting). Same as (A)—left, but the sequence feature of interest is the translated uORFs (i.e. supported by ribosome footprinting) with non‐canonical start codons. Same as (A)—left, but the sequence feature of interest is the translated out‐of‐frame upstream non‐canonical start codons (i.e. supported by ribosome footprinting). Data information: ** P < 0.01, *** P < 0.001; Mann–Whitney U ‐test. Box edges represent quantiles, whiskers represent extreme data points no more than 1.5 times the interquartile range.

Article Snippet: Both in vitro and in vivo analyses have demonstrated that different 5ʹUTR sequences derived from the same yeast genes can lead to large difference in translational efficiency (TE) (Rojas‐Duran & Gilbert, ).

Techniques: Sequencing, Footprinting, MANN-WHITNEY

Journal: Molecular Systems Biology

Article Title: Pervasive isoform‐specific translational regulation via alternative transcription start sites in mammals

doi: 10.15252/msb.20166941

Figure Lengend Snippet: Boxplots comparing the log2 TE fold changes between three groups of alternative isoform pairs, the first group with 5ʹ cap‐adjacent (50 nt to 5ʹ ends) stable RNA secondary structures (MFE < −30 kcal/mol) present only in long 5ʹUTR isoforms, the second group with 5ʹ cap‐adjacent stable RNA structure present/absent in both isoforms, and the last group with 5ʹ cap‐adjacent stable RNA structure present only in short 5ʹUTR isoforms. Boxplots comparing the log2 TE fold changes between two groups of alternative isoform pairs, one group with stable RNA secondary structures (MFE < −35 kcal/mol in any 50‐nt RNA fragments) present in the downstream divergent 5ʹUTR and the other without. Boxplots comparing the log2 TE fold changes between TOP genes and non‐TOP genes (controls). For TOP genes, the TE fold changes were the ratios between the isoforms with 5ʹ TOP sequences present and isoforms without, and for non‐TOP genes, isoforms were randomly assigned as numerators and denominators. Left: Boxplots comparing the log2 TE fold changes between two groups of alternative isoform pairs, one group with the motif AAUCCC present in divergent 5ʹUTRs and the other without. Right: Luciferase assay comparing the relative TE between reporter genes with five copies of motif AAUCCC, reverse complement of motif AAUCCC, and randomly shuffled sequences in their 5ʹUTRs ( n = 3; mean ± SEM; n.s. P > 0.05). Similar to (D), but the motif is CAAGAU ( n = 3; mean ± SEM; * P < 0.05; Student's t ‐test). Data information: In boxplots, * P < 0.05, ** P < 0.01, *** P < 0.001; Mann–Whitney U ‐test. Box edges represent quantiles, whiskers represent extreme data points no more than 1.5 times the interquartile range.

Article Snippet: Both in vitro and in vivo analyses have demonstrated that different 5ʹUTR sequences derived from the same yeast genes can lead to large difference in translational efficiency (TE) (Rojas‐Duran & Gilbert, ).

Techniques: Luciferase, MANN-WHITNEY

Journal: Molecular Systems Biology

Article Title: Pervasive isoform‐specific translational regulation via alternative transcription start sites in mammals

doi: 10.15252/msb.20166941

Figure Lengend Snippet: Same as Fig B, but in addition, we marked the six genes that were tested by luciferase reporter assay (Fig C) and containing unambiguously determined 5ʹUTR sequences (see ). The TE divergence values estimated based on 5ʹ end sequencing data are shown in cyan, and those based on reporter assay are shown in yellow.

Article Snippet: Both in vitro and in vivo analyses have demonstrated that different 5ʹUTR sequences derived from the same yeast genes can lead to large difference in translational efficiency (TE) (Rojas‐Duran & Gilbert, ).

Techniques: Luciferase, Reporter Assay, Sequencing