40S ribosome profiling reveals distinct roles for Tma20/Tma22 (MCT-1/DENR) and Tma64 (eIF2D) in 40S subunit recycling

The recycling of ribosomes at stop codons for use in further rounds of translation is critical for efficient protein synthesis. Removal of the 60S subunit is catalyzed by the ATPase Rli1 (ABCE1) while removal of the 40S is thought to require Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR). However, it remains unclear how these Tma proteins cause 40S removal and control reinitiation of downstream translation. Here we used a 40S ribosome footprinting strategy to directly observe intermediate steps of ribosome recycling in cells. Deletion of the genes encoding these Tma proteins resulted in broad accumulation of unrecycled 40S subunits at stop codons, directly establishing their role in 40S recycling. Furthermore, the Tma20/Tma22 heterodimer was responsible for a majority of 40S recycling events while Tma64 played a minor role. Introduction of an autism-associated mutation into TMA22 resulted in a loss of 40S recycling activity, linking ribosome recycling and neurological disease.

Phosphorylation of Ribosomal Protein S6 differentially affects mRNA translation based on ORF length

ABSTRACTPhosphorylation of Ribosomal Protein S6 (RPS6) was the first post-translational modification of the ribosome to be identified and is a commonly-used readout for mTORC1 activity. Although the cellular and organismal functions of RPS6 phosphorylation are known, its molecular consequences on translation are less well understood. Here we use selective ribosome footprinting to analyze the location of ribosomes containing phosphorylated RPS6 on endogenous mRNAs in cells. We find that RPS6 becomes progressively dephosphorylated on ribosomes as they translate an mRNA. As a consequence, average RPS6 phosphorylation is higher on mRNAs with short coding sequences (CDSs) compared to mRNAs with long CDSs. In particular, ribosomes translating on the endoplasmic reticulum are more rapidly dephosphorylated than cytosolic ribosomes. Loss of RPS6 phosphorylation causes a correspondingly larger drop in translation efficiency of mRNAs with short CDSs than long CDSs. Interestingly, mRNAs with 5’ TOP motifs are translated well also in the absence of RPS6 phosphorylation despite short CDS lengths, suggesting they are translated via a different mode. In sum this provides a dynamic view of RPS6 phosphorylation on ribosomes as they translate mRNAs in different subcellular localizations and the functional consequence on translation.

Identification and analysis of short open reading frames (sORFs) in the initially annotated noncoding RNA LINC00493 from human cells

Whole transcriptome analyses have revealed that mammalian genomes are massively transcribed, resulting in the production of huge numbers of transcripts with unknown functions (TUFs). Previous research has categorized most TUFs as noncoding RNAs (ncRNAs) because most previously studied TUFs do not encode open reading frames (ORFs) with biologically significant lengths (>100 amino acids). Recent studies, however, have reported that several transcripts harboring small ORFs (sORFs) that encode peptides shorter than 100 amino acids are translated and play important biological functions. Here, we examined the translational capacity of transcripts annotated as ncRNAs in human cells, and identified several hundreds of ribosome-associated transcripts previously annotated as ncRNAs. Ribosome footprinting and polysome profiling analyses revealed that 61 of them are potentially translatable. Among them, 45 were non-nonsense-mediated mRNA decay targets, suggesting that they are productive mRNAs. We confirmed the translation of one ncRNA, LINC00493, by luciferase reporter assaying and western blotting of a FLAG-tagged LINC00493 peptide. While proteomic analysis revealed that the LINC00493 peptide interacts with many mitochondrial proteins, immunofluorescence assays showed that its peptide is mitochondrially localized. Our findings indicate that some transcripts annotated as ncRNAs encode peptides and that unannotated peptides may perform important roles in cells.

Monosomes actively translate synaptic mRNAs in neuronal processes

To accommodate their complex morphology, neurons localize messenger RNAs (mRNAs) and ribosomes near synapses to produce proteins locally. However, a relative paucity of polysomes (considered the active sites of translation) detected in electron micrographs of neuronal processes has suggested a limited capacity for local protein synthesis. In this study, we used polysome profiling together with ribosome footprinting of microdissected rodent synaptic regions to reveal a surprisingly high number of dendritic and/or axonal transcripts preferentially associated with monosomes (single ribosomes). Furthermore, the neuronal monosomes were in the process of active protein synthesis. Most mRNAs showed a similar translational status in the cell bodies and neurites, but some transcripts exhibited differential ribosome occupancy in the compartments. Monosome-preferring transcripts often encoded high-abundance synaptic proteins. Thus, monosome translation contributes to the local neuronal proteome.

Full-length transcriptome reconstruction reveals a large diversity of RNA and protein isoforms in rat hippocampus

Gene annotation is a critical resource in genomics research. Many computational approaches have been developed to assemble transcriptomes based on high-throughput short-read sequencing, however, only with limited accuracy. Here, we combine next-generation and third-generation sequencing to reconstruct a full-length transcriptome in the rat hippocampus, which is further validated using independent 5´ and 3´-end profiling approaches. In total, we detect 28,268 full-length transcripts (FLTs), covering 6,380 RefSeq genes and 849 unannotated loci. Based on these FLTs, we discover co-occurring alternative RNA processing events. Integrating with polysome profiling and ribosome footprinting data, we predict isoform-specific translational status and reconstruct an open reading frame (ORF)-eome. Notably, a high proportion of the predicted ORFs are validated by mass spectrometry-based proteomics. Moreover, we identify isoforms with subcellular localization pattern in neurons. Collectively, our data advance our knowledge of RNA and protein isoform diversity in the rat brain and provide a rich resource for functional studies.

Protein synthesis rates and ribosome occupancies reveal determinants of translation elongation rates

Although protein synthesis dynamics has been studied both with theoretical models and by profiling ribosome footprints, the determinants of ribosome flux along open reading frames (ORFs) are not fully understood. Combining measurements of protein synthesis rate with ribosome footprinting data, we here inferred translation initiation and elongation rates for over a 1,000 ORFs in exponentially growing wild-type yeast cells. We found that the amino acid composition of synthesized proteins is as important a determinant of translation elongation rate as parameters related to codon and transfer RNA (tRNA) adaptation. We did not find evidence of ribosome collisions curbing the protein output of yeast transcripts, either in high translation conditions associated with exponential growth, or in strains in which deletion of individual ribosomal protein (RP) genes leads to globally increased or decreased translation. Slow translation elongation is characteristic of RP-encoding transcripts, which have markedly lower protein output compared with other transcripts with equally high ribosome densities.

Monosomes actively translate synaptic mRNAs in neuronal processes

In order to deal with their huge volume and complex morphology, neurons localize mRNAs and ribosomes near synapses to produce proteins locally. A relative paucity of polyribosomes (considered the active sites of translation) detected in electron micrographs of neuronal processes (axons and dendrites), however, has suggested a rather limited capacity for local protein synthesis. Polysome profiling together with ribosome footprinting of microdissected synaptic regions revealed that a surprisingly high number of dendritic and/or axonal transcripts were predominantly associated with monosomes (single ribosomes). Contrary to prevailing views, the neuronal monosomes were in the process of active protein synthesis (e.g. they exhibited elongation). Most mRNAs showed a similar translational status in both compartments, but some transcripts exhibited differential ribosome occupancy in the somata and neuropil. Strikingly, monosome-preferred transcripts often encoded high-abundance synaptic proteins. This work suggests a significant contribution of monosome translation to the maintenance of the local neuronal proteome. This mode of translation can presumably solve some of restricted space issues (given the large size of polysomes) and also increase the diversity of proteins made from a limited number of ribosomes available in dendrites and axons.

Cell type specific profiling of alternative translation identifies novel protein isoforms in the mouse brain

Translation canonically begins at a single AUG and terminates at the stop codon, generating one protein species per transcript. However, some transcripts may use alternative initiation sites or sustain translation past their stop codon, generating multiple protein isoforms. Through other mechanisms such as alternative splicing, both neurons and glia exhibit remarkable transcriptional diversity, and these other forms of post-transcriptional regulation are impacted by neural activity and disease. Here, using ribosome footprinting, we demonstrate that alternative translation is likewise abundant in the central nervous system and modulated by stimulation and disease. First, in neuron/glia mixed cultures we identify hundreds of transcripts with alternative initiation sites and confirm the protein isoforms corresponding to a subset of these sites by mass spectrometry. Many of them modulate their alternative initiation in response to KCl stimulation, indicating activity-dependent regulation of this phenomenon. Next, we detect several transcripts undergoing stop codon readthrough thus generating novel C-terminally-extended protein isoforms in vitro. Further, by coupling Translating Ribosome Affinity Purification to ribosome footprinting to enable cell-type specific analysis in vivo, we find that several of both neuronal and astrocytic transcripts undergo readthrough in the mouse brain. Functional analyses of one of these transcripts, Aqp4, reveals readthrough confers perivascular localization, indicating readthrough can be a conserved mechanism to modulate protein function. Finally, we show that AQP4 readthrough is disrupted in multiple gliotic disease models. Our study demonstrates the extensive and regulated use of alternative translational events in the brain and indicates that some of these events alter key protein properties.

A Zebrafish Model of Diamond-Blackfan Anemia Results in Bone Marrow Failure and Demonstrates Defective Translation in Erythroid Cells By Ribosome Footprinting

Diamond-Blackfan anemia (DBA) is a congenital bonemarrow failure syndrome that manifests as a profoundmacrocytic anemia, which classically presents within the first year of life. Heterozygous mutations or allelic loss of one of 12 ribosomal proteins (RP) leads to apoptosis of erythroid precursors and have been identified in over 50% of DBA patients, most commonly RPS19 that accounts for 25% of all cases. To study the role of Rps19 in erythroid development, we employed genome-editing tools to generate stable knockout line of Rps19 in zebrafish using TALENs targeting exon 1 of Rps19. We generated a stable mutant line and these mutants have a 5 bp complex indel in Exon 1 resulting in N13fs and truncated protein. Rps19 -/- embryos show profound developmental anomalies including a marked anemia and are embryonic lethal by 5dpf. Rps19 +/- heterozygotes were indistinguishable from their WT siblings during early development and had no erythroid defect detectable by whole mount in situ hybridization (WISH) or histochemical staining. To assess the effects of Rps19 on embryonic erythropoiesis more detail, we used Rps19 mutants carrying the Tg(gata1:dsRed) and Tg(globin:eGFP) transgenes. Rps19+/-fish were crossed and then exposed to cold stress from 12 hours post fertilization(hpf) continuously (22 degrees) and then analyzed by flow cytometry at the developmental age of 96hpf. GFP, dsRed and double GFP/dsRed expressing erythroid cells showed a reduction in cell number in Rps19+/- compared to Rps19+/+ siblings. Therefore, when stress is induced, the loss of one copy of rps19 is sufficient to induce anemia. To further study the effects of Rps19 on erythroid development, we investigated the phenotype of adult fish. Adult Rps19+/- fish at 6 months of age were significantly smaller than their siblings, weighed less and showed reduced hemoglobin levels in their peripheral blood. We then further characterized the adult rps19-deficient zebrafish phenotype by flow cytometry of whole kidney marrow using Tg(globin:eGFP);Rps19+/-animals. Rps19+/ - adults demonstrated pancytopenia, with a marked decrease in the erythroid lineage, similar to the phenotype observed in DBA patients. Recent data have suggested that tissue specific functions of ribosomes are common and we hypothesized that such an effect may explain the tropism of Rps19+/- phenotypes seen in DBA to erythroid and hematopoietic tissue. To test this hypothesis we assessed the effect of translation in Rps19+/- erythroid cells compared to Rps19+/+ using genome-wide analysis of translation by ribosome footprinting (RF) on sorted GFP+ cells from whole kidney marrow using Tg(globin:eGFP);Rps19 mutantzebrafish. In parallel we assessed RNAseq from the same samples. RNAseq showed differential expression of genes involved in mitochondrial cell death pathways including upregulation of caspase 9 and downregulation of p53 . Stabilization of p53 and consequent p53-dependent apoptosis is a well recognized mechanism of the anemia in DBA, however our data suggest that such stabilization results in transcriptional downregulation of p53 as a compensatory mechanism to maintain a steady state level of erythropoiesis. RF data by contrast showed that the principle genes with aberrant translation in Rps19+/- erythroid cells are other ribosomal protein genes, predominantly (although not exclusively) of the large subunit. In addition we observed that one of the most markedly upregulated footprints was on the branched chain keto acid dehydrogenase E1 subunit beta gene (encoded by bckdhb). This enzyme is part of a multimolecular complex that resides on the inner membrane of the mitochondria and is responsible for catabolism of branched chain amino acids L-Leucine and Valine, highlighting a potential underlying mechanism by which erythroid cells may be functionally leucine deficient, and thus respond to exogenous L-leucine as has previously been shown in zebrafish, murine and human cell models. In summary, we have generated a stable Rps19 mutant line and characterized the phenotype of heterozygous Rps19+/- mutants, which recapitulate features of DBA during developmental and adult hematopoiesis. In addition, we have used ribosome footprinting to study the effect of Rps19 heterozygosity on translation within erythroid cells of different genes, providing new insight for potential drug targets. Our on-going work is further studying the differentially expressed genes. Disclosures No relevant conflicts of interest to declare.

DHX36 binding at G-rich sites in mRNA untranslated regions promotes translation

ABSTRACTTranslation efficiency can be affected by mRNA stability and secondary structures, including so-called G-quadruplex (G4) structures. The highly conserved and essential DEAH-box helicase DHX36/RHAU is able to resolve G4 structures on DNA and RNA in vitro, however a system-wide analysis of DHX36 targets and function is lacking. We globally mapped DHX36 occupancy in human cell lines and found that it preferentially binds to G-rich sequences in the coding sequences (CDS) and 5' and 3' untranslated regions (UTR) of more than 4,500 mRNAs. Functional analyses, including RNA sequencing, ribosome footprinting, and quantitative mass spectrometry revealed that DHX36 decreased target mRNA stability. However, target mRNA accumulation in DHX36 KO cells did not lead to a significant increase in ribosome footprints or protein output indicating that they were translationally incompetent. We hypothesize that DHX36 resolves G4 and other structures that interfere with efficient translation initiation.