Heterogeneity and specialized functions of translation machinery: from genes to organisms

Abstract

Regulation of mRNA translation offers the opportunity to diversify the expression and abundance of proteins made from individual gene products in cells, tissues and organisms. Emerging evidence has highlighted variation in the composition and activity of several large, highly conserved translation complexes as a means to differentially control gene expression. Heterogeneity and specialized functions of individual components of the ribosome and of the translation initiation factor complexes eIF3 and eIF4F, which are required for recruitment of the ribosome to the mRNA 5' untranslated region, have been identified. In this Review, we summarize the evidence for selective mRNA translation by components of these macromolecular complexes as a means to dynamically control the translation of the proteome in time and space. We further discuss the implications of this form of gene expression regulation for a growing number of human genetic disorders associated with mutations in the translation machinery.

Fig. 1 |. Technologies for the quantification and characterization of translation machinery heterogeneity and functional specialization.

a | Translation efficiency measured by polysome profiling: lysates from cells treated with the drugs that immobilize elongating ribosomes on mRNAs are loaded onto sucrose gradients and spun in an ultracentrifuge. The free subunits, 80S ribosome and polysomes are separated into defined fractions in the gradient. RNA is isolated from each individual fraction, and the abundance of genes of interest is determined by quantitative PCR (qPCR) or RNA sequencing (RNA-seq). Highly translated mRNAs are enriched in the heavier polysome fractions (blue line), while poorly translated mRNAs are enriched in lighter fractions (red line). b | Translation efficiency measured by ribosome profiling: ribosomes are immobilized on mRNA, isolated by density centrifugation and treated with nucleases to digest unprotected mRNA, and then the ~30-nucleotide ribosome-protected mRNA fragments are sequenced. Mapping of these fragments to the transcriptome calculates the number of ribosomes at each position on mRNAs genome-wide. Translation efficiency is determined by comparing ribosome occupancy to mRNA abundance. Genes with the same transcript levels but different ribosome footprints are under different forms of translational regulation. c | Absolute protein quantification by selected or parallel reaction monitoring (SRM or PRM, respectively): complexes of interest, such as the ribosome, are isolated, digested into peptides, spiked with a known quantity of heavy-labelled peptides from the ribosomal proteins (RPs) of interest and analysed by mass spectrometry (MS). The absolute quantity of each RP is calculated by comparing the intensity of its peptides relative to the heavy-labelled standards. RPs present at one copy on every ribosome would have the same abundance; a substoichiometric RP found only on half of ribosomes would have a 50% lower abundance. d | Relative protein quantification by stable isotope labelling with amino acids in cell culture (SILAC): two cell populations, such as those containing a tagged RP or a tagged PKM2 allele, are treated with light (blue) or heavy (red) isotopes, respectively, and then pooled. The resulting heavy or light ribosomes are isolated by immunoprecipitation (IP) and digested into peptides, and the component proteins are identified by MS. The heavy/light ratio for each protein equals the relative abundance of the protein in the PKM2-containing ribosomes compared with total ribosomes. e | Relative protein quantification by tandem mass tags (TMTs): protein samples of interest, such as the individual ribosomal subunits and polysomes, are digested into peptides, are labelled with unique TMT reagents, are pooled and undergo MS. For each protein of interest, the ratios of the TMT labels determine the relative abundance of that protein in the free subunits compared with the polysomes.

Fig. 2 |. Ribosome heterogeneity and specialization tune genetic networks.

Ribosome heterogeneity exists at multiple levels. a | Ribosomes containing or lacking specific core RPs, such as RPL10a/uL1 or RPS25/eS25, are specialized for the translation of mRNAs from specific cellular pathways. b | Ribosomes can contain paralogues of core ribosomal proteins (RPs). RPL22/eL22 represses expression of its paralogue, RPL22L1/eL22L1, as well as the important developmental regulator SMAD1. A switch from RPL22L1/eL22L1 to RPL22/eL22 expression is required for haematopoietic stem cell differentiation into T cell progenitors. c | Ribosomes are extensively post-translationally modified. Phosphorylation (P) of RPL13a/uL13 upon interferon-γ signalling causes its removal from the ribosome and incorporation into the γ-interferon inhibitor of translation (GAIT) complex, which binds the 3′ UTR of ceruloplasmin (Cp) mRNA and inhibits its translation. d | Ribosomal RNA (rRNA) is extensively modified. Small nucleolar RNAs (snoRNAs) can direct pseudouridylation (Ψ) of rRNA, and preventing this modification inhibits translation of several cellular and viral internal ribosome entry sites (IRESs). e | RNA can vary in sequence. During zebrafish development, ribosomes switch from an oocyte-specific rRNA sequence variant to a somatic sequence variant. These different rRNA sequences may promote translation of maternal or somatic mRNAs, respectively. f | Ribosome-associated proteins (RAPs) have many functions. One RAP, PKM2, is a metabolism enzyme that binds to the 28S rRNA and is enriched on ribosomes found at the endoplasmic reticulum (ER). PKM2 additionally binds to transcripts of ER-associated and membrane-associated proteins to upregulate their translation. CrPV, cricket paralysis virus; ECM, extracellular matrix; PTM, post-translational modification.

Fig. 3 |. eIF3 is required for translation of specific cellular mRNAs.

a | Schematic of the mammalian eIF3 complex, which consists of 13 subunits, 8 of which form a core octamer (blue), while the remaining 5 peripheral subunits (green) seem to be more conformationally flexible. Post-translational modifications (PTMs) identified on individual subunits are labelled (P, phosphorylation; Ac, acetylation). b | eIF3 binds to N⁶-methyladenosine (m⁶A) modifications (red) in the 5′ untranslated regions (UTRs) of mRNAs involved in cell stress, including HSP70 (blue circles), and in additional pathways to promote recruitment of the 40S ribosomal subunit to the start codon (AUG). This process is cap-independent, but the precise mechanism is unknown. c | eIF3 regulates cellular proliferation by repressing expression of BTG1 and activating expression of JUN in a cap-dependent manner. Regulation of both of these genes requires eIF3 binding to a hairpin structure (in red) in their 5′ UTRs. In the case of JUN, the 5′ cap is additionally bound by eIF3 (via the eIF3d subunit) instead of the canonical eIF4F cap-binding complex. d | In zebrafish, eIF3h regulates translation of crystallin proteins (blue circles), which are required for proper development of the eye lens. Whether eIF3h makes direct contact with these mRNAs is unknown, but both the 5′ UTR and the 3′ UTR are required for eIF3h-mediated translational activation.

Fig. 4 |. eIF4F specialization is regulated by cellular stimuli.

a | eIF4E promotes translation of mRNAs containing the cytosine-enriched regulator of translation (CERT) element in their 5′ untranslated regions (UTRs). These mRNAs encode genes involved in multiple genetic networks that promote tumour development. b | Upon MAPK signalling inhibition, the eIF4E3 homologue is incorporated into eIF4F and promotes translation of mRNAs encoding RNA editing and transcription regulators with particular sequence motifs in their 5′ UTRs. c | During hypoxia, when cap-dependent translation initiation is downregulated, eIF4F complexes containing eIF4G3 initiate translation of mRNAs containing internal ribosome entry site (IRESs) in their 5′ UTRs. d | During apoptosis, eIF4G1 is cleaved by caspases into several fragments, each containing different initiation factor binding domains. While this effectively eliminates formation of the preinitiation complexes required for cap-dependent translation initiation, the eIF4G1 fragment that retains the binding domains for eIF4A and eIF3 can initiate translation at cellular IRESs. eIF4G2, which is homologous to this eIF4G1 fragment, can similarly activate cellular IRES translation initiation during apoptosis after cleavage by caspases removes an inhibitory domain.