The Discovery of Ribosome Heterogeneity and Its Implications for Gene Regulation and Organismal Life
2018; Elsevier BV; Volume: 71; Issue: 3 Linguagem: Inglês
10.1016/j.molcel.2018.07.018
ISSN1097-4164
Autores Tópico(s)RNA Research and Splicing
ResumoThe ribosome has recently transitioned from being viewed as a passive, indiscriminate machine to a more dynamic macromolecular complex with specialized roles in the cell. Here, we discuss the historical milestones from the discovery of the ribosome itself to how this ancient machinery has gained newfound appreciation as a more regulatory participant in the central dogma of gene expression. The first emerging examples of direct changes in ribosome composition at the RNA and protein level, coupled with an increased awareness of the role individual ribosomal components play in the translation of specific mRNAs, is opening a new field of study centered on ribosome-mediated control of gene regulation. In this Perspective, we discuss our current understanding of the known functions for ribosome heterogeneity, including specialized translation of individual transcripts, and its implications for the regulation and expression of key gene regulatory networks. In addition, we suggest what the crucial next steps are to ascertain the extent of ribosome heterogeneity and specialization and its importance for regulation of the proteome within subcellular space, across different cell types, and during multi-cellular organismal development. The ribosome has recently transitioned from being viewed as a passive, indiscriminate machine to a more dynamic macromolecular complex with specialized roles in the cell. Here, we discuss the historical milestones from the discovery of the ribosome itself to how this ancient machinery has gained newfound appreciation as a more regulatory participant in the central dogma of gene expression. The first emerging examples of direct changes in ribosome composition at the RNA and protein level, coupled with an increased awareness of the role individual ribosomal components play in the translation of specific mRNAs, is opening a new field of study centered on ribosome-mediated control of gene regulation. In this Perspective, we discuss our current understanding of the known functions for ribosome heterogeneity, including specialized translation of individual transcripts, and its implications for the regulation and expression of key gene regulatory networks. In addition, we suggest what the crucial next steps are to ascertain the extent of ribosome heterogeneity and specialization and its importance for regulation of the proteome within subcellular space, across different cell types, and during multi-cellular organismal development. In the mid-1950s, future Nobel Prize winner George Palade became fascinated by “a small particulate component of the cytoplasm” (Palade, 1955Palade G.E. A small particulate component of the cytoplasm.J. Biophys. Biochem. Cytol. 1955; 1: 59-68Crossref PubMed Scopus (239) Google Scholar; Figure 1). While examining the structure of the cell by electron microscopy, he noticed small, roughly spherical granules both free-floating in the cytoplasm and associated with the endoplasmic reticulum (ER) membrane. These “microsomes,” as the ER-bound granules were originally termed, were determined to be composed of both protein and RNA and to be the sites of protein synthesis in the cell (reviewed in Palade, 1958Palade G.E. Microsomes and ribonucleoprotein particles.in: Microsomal Particles and Protein Synthesis. 1958: 36-61Google Scholar). Because of confusion in the field over whether microsomes referred to just granules or also to the ER lipid membrane, a new name was suggested at a 1958 symposium of the Biophysical Society: “The phrase ‘microsomal particles’ does not seem adequate, and ‘ribonucleoprotein particles of the microsome fraction’ is much too awkward. During the meeting the word ‘ribosome’ was suggested: this seems a very satisfactory name, and it has a pleasant sound” (Roberts, 1958Roberts R.B. Introduction.in: Microsomal Particles and Protein Synthesis. Pergamon Press, 1958: 7-8Google Scholar). Almost immediately subsequent to the discovery of the ribosome, several scientists postulated that there may be diversity in ribosome composition. Palade noted small differences in the size and shape of microsomes and suggested that there may be further heterogeneity that couldn’t be seen with the current resolution of electron microscopy (Palade, 1958Palade G.E. Microsomes and ribonucleoprotein particles.in: Microsomal Particles and Protein Synthesis. 1958: 36-61Google Scholar). Perhaps the greatest proponent of ribosome heterogeneity in composition and activity at that time was Francis Crick, who after his discovery of the structure of DNA was eagerly discussing the genetic code with the RNA Tie Club, a private scientific social group with twenty primary members—one for each amino acid—and up to four honorary members—each designated by a nucleotide. In 1958 he sparked debate with his bold model for the flow of genetic information known as the “one gene-one ribosome-one protein hypothesis,” wherein each ribosome carries the genetic information required to encode a single protein (Crick, 1958Crick F.H.C. On protein synthesis.Symp. Soc. Exp. Biol. 1958; 12: 138-163PubMed Google Scholar). This model suggested that the cell contains thousands of distinct ribosomes, each tailored to the production of a single protein. However, this view rapidly fell out of favor when further biochemical characterization of the ribosome did not reveal pronounced differences in ribosomal rRNA (rRNA) size across individual ribosomes, so Crick altered his model “to the idea that only part of the ribosomal RNA acts as a template” (Crick and Brenner, 1959Crick, F.H.C., and Brenner, S. (1959). Some Footnotes on Protein Synthesis: a Note for the RNA TIE club. Profiles in Medicine. https://profiles.nlm.nih.gov/ps/access/SCBBFV.pdf.Google Scholar). However, work by Francois Jacob and Jacques Monod on the kinetics of β-galactosidase synthesis led Crick and his fellow RNA Tie club member Sydney Brenner to conclude that the template for protein synthesis is likely an unstable “genetic RNA” that allows the ribosome to make “one protein at one moment, and a quite different protein a few minutes later” (Brenner and Crick, 1960Brenner, S., and Crick, F.H.C. (1960). What are the properties of genetic RNA? A note for the RNA Tie Club. Profiles in Medicine. https://profiles.nlm.nih.gov/ps/access/SCBBFZ.pdf.Google Scholar). In a series of elegant experiments, Brenner, Jacob, and Matthew Meselson showed that in E. coli after bacteriophage infection, new RNA corresponding to the phage genome was synthesized but no new ribosomes were made (Brenner et al., 1961Brenner S. Jacob F. Meselson M. An unstable intermediate carrying information from genes to ribosomes for protein synthesis.Nature. 1961; 190: 576-581Crossref PubMed Scopus (374) Google Scholar). The phage RNA that was made post-infection associated with actively translating bacterial ribosomes that had been made prior to infection, revealing that bacterial ribosomes are capable of synthesizing phage proteins and leading to the conclusion that “ribosomes are non-specialized structures which synthesize, at a given time, the protein dictated by the messenger they happen to contain.” In three short years, the field had vacillated from the most extreme view of ribosome specialization—in which each protein is translated by a different type of ribosome—to the most extreme view of ribosome homogeneity—in which ribosomes are passive machines with no regulatory function. Over the next few decades, the idea that all ribosomes are exactly the same largely prevailed, and is in fact a notion still prevalent in most modern biology textbooks. Any subsequent studies suggesting greater modularity in ribosome composition or activity were met with skepticism, as differences in laboratory procedures made published results from one group difficult to replicate by others, leading to confusion and a general disregard for proponents of ribosome heterogeneity (reviewed in Dinman, 2016Dinman J.D. Pathways to specialized ribosomes: the Brussels lecture.J. Mol. Biol. 2016; 428: 2186-2194Crossref PubMed Scopus (74) Google Scholar, Shi and Barna, 2015Shi Z. Barna M. Translating the genome in time and space: specialized ribosomes, RNA regulons, and RNA-binding proteins.Annu. Rev. Cell Dev. Biol. 2015; 31: 31-54Crossref PubMed Scopus (128) Google Scholar, Xue and Barna, 2012Xue S. Barna M. Specialized ribosomes: a new frontier in gene regulation and organismal biology.Nat. Rev. Mol. Cell Biol. 2012; 13: 355-369Crossref PubMed Scopus (448) Google Scholar). However, inklings of greater dynamics to ribosomes began to arise again in the 1980s and 1990s as studies in multiple, diverse invertebrate model organisms observed differential expression of individual ribosome components, albeit without a direct measure of whether such changes actually produced mature ribosomes that were of a distinct composition. For example, in the slime mold Dictyostelium discoideum, changes in the modification of several ribosomal proteins (RPs) occurred during the transition from unicellular growth to a multicellular body (Ramagopal, 1990Ramagopal S. Induction of cell-specific ribosomal proteins in aggregation-competent nonmorphogenetic Dictyostelium discoideum.Biochem. Cell Biol. 1990; 68: 1281-1287Crossref PubMed Scopus (21) Google Scholar). In Arabidopsis thaliana and Brassica napus, where RPs typically have multiple paralogs due to genome duplication events, RP paralogs are expressed in different regions of the plant (Weijers et al., 2001Weijers D. Franke-van Dijk M. Vencken R.J. Quint A. Hooykaas P. Offringa R. An Arabidopsis Minute-like phenotype caused by a semi-dominant mutation in a RIBOSOMAL PROTEIN S5 gene.Development. 2001; 128: 4289-4299Crossref PubMed Google Scholar, Whittle and Krochko, 2009Whittle C.A. Krochko J.E. Transcript profiling provides evidence of functional divergence and expression networks among ribosomal protein gene paralogs in Brassica napus.Plant Cell. 2009; 21: 2203-2219Crossref PubMed Scopus (65) Google Scholar, Williams and Sussex, 1995Williams M.E. Sussex I.M. Developmental regulation of ribosomal protein L16 genes in Arabidopsis thaliana.Plant J. 1995; 8: 65-76Crossref PubMed Scopus (85) Google Scholar). Different rRNA sequences were also identified in Plasmodium berghei, a parasite that causes malaria in rodents, at different stages of its life cycle (Gunderson et al., 1987Gunderson J.H. Sogin M.L. Wollett G. Hollingdale M. de la Cruz V.F. Waters A.P. McCutchan T.F. Structurally distinct, stage-specific ribosomes occur in Plasmodium.Science. 1987; 238: 933-937Crossref PubMed Scopus (258) Google Scholar). Mutations in ribosomal components also unexpectedly produced tissue-specific phenotypes in fruit flies (Kongsuwan et al., 1985Kongsuwan K. Yu Q. Vincent A. Frisardi M.C. Rosbash M. Lengyel J.A. Merriam J. A Drosophila Minute gene encodes a ribosomal protein.Nature. 1985; 317: 555-558Crossref PubMed Scopus (156) Google Scholar, Marygold et al., 2005Marygold S.J. Coelho C.M.A. Leevers S.J. Genetic analysis of RpL38 and RpL5, two minute genes located in the centric heterochromatin of chromosome 2 of Drosophila melanogaster.Genetics. 2005; 169: 683-695Crossref PubMed Scopus (52) Google Scholar), zebrafish, and mice (reviewed in Shi and Barna, 2015Shi Z. Barna M. Translating the genome in time and space: specialized ribosomes, RNA regulons, and RNA-binding proteins.Annu. Rev. Cell Dev. Biol. 2015; 31: 31-54Crossref PubMed Scopus (128) Google Scholar), as well as in human disease (Draptchinskaia et al., 1999Draptchinskaia N. Gustavsson P. Andersson B. Pettersson M. Willig T.N. Dianzani I. Ball S. Tchernia G. Klar J. Matsson H. et al.The gene encoding ribosomal protein S19 is mutated in Diamond-Blackfan anaemia.Nat. Genet. 1999; 21: 169-175Crossref PubMed Scopus (666) Google Scholar). Additional studies suggested RP-paralog-specific differences in control of cell function in S. cerevisiae, although additional moonlighting functions of RPs outside of the ribosome made such observations difficult to attribute to selective changes in the ribosome itself (Komili et al., 2007Komili S. Farny N.G. Roth F.P. Silver P.A. Functional specificity among ribosomal proteins regulates gene expression.Cell. 2007; 131: 557-571Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, Ni and Snyder, 2001Ni L. Snyder M. A genomic study of the bipolar bud site selection pattern in Saccharomyces cerevisiae.Mol. Biol. Cell. 2001; 12: 2147-2170Crossref PubMed Scopus (238) Google Scholar, Segev and Gerst, 2018Segev N. Gerst J.E. Specialized ribosomes and specific ribosomal protein paralogs control translation of mitochondrial proteins.J. Cell Biol. 2018; 217: 117-126Crossref PubMed Scopus (48) Google Scholar). While these correlative findings were suggestive of possibly greater regulation to the ribosome itself, it is only in the last few years that ribosome heterogeneity and specialization has undergone a renaissance, with modern techniques finally permitting the discovery of ribosomes with distinct compositions performing unique cellular functions. Evidence of physical ribosome heterogeneity has been technologically challenging to obtain. Definitive determination of the stoichiometry of a macromolecular complex requires state-of-the-art quantitative mass spectrometry measurements, ideally of all protein components. Our lab recently measured the absolute abundance of 15 of the 80 core RPs in polysomes (actively translating ribosomes) from mouse embryonic stem cells (mESCs) using selected reaction monitoring (SRM) mass spectrometry, a technique in which the abundance of a peptide from the protein of interest is measured relative to a heavy-labeled peptide spiked in at a known concentration (Shi et al., 2017Shi Z. Fujii K. Kovary K.M. Genuth N.R. Röst H.L. Teruel M.N. Barna M. Heterogeneous ribosomes preferentially translate distinct subpools of mRNAs genome-wide.Mol. Cell. 2017; 67: 71-83.e7Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). Six of the 15 RPs measured were substoichiometric, with four of those present on only 60%–70% of polysomal ribosomes, revealing for the first time that there are indeed actively translating ribosomes lacking at least one core RP. Importantly, these findings were also observed upon treatment with formaldehyde, which cross-links RPs onto rRNA prior to cell lysis. While this is direct evidence of ribosome heterogeneity, the stoichiometry of the other 65 core RPs has yet to be quantified, and even small changes in abundance (i.e. approximately 10%) could have large effects on translation given that millions of ribosomes are present in a single mammalian cell. As this data is also from a single cell type, there may be additional changes in ribosome composition across cell types. Proteomics methods have yet to be brought to bear on this question, but RNA-seq of RP transcripts across mouse tissues (Kondrashov et al., 2011Kondrashov N. Pusic A. Stumpf C.R. Shimizu K. Hsieh A.C. Ishijima J. Shiroishi T. Barna M. Ribosome-mediated specificity in Hox mRNA translation and vertebrate tissue patterning.Cell. 2011; 145: 383-397Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar), human tissues (Guimaraes and Zavolan, 2016Guimaraes J.C. Zavolan M. Patterns of ribosomal protein expression specify normal and malignant human cells.Genome Biol. 2016; 17: 236Crossref PubMed Scopus (101) Google Scholar, Gupta and Warner, 2014Gupta V. Warner J.R. Ribosome-omics of the human ribosome.RNA. 2014; 20: 1004-1013Crossref PubMed Scopus (47) Google Scholar), human hematopoietic cell types (Guimaraes and Zavolan, 2016Guimaraes J.C. Zavolan M. Patterns of ribosomal protein expression specify normal and malignant human cells.Genome Biol. 2016; 17: 236Crossref PubMed Scopus (101) Google Scholar), and human cancer cell lines (Guimaraes and Zavolan, 2016Guimaraes J.C. Zavolan M. Patterns of ribosomal protein expression specify normal and malignant human cells.Genome Biol. 2016; 17: 236Crossref PubMed Scopus (101) Google Scholar) suggests that RPs have many more cell-type-specific expression patterns than would be expected for a machine deemed invariable across all cell types. However, more work at the protein level, and in particular at the level of the ribosome itself, is required to confirm whether these changes produce ribosomes which are heterogeneous, as RNA abundance may not correlate with protein amount or incorporation into functional ribosomes. Tissue-specific expression patterns are also frequently observed for the paralogs of core RPs (Gupta and Warner, 2014Gupta V. Warner J.R. Ribosome-omics of the human ribosome.RNA. 2014; 20: 1004-1013Crossref PubMed Scopus (47) Google Scholar, Wong et al., 2014Wong Q.W.-L. Li J. Ng S.R. Lim S.G. Yang H. Vardy L.A. RPL39L is an example of a recently evolved ribosomal protein paralog that shows highly specific tissue expression patterns and is upregulated in ESCs and HCC tumors.RNA Biol. 2014; 11: 33-41Crossref PubMed Scopus (36) Google Scholar). Unlike yeast or plants, in which the majority of RPs have paralogs due to genome duplications, only a handful of RPs have paralogs in metazoans. As RP paralogs are highly homologous to each other, they are likely to compete for the same position on the ribosome, suggesting that different paralogs could be swapped out on the ribosome to varying degrees depending on the cell type. For some RPs, such as RPL22/eL22 and RPL22L/eL22L in Drosophila, one paralog appears to be ubiquitous, while the other has tissue-specific expression (Kearse et al., 2017Kearse M.G. Chen A.S. Ware V.C. Expression of ribosomal protein L22e family members in Drosophila melanogaster : rpL22-like is differentially expressed and alternatively spliced.Nucleic Acids Res. 2017; 39: 2701-2716Crossref Scopus (24) Google Scholar). In other cases, RP paralogs actually appear to anti-correlate in expression, suggesting coordinated regulation of their abundance on the ribosome. For instance, the paralog RPL3L/uL3L is most abundant at the RNA level in the human heart and skeletal muscle; in those same tissues, the canonical RP RPL3/uL3 mRNA is decreased in abundance relative to other regions of the body (Gupta and Warner, 2014Gupta V. Warner J.R. Ribosome-omics of the human ribosome.RNA. 2014; 20: 1004-1013Crossref PubMed Scopus (47) Google Scholar). In fact, in response to a hypertrophic stimulus, RPL3L/uL3 mRNA abundance is decreased in skeletal muscle coordinately with an increase in RPL3/uL3 mRNA (Chaillou et al., 2014Chaillou T. Kirby T.J. McCarthy J.J. Ribosome biogenesis: emerging evidence for a central role in the regulation of skeletal muscle mass.J. Cell. Physiol. 2014; 229: 1584-1594Crossref PubMed Scopus (118) Google Scholar, Chaillou et al., 2016Chaillou T. Zhang X. McCarthy J.J. Expression of muscle-specific ribosomal protein L3-like impairs myotube growth.J. Cell. Physiol. 2016; 231: 1894-1902Crossref PubMed Scopus (27) Google Scholar, Kirby et al., 2015Kirby T.J. Lee J.D. England J.H. Chaillou T. Esser K.A. McCarthy J.J. Blunted hypertrophic response in aged skeletal muscle is associated with decreased ribosome biogenesis.J. Appl. Physiol. 2015; 119: 321-327Crossref PubMed Scopus (61) Google Scholar). While the mechanism underlying this regulation of RPL3/uL3 and RPL3L/uL3L levels is unknown, RP paralogs have been known to regulate each other’s expression directly. For instance, RPL22/eL22 destabilizes the transcript of its paralog RPL22L1/eL22L1 via direct binding to a stem loop in its mRNA (O’Leary et al., 2013O’Leary M.N. Schreiber K.H. Zhang Y. Duc A.C.E. Rao S. Hale J.S. Academia E.C. Shah S.R. Morton J.F. Holstein C.A. et al.The ribosomal protein Rpl22 controls ribosome composition by directly repressing expression of its own paralog, Rpl22l1.PLoS Genet. 2013; 9: e1003708Crossref PubMed Scopus (72) Google Scholar). Interestingly, GWAS studies have linked RPL3L/uL3 mutations to a remarkably specific disease state leading to increased risk of atrial fibrillation, but the consequence of these mutations on the relative activities of RPL3L/uL3L and RPL3/uL3 or on translational control are yet to be determined (Thorolfsdottir et al., 2018Thorolfsdottir R.B. Sveinbjornsson G. Sulem P. Nielsen J. Jonsson S. Halldorsson G.H. Melsted P. Ivarsdottir E.V. Davidsson O.B. Kristjansson R.P. et al.Coding variants in RPL3L and MYZAP increase risk of atrial fibrillation.Commun. Biol. 2018; 1https://doi.org/10.1038/s42003-018-0068-9Crossref PubMed Scopus (22) Google Scholar). In addition to the core RPs, many other proteins associate with the ribosome and may be sources of heterogeneity. A recent study from our lab developed an affinity enrichment methodology for ribosome isolation from mESCs and used mass spectrometry to identify the “ribo-interactome,” which is comprised of hundreds of ribosomal associated proteins (RAPs) that fall into diverse functional categories such as mRNA binding proteins, mRNA/tRNA modifiers, RNA helicases, and regulators of metabolism and cell cycle control (Simsek et al., 2017Simsek D. Tiu G.C. Flynn R.A. Byeon G.W. Leppek K. Xu A.F. Chang H.Y. Barna M. The mammalian ribo-interactome reveals ribosome functional diversity and heterogeneity.Cell. 2017; 169: 1051-1065.e18Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Intriguingly, one RAP, the glycolytic enzyme PKM2, was enriched on ribosomes at the ER compared to ribosomes in the cytosol, revealing the existence of subcellular ribosome heterogeneity within mESCs. Subcellular heterogeneity is an area requiring further investigation, not only in mESCs but particularly in polarized cells with localized translation like the intestinal epithelium (Moor et al., 2017Moor A.E. Golan M. Massasa E.E. Lemze D. Weizman T. Shenhav R. Baydatch S. Mizrahi O. Winkler R. Golani O. et al.Global mRNA polarization regulates translation efficiency in the intestinal epithelium.Science. 2017; 357: 1299-1303Crossref PubMed Scopus (94) Google Scholar) and neurons (reviewed in Jung et al., 2014Jung H. Gkogkas C.G. Sonenberg N. Holt C.E. Remote control of gene function by local translation.Cell. 2014; 157: 26-40Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Ribosomes are in fact located in the dendrites as well as in the soma of neurons, and another RAP, Fragile X mental retardation protein (FMRP), has been shown to localize to synaptosomal polysomes (Feng et al., 1997Feng Y. Gutekunst C.-A. Eberhart D.E. Yi H. Warren S.T. Hersch S.M. Fragile X mental retardation protein: nucleocytoplasmic shuttling and association with somatodendritic ribosomes.J. Neurosci. 1997; 17: 1539-1547Crossref PubMed Google Scholar). What percentage of ribosomes contain FMRP, and whether FMRP is particularly enriched on ribosomes in certain cellular compartments, is not yet established; however, given FMRP’s known regulation of the translation of transcripts associated with synaptic signaling (Chen et al., 2014Chen E. Sharma M.R. Shi X. Agrawal R.K. Joseph S. Fragile X mental retardation protein regulates translation by binding directly to the ribosome.Mol. Cell. 2014; 54: 407-417Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, Darnell et al., 2011Darnell J.C. Van Driesche S.J. Zhang C. Hung K.Y.S. Mele A. Fraser C.E. Stone E.F. Chen C. Fak J.J. Chi S.W. et al.FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism.Cell. 2011; 146: 247-261Abstract Full Text Full Text PDF PubMed Scopus (1419) Google Scholar), these findings suggest that FMRP could specialize ribosomes in key functional cellular regions for the translation of particular mRNAs. Ribosome heterogeneity is not unique to the protein components of the ribosome: it can also occur at the level of rRNA. The mammalian ribosome contains four rRNAs (18S, 28S, 5.8S, 5S), and these are encoded by multiple copies of ribosomal DNA (rDNA) on different chromosomes (Henderson et al., 1972Henderson A.S. Warburton D. Atwood K.C. 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In fact, all four rRNAs have also been shown to change from maternal to somatic ribosomes over the course of embryonic development in zebrafish (Locati et al., 2017aLocati M.D. Pagano J.F.B. Girard G. Ensink W.A. van Olst M. van Leeuwen S. Nehrdich U. Spaink H.P. Rauwerda H. Jonker M.J. et al.Expression of distinct maternal and somatic 5.8S, 18S, and 28S rRNA types during zebrafish development.RNA. 2017; 23: 1188-1199Crossref PubMed Scopus (54) Google Scholar, Locati et al., 2017bLocati M.D. Pagano J.F.B. Ensink W.A. van Olst M. van Leeuwen S. Nehrdich U. Zhu K. Spaink H.P. Girard G. Rauwerda H. et al.Linking maternal and somatic 5S rRNA types with different sequence-specific non-LTR retrotransposons.RNA. 2017; 23: 446-456Crossref PubMed Scopus (22) Google Scholar). While the functional consequences of this rRNA variation is not yet established, in silico modeling has suggested that maternal and somatic rRNA variants may preferentially bind different mRNAs (Locati et al., 2017aLocati M.D. Pagano J.F.B. Girard G. Ensink W.A. van Olst M. van Leeuwen S. Nehrdich U. Spaink H.P. Rauwerda H. Jonker M.J. et al.Expression of distinct maternal and somatic 5.8S, 18S, and 28S rRNA types during zebrafish development.RNA. 2017; 23: 1188-1199Crossref PubMed Scopus (54) Google Scholar), suggesting that rRNA may play a direct role in the regulation of translation, as was initially proposed by the ribosome filter hypothesis (Mauro and Edelman, 2002Mauro V.P. Edelman G.M. The ribosome filter hypothesis.Proc. Natl. Acad. Sci. USA. 2002; 99: 12031-12036Crossref PubMed Scopus (195) Google Scholar). In addition to mRNA selection, rRNA variation may also specialize ribosomes for programmed frameshifting, as was suggested for several yeast and Xenopus laevis rRNA alleles (Kiparisov et al., 2005Kiparisov S. Petrov A. Meskauskas A. Sergiev P.V. Dontsova O.A. Dinman J.D. Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae.Mol. Genet. Genomics. 2005; 274: 235-247Crossref PubMed Scopus (33) Google Scholar). Furthermore, rRNA is extensively modified, adding an additional layer of heterogeneity, as described in other recent reviews on the topic (McMahon et al., 2015McMahon M. Contreras A. Ruggero D. Small RNAs with big implications: new insights into H/ACA snoRNA function and their role in human disease.Wiley Interdiscip. Rev. RNA. 2015; 6: 173-189Crossref PubMed Scopus (88) Google Scholar, Roundtree et al., 2017Roundtree I.A. Evans M.E. Pan T. He C. Dynamic RNA modifications in gene expression regulation.Cell. 2017; 169: 1187-1200Abstract Full Text Full Text PDF PubMed Scopus (1447) Google Scholar, Xue and Barna, 2012Xue S. Barna M. Specialized ribosomes: a new frontier in gene regulation and organismal biology.Nat. Rev. Mol. Cell Biol. 2012; 13: 355-369Crossref PubMed Scopus (448) Google Scholar). While there is evidence of ribosomes with varying composition, what is the actual extent of ribosome heterogeneity? A candid answer to this question is that it really remains largely unknown. The stoichiometry of 15 RPs has been calculated in mESCs, and six RPs have been identified as substoichiometric; these alone could produce dozens of distinct ribosome compositions lacking one or more of these RPs. It is not yet clear whether this handful of variations in the ribosome is all that can be tolerated. If one considers not only the other 65 core RPs but also the additional hundreds of RAPs and rRNA sequence variants, the complexity increases exponentially. While systematic assessment of the stoichiometry of each of these variants, using SRM or similar mass spectrometry methods, would be helpful for understanding ribosome heterogeneity, determining the actual number of ribosome types requires examination of their composition on a ribosome-by-ribosome basis. One potential strategy to accomplish this is to use native mass spectrometry, in which intact macromolecular complexes as large as 9 MDa are run on the spectrometer. This method ha
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