Tag Archives: Translation

A Ribosome Code?

The ribosome, a universally conserved molecular machine that catalyses protein synthesis, has generally been considered to act constitutively. That is to say, that ribosomes act to translate mRNAs in the same way across all cells and developmental stages. Regulatory control of translation is predominantly exerted by the action of translation initiation factors, which guide the association of the ribosome with target mRNAs. The eukaryotic ribosome is composed of 4 RNA molecules and 79 different ribosomal proteins (RPs). A paper published last year by Kondrashov et al. has shown one RP (RPL38) specifically regulates the expression of a subset of mRNAs during embryonic development in the mouse. Together with findings from human genetic diseases and from other organisms, this data is suggestive of a ‘ribosomal code’ regulating translation.

Kondrashov et al. set out to discover what gene was responsible for causing the morphological defects found in a spontaneous mouse mutant, tail-short (Ts). These mice display skeletal patterning defects, including homeotic transformations (ie. the conversion of a tissue’s identity to that of a different tissue; in this case changes between the segmental identities of vertebrae and ribs). They also display eye and craniofacial defects, short and kinky tails, and wavy neural tubes. These phenotypes are only found in heterozygous mice (Ts/+); homozygotes die at implantation stages. By positional cloning, Kondrashov et al. found that the gene responsible for Ts was Rpl38.

Ts/+ mice display skeletal defects and transformations along the entire length of the anterior-posterior body axis. The key regulators of morphological identity along the A-P axis are Hox genes. Hox genes encode homeodomain-containing transcription factors, and are found in four genomic clusters in vertebrates. Loss of function mutations in, or missexpression of, Hox genes generally leads to homeotic transformations (most shockingly seen in the Drosophila mutants antennapedia and ultrabithorax). Kondrashov et al. therefore examined the expression of Hox gene transcripts in Ts/+ mouse embryos. Surprisingly, they found no changes in the levels or expression domains of the Hox genes.

Schematic representation of the axial skeleton of WT and Ts/+ mice. Defects are explained by the effects of corresponding Hox gene mutants.

The researchers then asked whether changes in translational control of Hox genes were responsible for the Ts/+ phenotypes. Using various techniques they showed that there were no changes in global protein synthesis. However, by using quantitative PCR on mRNAs that were purified with active ribosomes, they identified a subset of Hox genes that were translationally deregulated in Ts/+ embryos (Hoxa4; a5; a9; a11; b3; b13; c8; d11).  These findings were confirmed by observing protein levels for HOXA5, A11, and B13 in the Ts/+ mouse embryos. The majority of the Ts/+ axial skeleton phenotypes could be accounting for by the known effects of loss of function mutations in the Hox genes that were translationally deregulated.

It therefore appears that RPL38 is exerting a specialised control on the translation of specific Hox genes. In further experiments Kondrashov et al. find that RPL38 is likely facilitating the formation of the 80S (complete) ribosomal complex on specific mRNAs (the ribosome is made up of two subunits, the 40S subunit associates with the 5’UTR of the target mRNA first and is then joined by the 60S subunit to make a translationally competent ribosome). An important question is whether RPL38 exerts it’s function as part of the ribosome, or whether it has extra-ribosomal roles as well? By separating ribosomal from ribosome-free cytosolic fractions, Kondrashov et al, found that RPL38 was only ever found in the ribosome.

Ribosomal proteins have generally been considered as ubiquitously expressed cellular ‘housekeeping’ proteins. However, when the researchers examined Rpl38 expression, they found that transcripts were enriched in specific tissues. For instance, embryonic tissues that give rise to facial structures, as well as the neural retina, showed high levels of Rpl38 expression, correlating with the craniofacial and eye defects in Ts/+ mice. Likewise, Rpl38 was strongly expressed in the somites and the neural tube, the embryonic tissues giving rise to the vertebrae and the spinal cord respectively. Kondrashov et al. went on to examine the expression of 72 different ribosomal proteins in 14 different tissue and cell types. They found a large amount of heterogeneity in RP expression, suggesting that many have specialised, tissue specific roles.

A few obvious outstanding questions for future studies should be noted; Does RPL38 bind cis-regulatory sequence or structure elements within target mRNAs? and what are they? Do trans-acting factors also play a role? Other developmental questions also stand out. Hox genes are not involved in eye development, and it also seems unlikely that the Hox genes implicated in the trunk segmental effects are also responsible for the craniofacial defects. What other RPL38 mRNA targets are responsible for these phenotypes?

These experiments have therefore shown that RPL38 has transcript-specific roles in the control of translation, and that many RPs display heterogeneous expression patterns rather than the previously assumed ubiquity. Together these findings suggest that RPs are imparting a new level of specificity in the control of gene expression. They fit into a broader array of observations that hint at the existence of a ‘ribosome code’ in which alterations in the composition of ribosomes leads to their translational specialisation towards subsets of mRNAs. Diamond-Blackfan Anaemia is a human genetic disease caused by mutations in a number of ribosomal proteins. Patients display limb defects, cleft palates, growth failures and cancer predisposition. Likewise knockdown of multiple distinct RPs in zebrafish leads to a wide range of developmental defects and a high incidence of cancer. A possible explanation for these types of finding, is that highly proliferating tissues may be more sensitive to differences in the rate of protein synthesis. Hence, indirect effects on cell proliferation and apoptosis may lead to the morphological abnormalities. However, Kondrashov et al. have shown in this study of Ts/+, overall protein synthesis is not affected, and the effects on a subset of developmental patterning genes are responsible for the bulk of the phenotypes.

Ribosomal RNAs and proteins are also targets for extensive chemical modifications such as phosphorylation and methylation, most of which are as yet uncharacterised. Interestingly, another human genetic disease, X-linked Dyskeratosis Congenita, is probably caused by failures of rRNA modifications. By analogy with the levels of complexity see with regard to modifications and combinations of chromatin-associated histones, a ‘ribosome code’ imparting translational specificity by heterogeneity of RPs and modifications has the potential to be a hugely important level of regulatory control.

Kondrashov, N., Pusic, A., Stumpf, C., Shimizu, K., Hsieh, A., Xue, S., Ishijima, J., Shiroishi, T., & Barna, M. (2011). Ribosome-Mediated Specificity in Hox mRNA Translation and Vertebrate Tissue Patterning Cell, 145 (3), 383-397 DOI: 10.1016/j.cell.2011.03.028

Topisirovic, I., & Sonenberg, N. (2011). Translational Control by the Eukaryotic Ribosome Cell, 145 (3), 333-334 DOI: 10.1016/j.cell.2011.04.006

On Ribosomal Pausing

A new paper in Nature, describes how Shine-Dalgarno-like features in protein coding sequences, leads to bacterial ribosomes pausing during translation. Selection against ribosomal pausing leads to biases in codon usage and coding sequence evolution. Translational pausing represents a new level of regulatory control of gene expression.

Translation, the process by which the nucleotide sequence of mRNA transcripts is decoded and converted into amino acid sequence during protein synthesis, is carried out by ribosomes. Within the ribosome, transfer RNA molecules recognise specific trinucleotide codons on the mRNA, and add their cognate amino acids to nascent protein chains. In bacteria and archaea, ribosomes often recognise the translation start site with the help of a sequence 8 to 12 nucleotides upstream of it – the Shine-Dalgarno sequence (SD). It’s been known since the 1980s that different mRNAs are translated at different rates. The main reason for these differences was thought to be the concentration of rarer varieties of tRNA limiting the rate at which some transcripts could be decoded.

Li et al. have used a new technique, ribosome profiling, that maps ribosome occupancy along mRNAs. This has yielded high-resolution views of local translation rates on the entire protein coding transcriptome of E. coli and Bacillus subtilis.  Briefly put, mRNA fragments that have been protected from nuclease digestion by ribosomal binding, are ‘deep sequenced’. The concentration of these ribosome footprints equates to the ribosome occupancy throughout the transcriptome. The local translation rate is inversely related to ribosome occupancy.

Using this technique, Li et al. found many sites where ribosomal density is ten fold or more than the mean. They sought to correlate these translational pauses with specific codons. However, there was little link between occupancy of specifc codons and the abundance of their corresponding tRNAs. Therefore, the concentration of rare tRNAs is not responsible for much translational pausing under normal growth conditions.

To try to find sequence features that were linked to ribosomal pausing, the researchers then tried to correlate any trinucleotide sequences (independently of reading frame) with ribosome occupancy. They found that 6 different trinucleotide sequences, with features resembling Shine-Dalgarno sequences, did correlate with the position of paused ribosomes. This correlation was not found in the yeast, Saccharomyces cerevisiae; in agreement with eukaryotic ribosomes not using SD- anti-SD interactions.

Li et al. went on to show definitively that internal SD-like sequences are linked to translational pausing, by introducing a mutation into one such site and showing that ribosome occupancy was reduced. They also showed that peaks of ribosome occupancy, were caused by translational pausing, rather than attempted internal translational initiation.

As translational pausing limits the amount of free ribosomes, widespread internal SD-like sequences would limit the rate of protein synthesis, and hence the potential bacterial growth rate. In line with this, SD-like sequences in protein coding genes are disfavoured. Selection pressure against SD-like sequences is therefore a major factor in determining codon usage, and more especially the usage of codon pairs (SD sequences are 6/7 nt long).

Interestingly, the authors found that patterns of ribosome occupancy were conserved between orthologous genes in E. coli and B. subtilis. This reflects two different factors; firstly, coding is obviously constrained by protein’s functionality, but secondly it’s suggestive of translational pausing being exploited for functional purposes. Li et al. suggest a number of different ways in which ribosomal pausing can regulate gene expression. It’s known that internal SD-like sequences can promote regulated shifting of reading frame. Ribosome pausing may also modulate folding of nascent protein chains. Lastly, as transcription and translation are closely coupled in bacteria, ribosome occupancy may inhibit the formation of stem-loop structures that prevent transcriptional termination. It will be exciting to work out the extents to which these potential regulatory systems are active. Eukaryotic ribosomes do not use recognition of SD sequences, instead using the 5’ mRNA cap and the less well defined Kozak sequence for translational initiation. Does ribosome pausing occur in eukaryotes? and does it have functional significance?

Li, G., Oh, E., & Weissman, J. (2012). The anti-Shine–Dalgarno sequence drives translational pausing and codon choice in bacteria Nature, 484 (7395), 538-541 DOI: 10.1038/nature10965