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Control of translation by eukaryotic mRNA transcript leaders—Insights from high‐throughput assays and computational modeling

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Abstract Eukaryotic gene expression is tightly regulated during translation of mRNA to protein. Mis‐regulation of translation can lead to aberrant proteins which accumulate in cancers and cause neurodegenerative diseases. Foundational studies on model genes established fundamental roles for mRNA 5′ transcript leader (TL) sequences in controlling ribosome recruitment, scanning, and initiation. TL cis‐regulatory elements and their corresponding trans‐acting factors control cap‐dependent initiation under unstressed conditions. Under stress, cap‐dependent initiation is suppressed, and specific mRNA structures and sequences promote translation of stress‐responsive transcripts to remodel the proteome. In this review, we summarize current knowledge of TL functions in translation initiation. We focus on insights from high‐throughput analyses of ribosome occupancy, mRNA structure, RNA Binding Protein occupancy, and massively parallel reporter assays. These data‐driven approaches, coupled with computational analyses and modeling, have paved the way for a comprehensive understanding of TL functions. Finally, we will discuss areas of future research on the roles of mRNA sequences and structures in translation. This article is categorized under: Translation > Translation Mechanisms RNA Evolution and Genomics > Computational Analyses of RNA RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems
Preinitiation complex (PIC) recruitment to 5′ TL in eukaryotic translation. (a) Canonical cap dependent initiation. The cap‐binding protein complex or eukaryotic initiation factor 4F (eIF4F) is made up of eIFs 4A, 4E, 4G. eIF4F which attaches to the 5′ m7G cap, and the poly(A)‐binding protein (PABP), which binds the 3′ poly(A) stretch come together in a closed‐loop formation. Additional RNA binding proteins (RBP) regulate and stabilize the mRNA. The ternary complex (TC) is made up of eIF2, an initiator methionyl‐tRNA (Met‐tRNA), and GTP. The TC then binds the 40S ribosomal subunit along with eIFs 1, 1A, 3, and 5 to form the 43S preinitiation complex (43S PIC). The cap recruits the 43S PIC to the 5′ end resulting in a 48S PIC. Scanning of the 48S PIC proceeds 5′–3′ along the mRNA until a suitable start codon is reached. (b) Direct recruitment of eIF3. In repressed conditions of eIF2, the eIF3 complex binds the 5′ m7G cap via the eIF3d subunit and recruits the 40S for mRNA scanning and initiation (top). m6A chemical modification may be recognized by eIF3, which in turn recruits PIC for scanning (bottom). (c) Translation Initiation of Short 5′UTR (TISU). A cap‐dependent scanning‐free alternative for translation initiation, in which the ribosome directly identifies the Translation Initiation of Short 5′ UTR (TISU) and begins translation. Usually around +5 to +30 relative to TSS. TISU is optimal for short 5′ TLs, in which scanning may be limited. The TISU sequence is “SAASAUGGCGGC”, with most important nucleotides being at position −3, +4, +5, and +6, and S standing for C/G. (d) Structure mediated PIC recruitment. Cap independent mechanism for translation initiation (top) An IRES in the 5′ TL promotes direct recruitment of a 43S PIC to the mRNA at its location. This process may be regulated by IRES trans‐acting factors (ITAFs). (bottom) Cap‐Independent Translation Enhancers (CITES), found in the 3′ UTR, form a closed loop with RNA interactions from the 5′ end. The CITE promotes 43S PIC recruitment and initiation at the AUG start codon
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Polysome profiling analysis of alternative transcript isoforms. (a) Schematic showing preparation of a sucrose gradient for polysome fractionation. The sucrose density increases from top to bottom to allow for polysome separation. (b) Top layer indicates cell extract containing TL library of interest. The centrifuge tube containing cell extract is spun in an ultracentrifuge. The dense sequences (containing polysomes) sediment more rapidly than the less dense fractions containing individual ribosomal subunits and monosomes. The polysome fractions are then separated by bin and prepared for sequencing. (c) Graph of the polysome fractions (UV absorbance by RNA). The first peak (40S), second peak (60S), third peak (80S) all represent primarily nontranslating mRNAs. The fourth peak (2 polysomes), fifth peak (3 polysomes), sixth peak (4 polysomes), seventh peak and beyond (5+ polysomes) represent translating mRNAs. Alternative splicing generates mRNA transcript isoforms containing different functional elements in their transcript leaders and 3′ UTRs. These isoforms are differentially translated and, consequently, differentially distributed throughout the polysomal fractions
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Ribosome footprint profiling to quantify translation and identify novel open reading frames. (a) First cells are lysed, and mRNA molecules isolated and bound to ribosomes via cycloheximide or other chemical inhibitors. (b) Ribonucleases are used to digest mRNA that is not protected by ribosomes. Ribosomes protect approximately 30 nucleotide mRNA fragments. (c) A density gradient is used to size‐select ribosome‐protected mRNA fragments (RPFs) protected by the ribosomes. (d) After sequencing the RPFs, a ribosome footprinting profile is generated which plots footprints vs. mRNA position. From these data, novel uORFs and start sites are identified. Start codon indicated in green and stop codon in pink
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Impact of cis‐regulatory elements and mRNA structures on translation initiation via directional scanning. (a) 5′ regulatory elements. The 48S PIC scans along the 5′ TL for an appropriate start codon. In addition to the 5′ m7G cap, the 5′ TL contains other regulatory elements. Upstream open reading frames (uORFs), stem loops and other mRNA structures regulate the flow of PICs to the CDS. The Kozak sequence modulates start codon selection, with GCCRCCAUGC representing the favorable consensus sequence for higher eukaryotes. Shown besides the Kozak sequence are non‐canonical start codons or near‐cognate codons (NCCs), that differ from AUG by one nucleotide and can be used for alternative initiation. The main coding region is labeled as CDS. (b) uORF regulation and translation. (a) PIC translates uORF. Typically, uORF translation represses translation of the main CDS. (b) Alternatively, a premature translation‐termination codon from uORF translation destabilizes the mRNA and triggers nonsense‐mediated mRNA decay (NMD). (c) A PIC initiating in‐frame with the main CDS can continue translation and produce an alternative N‐terminal extension (NTE) protein isoform. The new N‐terminus is indicated in gray of the resulting protein. (c) Scanning roadblocks. (a) Structured mRNA within the 5′ TL blocks and slows down PIC scanning. This can increase uORF translation and decrease translation at the main CDS. (b) uORFs buried in stable mRNA structures can be skipped via ribosome shunting. Here the PIC bypasses the structure and continues scanning and initiates translation downstream
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Analysis of translation cis‐regulatory sequence functions using massively parallel reporter assays (MPRAs). (a–c) Different methods of preparing dual‐fluorescent reporter libraries. Oligonucleotides of interest are designed and synthesized via microarray. (a) Screen in which oligos are inserted between red fluorescence protein (RFP) and green fluorescence protein (GFP) to test the activity of putative IRESs on translation of GFP relative to RFP. Production of GFP is directly regulated by the ability of an IRES to recruit a PIC. (b) A control promoter is fused to RFP. The TL of interest, in this example containing 10 random nucleotides before the start codon (N(10)ATG) is fused to GFP and controls its translation. Both RFP and GFP are directly integrated at a chromosomal locus. (c) Sequences are inserted downstream a promoter and upstream GFP, to measure effects of 5′ TL on translation while RFP acts as an internal control, both on a single copy yeast plasmid. The influence of uORFs on translation efficiency is assayed by comparing wildtype (AUG) uORF containing TLs and mutant (AGG) TLs. (d) Cells are grown in organism being tested (e.g., yeast, human). The cell population is sorted via flow cytometry (FACS). The flow cytometer laser detects GFP and RFP and sorts cells into bins by the GFP/RFP ratio. Bins on the left end represent low GFP expression levels, while bins on the right signify high GFP expression levels. (e) Cells are collected from each bin. (f) DNA is extracted from the cells and each bin population is uniquely barcoded for sequencing. Sequencing the library results in a distribution which is used to calculate the mean GFP expression level for each sequence of interest. Alternatively, reporter constructs can be separated by polysome profiling (see Figure 6 and Sample et al., 2019)
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Identification of RNA binding protein binding sites using high‐througput CLIP assays. (a) Cells are treated with a photoreactive ribonucleoside analog such as 4‐thiouridine (4SU), which is incorporated into cellular RNA and facilitates crosslinking. Ultraviolet light irradiation at 365 nm is used to stimulate protein‐RNA crosslinks. (b) Cartoon depiction of a ribonucleoprotein complex (RNP). Protein‐RNA crosslinks (indicated by X), 4SU or equivalent are indicated by pink patches on mRNA. (c) Cell lysates are prepared and RNPs are immunoprecipitated using antibodies specific to the RBP of interest. Crosslinked RNA is separated using SDS‐PAGE. RNPs are identified from the expected size (circled), while unbound RNAs run to the bottom of the gel. (d) The RNAs from RNPs are isolated and prepared for sequencing. The resulting reads map the location of RBP sites onto target mRNA. Specific crosslink sites introduce mutations (gray box) in read aligntments. Note that several variants of CLIP assays have been developed, including HiTS‐CLIP, PAR‐CLIP, and eCLIP
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RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems
RNA Evolution and Genomics > Computational Analyses of RNA
Translation > Translation Mechanisms

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