Regulation of protein synthesis is a vital step in controlling gene expression, especially during development. Over the last 10 years, it has become clear that rather than being homogeneous machines responsible for mRNA translation, ribosomes are highly heterogeneous and can play an active part in translational regulation. These “specialized ribosomes” comprise of specific protein and/or rRNA components, which are required for the translation of particular mRNAs. However, while there is extensive evidence for ribosome heterogeneity, support for specialized functions is limited. Recent work in a variety of developmental model organisms has shed some light on the biological relevance of ribosome heterogeneity. Tissue‐specific expression of ribosomal components along with phenotypic analysis of ribosomal gene mutations indicate that ribosome heterogeneity and potentially specialization are common in key development processes like embryogenesis, spermatogenesis, oogenesis, body patterning, and neurogenesis. Several examples of ribosome specialization have now been proposed but strong links between ribosome heterogeneity, translation of specific mRNAs by defined mechanisms, and role of these translation events remain elusive. Furthermore, several studies have indicated that heterogeneous ribosome populations are a product of tissue‐specific expression rather than specialized function and that ribosomal protein phenotypes are the result of extra‐ribosomal function or overall reduced ribosome levels. Many important questions still need to be addressed in order to determine the functional importance of ribosome heterogeneity to development and disease, which is likely to vary across systems. It will be essential to dissect these issues to fully understand diseases caused by disruptions to ribosomal composition, such as ribosomopathies. This article is categorized under: Translation > Translation Regulation Translation > Ribosome Structure/Function RNA in Disease and Development > RNA in Development

Nanopores hold great potential for the analysis of complex biological molecules at the single-entity level. One particularly interesting macromolecular machine is the ribosome, responsible for translating mRNAs into proteins. In this study, we use a solid-state nanopore to fingerprint 80S ribosomes and polysomes from a human neuronal cell line and Drosophila melanogaster cultured cells and ovaries. Specifically, we show that the peak amplitude and dwell time characteristics of 80S ribosomes are distinct from polysomes and can be used to discriminate ribosomes from polysomes in mixed samples. Moreover, we are able to distinguish large polysomes, containing more than seven ribosomes, from those containing two to three ribosomes, and demonstrate a correlation between polysome size and peak amplitude. This study highlights the application of solid-state nanopores as a rapid analytical tool for the detection and characterization of ribosomal complexes.

Ribosomes have long been thought of as homogeneous macromolecular machines, but recent evidence suggests they are heterogeneous and could be specialised to regulate translation. Here, we have characterised ribosomal protein heterogeneity across 4 tissues of Drosophila melanogaster. We find that testes and ovaries contain the most heterogeneous ribosome populations, which occurs through a combination of paralog-enrichment and paralog-switching. We have solved structures of ribosomes purified from in vivo tissues by cryo-EM, revealing differences in precise ribosomal arrangement for testis and ovary 80S ribosomes. Differences in the amino acid composition of paralog pairs and their localisation on the ribosome exterior indicate paralog-switching could alter the ribosome surface, enabling different proteins to regulate translation. One testis-specific paralog-switching pair is also found in humans, suggesting this is a conserved site of ribosome heterogeneity. Overall, this work allows us to propose that mRNA translation might be regulated in the gonads through ribosome heterogeneity, providing a potential means of ribosome specialisation.

13Ribosomes have long been thought of as homogeneous, macromolecular machines but 14 recent evidence suggests they are heterogeneous and their specialisation can regulate translation. 15Here, we have characterised ribosomal protein heterogeneity across 5 tissues of Drosophila 16 melanogaster. We find that testis and ovary contain the most heterogeneous ribosome populations, 17 and that specialisation in these tissues occurs through paralog-switching. For the first time, we have 18 solved structures of ribosomes purified from in vivo tissues by cryo-EM, revealing differences in 19 precise ribosomal arrangement for testis and ovary 80S ribosomes. Differences in the amino acid 20 composition of paralog pairs and their localisation on the ribosome exterior indicate paralog-21 switching could alter the ribosome surface, enabling different proteins to regulate translation. One 22 testis-specific paralog-switching pair is also found in humans, suggesting this is a conserved site of 23 ribosome specialisation. Overall, this work allows us to propose possible mechanisms by which 24 ribosome specialisation can regulate translation. 25 26 27 48 effects [1], whilst RpL38 mutants in D. melanogaster exhibit large wings, small bristles, delayed 49 development and disorganised wing hair polarity [14]. 50 2 Human cytoplasmic ribosomes usually comprise of 80 RPs and 4 rRNAs. This is similar across 51 the majority of multicellular eukaryotes including D. melanogaster with 80 RPs and 5 rRNAs. 52 However, annotated in FlyBase there are 93 cytoplasmic RP genes, including 39 small subunit 53 proteins and 54 large subunit proteins [15]. These additional genes code for 13 paralogs in D. 54 melanogaster. In fact, across eukaryotes many RP genes possess paralogs, for example human RpL3 55 and RpL3L [11] and Arabidopsis RpS8A and RpS8B [13]. In total, there are 19 pairs of paralogs in 56 humans [4] and all 80 RPs in Arabidopsis thaliana have paralogs [16].57 To dissect the function of ribosome heterogeneity it is necessary to understand biological 58 importance within context of whole organisms. Within the developmental biology field, a large 59 proportion of research focuses on the contribution of transcription to gene expression control.60 However, during development a variety of processes and key time points are highly dependent on 61 the regulation of mRNA translation (oogenesis in Xenopus [17], early embryo development in 62 Drosophila [18] and mammalian erythropoiesis [19]). The balance between self-renewal and 63 differentiation at the stem cell niche is highly dependent on translation in both the ovary and the 64 testis [20]. This is exemplified by disruptions to the stem cell niche in the testis when RPs are 65 knocked down e.g. RpL19 RNAi results in over-proliferation of early germ cells in D. melanogaster 66 [21]. During the meiotic phase of gametogenesis, transcription does not occur [22]; therefore 67 meiotic cells rely on post-transcriptional gene regulation [23]. The translational machinery has 68 evolved to become specialise...

The regulation of protein synthesis plays an important role in the growth and development of all organisms. Upstream open reading frames (uORFs) are commonly found in eukaryotic messenger RNA transcripts and typically attenuate the translation of associated downstream main ORFs (mORFs). Conserved peptide uORFs (CPuORFs) are a rare subset of uORFs, some of which have been shown to conditionally regulate translation by ribosome stalling. Here, we show that Arabidopsis CPuORF19, CPuORF46 and CPuORF47, which are ancient in origin, regulate translation of any downstream ORF, in response to the agriculturally significant environmental signals, heat stress and water limitation. Consequently, these CPuORFs represent a versatile toolkit for inducible gene expression with broad applications. Finally, we note that different classes of CPuORFs may operate during distinct phases of translation, which has implications for the bioengineering of these regulatory factors.