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Utrs in Translation Initiation bioRxiv preprint doi: https://doi.org/10.1101/2021.05.28.446092; this version posted May 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Unbiased screen of human transcriptome reveals an unexpected role 2 of 3ʹUTRs in translation initiation 3 4 5 Yun Yang 1, *, #, Xiaojuan Fan1, *, Yanwen Ye1, 2, Chuyun Chen1, 2, Sebastian E.J. Ludwig 3, Sirui 6 Zhang1, 2, Qianyun Lu1, 2, Cindy L. Will3, Henning Urlaub4, Jing Sun1, Reinhard Lü hrmann3, Zefeng 7 Wang1, 2, 5, # 8 9 1 Bio-med Big Data Center, CAS Key Laboratory of Computational Biology, CAS Center for 10 Excellence in Molecular Cell Science, Shanghai Institute of Nutrition and Health, Chinese Academy 11 of Sciences, China 12 2 University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, 13 China 14 3 Max Planck Institute for Biophysical Chemistry, Cellular Biochemistry, Am Fassberg 11, D- 15 37077, Göttingen, Germany 16 4 Bioanalytical Mass Spectrometry, MPI for Biophysical Chemistry, Am Fassberg 11, D-37077 17 Göttingen, Germany 18 5Lead Contact 19 20 * These authors contributed equally to this work 21 # Corresponding to: [email protected]; [email protected] 22 23 24 Running title: Translational enhancement from 3ʹUTR 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.28.446092; this version posted May 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 25 Abstract 26 Although most eukaryotic mRNAs require a 5ʹ-cap for translation initiation, some can 27 also be translated through a poorly studied cap-independent pathway. Here we 28 developed a circRNA-based system and unbiasedly identified more than 10,000 29 sequences in the human transcriptome that contain Cap-independent Translation 30 Initiators (CiTIs). Surprisingly, most of the identified CiTIs are located in 3ʹUTRs, 31 which mainly promote translation initiation in mRNAs bearing highly structured 32 5ʹUTR. Mechanistically, CiTI recruits several translation initiation factors including 33 eIF3 and DHX29, which in turn unwind 5ʹUTR structures and facilitate ribosome 34 scanning. Functionally, we showed that the translation of HIF1A mRNA, an 35 endogenous DHX29 target, is antagonistically regulated by its 5ʹUTR structure and a 36 new 3ʹ-CiTI in response to hypoxia. Therefore, deletion of 3ʹ-CiTI suppresses cell 37 growth in hypoxia and tumor progression in vivo. Collectively, our study uncovers a 38 new regulatory mode for translation where the 3ʹUTR actively participate in the 39 translation initiation. 40 41 42 43 44 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.28.446092; this version posted May 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 45 Introduction 46 The mRNA translation is an essential and energetically expensive process tightly 47 regulated at the initiation stage. In eukaryotic cells, canonical translation initiation is 48 mediated by the formation of eIF4F complex on the 5ʹ-cap (7-methylguanosine), 49 followed by the attachment of the ribosomal 43S preinitiation complex that scans 50 along the mRNA in a 5ʹ-to-3ʹ direction for a start codon 1,2. Multiple cis-elements and 51 trans-factors are employed to accurately regulate mRNA translation initiation. For 52 example, the RNA structures and upstream ORF (uORF) in 5ʹUTRs are well known 53 cis-elements that regulate mRNA translation, whereas many protein factors or 54 miRNAs can control translation in trans 1-4. 55 In addition, many genes can also be translated in a cap-independent fashion, 56 mostly through direct recruitment of ribosomes to the internal ribosome entry site 57 (IRES), which often occurs during stress conditions (like virus infection, heat shock, 58 or apoptosis) 5,6. It has been estimated that up to 10-15% of cellular mRNAs undergo 59 cap-independent translation under various conditions where cap-dependent translation 60 is suppressed 7. A recent high-throughput screen targeting the viral RNAs and 5ʹUTRs 61 of human mRNAs revealed that many human genes (583 out of 5058 genes tested) 62 harbor the “cap-independent translation elements” that function as IRESs to drive 63 mRNA translation 8. However, an unbiased screen of the translation initiation 64 elements remains absent. Recently, thousands of circular RNAs (circRNAs) have 65 been identified in eukaryotic cells 9-11, many of which can be translated in vitro and in 66 vivo 12-16. Since circRNAs lack free ends and can only be translated cap- 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.28.446092; this version posted May 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 67 independently, they may serve as an improved system to study this noncanonical 68 translation pathway. 69 70 Results 71 Identification of cap-independent translation initiation elements with a circRNA- 72 based translation system 73 To unbiasedly screen the human transcriptome for endogenous sequences that 74 drive cap-independent translation, we developed an efficient cell-based circRNA 75 translation system (Fig. 1a) that has been extensively validated 13,15-17. We inserted a 76 library of cDNA short fragments from normalized human transcriptome before start 77 codon of the “split” EGFP (Fig. 1a) 13,16, and transfected the library to generate ~3 78 million stable clones. The green cells were selected by FACS (Extended data Fig. 1a), 79 and the insertion libraries were sequenced at each stage (supplementary information). 80 The fragment abundance in this library correlated poorly with gene levels in 81 HeLa cells (r<0.09, Extended data Fig. 1b), with rRNAs representing <10% of the 82 total fragments (Extended data Fig. 1c), indicative of an effective transcriptome 83 normalization. After sorting, we recovered 82,367 unique fragments from the green 84 cells with similar length distribution as the input (Extended data Fig. 1d and Extended 85 data Table 1). The recovered fragments were not dominated by the pre-sorting library 86 (Extended data Fig. 1e), suggesting the screen was unbiased. The majority of the 87 reads from the pre- and post-sorting libraries were located in protein coding genes 88 (Fig. 1b), with two examples shown in read density plots (Fig. 1c). Surprisingly, the 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.28.446092; this version posted May 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 89 percentage of positive reads from 3ʹUTRs was substantially increased whereas the 90 reads from 5ʹUTRs and coding sequences (CDS) was decreased after sorting (Fig. 91 1b), suggesting a prominent role of 3ʹUTRs in regulation of cap-independent 92 translation. 93 We further validated 14 out of 15 randomly selected fragments for their activity in 94 driving circRNA translation (Fig. 1d, activities are independent on their read depth), 95 indicating a low false positive rate. The IRES-like activities in 8 out of 9 positive 96 fragments were also confirmed using a bicistronic translation reporter (Fig. 1e). 97 Conversely, 3 out of the randomly selected 12 single clones from the input library 98 promoted circRNA translation (Extended data Fig. 1f), suggesting that our screen had 99 not reached saturation. 100 The overlapping positive fragments were merged into 10,455 peaks in human 101 transcriptome, which were defined as cap-independent translation initiators (CiTIs). 102 Most CiTIs were recovered only by a small number of reads in the post-sorting library 103 while several CiTIs have exceedingly high read depth (Extended data Fig. 1g). The 104 CiTIs were widely distributed across all human chromosomes (Extended data Fig. 105 1h), and their abundance correlated positively with the gene numbers (r=0.96) and the 106 size (r=0.76) of each chromosome (Extended data Fig. 1i). The newly identified CiTIs 107 included many previously reported IRESs 8,18 (Extended data Fig. 1j), including ~10% 108 of IRESs identified from a screen biased towards the 5ʹUTRs and viral sequences 8. 109 The CiTI-containing genes were more conserved than an average human gene 110 (Extended data Fig. 2a), and were actively expressed and translated in cultured cells 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.28.446092; this version posted May 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 111 (Extended data Fig. 2b). Interestingly, CiTI containing genes were substantially 112 enriched in functional categories of RNA translation, splicing, cell-cell adhesion and 113 viral processes (Extended data Fig. 2c). Particularly, the CiTIs were enriched in genes 114 encoding ribosomal proteins and translation initiation factors, suggesting a potential 115 positive feedback regulation of translation. 116 Although the canonical IRESs are mostly located in the 5ʹUTR, the newly 117 identified CiTIs were found in all gene regions including 5ʹUTRs (termed 5ʹ-CiTI), 118 CDS (termed C-CiTI), and 3ʹUTRs (termed 3ʹ-CiTI). Consistent with our earlier 119 finding, the CiTIs were enriched in 3ʹUTRs and peaked in the CDS near the start and 120 stop codons (Fig. 1f). Compared to the cognate controls, we found a significant 121 increase of sequence conservation in 3ʹ-CiTIs, a modest increase in 5ʹ-CiTIs, and no 122 increase in C-CiTIs (Extended data Fig. 2d). The CiTIs generally had less structures 123 as judged by higher minimal free energy (MFE) (Extended data Fig.
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