Developmental regulation of Canonical and small ORF translation from mRNAs
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ABSTRACT:
PROVIDER: PRJNA615808 | ENA |
REPOSITORIES: ENA
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Project description:We present a genome-wide assessment of the translation of small open reading frames (smORF) in Drosophila melanogaster mRNAs, using ribosomal profiling of polysomal fractions in three contiguous temporal windows, which encopass all of embryogenesis. We also performed the same protocol using S2 cells. In this way, mRNAs bound by multiple ribosomes can be isolated and distinguished from mRNAs bound by sporadic, putatively non-productive single ribosomes or ribosomal subunits.
2020-03-27 | GSE147619 | GEO
Project description:Bacterial mRNAs are organized into operons consisting of discrete open reading frames (ORFs) in a single polycistronic mRNA. Individual ORFs on the mRNA are differentially translated, with rates varying as much as 100-fold. The signals controlling differential translation are poorly understood. Our genome-wide mRNA secondary structure analysis indicated that operonic mRNAs are comprised of ORF-wide units of secondary structure that vary across ORF boundaries such that adjacent ORFs on the same mRNA molecule are structurally distinct. ORF translation rate is strongly correlated with its mRNA structure in vivo, and correlation persists, albeit in a reduced form, with its structure when translation is inhibited and with that of in vitro refolded mRNA. These data suggests that intrinsic ORF mRNA structure encodes a rough blueprint for translation efficiency. This structure is then amplified by translation, in a self-reinforcing loop, to provide the structure that ultimately specifies the translation of each ORF.
2017-01-30 | GSE77617 | GEO
Project description: Not available
| S-EPMC7260771 | biostudies-literature
Project description:Half of mammalian transcripts contain short upstream open reading frames (uORFs) that potentially regulate translation of the downstream coding sequence (CDS). The molecular mechanisms governing these events remain poorly understood. Here, we find that the non-canonical initiation factor Death-associated protein 5 (DAP5 or eIF4G2) is required for translation initiation on select transcripts. Using ribosome profiling and luciferase-based reporters coupled with mutational analysis we show that DAP5-dependent translation occurs on messenger RNAs (mRNAs) with long, structure-prone 5′ leader sequences and persistent uORF translation. These mRNAs preferentially code for signalling factors such as kinases and phosphatases. We also report that cap/eIF4F- and eIF4A-dependent recruitment of DAP5 to the mRNA facilitates main CDS, but not uORF, translation suggesting a role for DAP5 in translation re-initiation. Our study reveals important mechanistic insights into how a non-canonical translation initiation factor involved in stem cell fate shapes the synthesis of specific signalling factors.
2022-11-07 | GSE155854 | GEO
Project description:A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames. To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a step-wise approach to employ multiple CRISPR-Cas9 screens to elucidate functional non-canonical ORFs implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream open reading frames (uORFs) exhibited selective functionality independent of the main coding sequence. One of these, ASNSD1-uORF or ASDURF, was upregulated, associated with the MYC family oncogenes, and was required for medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future cancer genomics studies seeking to define new cancer targets.
| EGAS00001007426 | EGA
Project description:The production of mitochondrial OXPHOS complexes is central to cellular metabolism, although the molecular details of mitochondrial translation remain enigmatic. It is widely held that translation initiation in human mitochondria proceeds similarly to bacterial systems, with mRNA binding the mitoribosomal small subunit in the presence of initiation factors, mtIF2and mtIF3, and initiator tRNA. However, unlike in bacteria, most human mitochondrial mRNAs do not possess 5′ leader sequences that mediate binding to the small subunit. Thus, how leaderless mRNAs are recognized by the mitoribosome is not known. By developing a single-molecule, fluorescence-based in vitro translation initiation assay, alongside the biochemical and genetic characterization of cellular knockouts of mitochondrial translation factors, we describe a mechanism for non-canonical translation initiation in human mitochondria. We show leaderless mt-mRNAs can be loaded onto 55S monosomes and translated independently of mtIF3 activity. However, in the case of the bicistronic ATP8/ATP6 transcript, translation of the downstream open reading frame (ORF) is dependent upon mtIF3 and is uncoupled from the upstream leaderless ORF, highlighting distinct role for the human initiation factor. Furthermore, we found mtIF2 to be essential for mitochondrial protein synthesis, but not monosome formation, while mitoribosome recycling was important for mitoribosome homeostasis. These data define an important evolutionary diversion of mitochondrial translation system, and further our fundamental understanding of a process central to eukaryotic metabolism.
2022-11-29 | PXD021015 | Pride
Project description:Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides but lacking canonical coding sequences. Apparently unable to produce peptides, lncRNA function seems to only involve RNA sequence and structure. Here, we exhaustively detect in-vivo translation of small open reading frames (small ORFs) within lncRNAs using Ribosomal profiling during Drosophila melanogaster embryogenesis. We show that around 30% of lncRNAs contain small ORFs engaged by ribosomes, leading to regulated translation of 100 to 300 micropeptides. We identify lncRNA features that favour translation, such as cistronicity, Kozak sequences, and conservation. For this latter, we develop a bioinformatics pipeline to detect small ORF homologues, and we reveal evidence of natural selection favouring the conservation of micropeptide sequence and function across evolution. Our results expand the repertoire of lncRNA functions, and suggest that lncRNAs give rise to novel coding genes throughout evolution. Since most lncRNAs contain small ORFs with as yet unknown translation potential, we propose to rename them “long non-canonical RNAs”.
2022-11-02 | GSE204739 | GEO
Project description:Translation is a fundamental step in gene expression that regulates multiple developmental and stress responses. One key step of translation is the association between eIF4E and eIF4G. This process is regulated in different eukaryotes by proteins which bind to eIF4E and block the formation of the eIF4E/eIF4G complex. Here, we report the discovery of CERES, the first functional eIF4E regulator described in plants. CERES is a modular protein that contains a LRR domain and a canonical eIF4E binding site (4E-BS), critical for CERES interaction with eIF4E in planta. CERES/eIF4E interaction excludes eIF4G from the complex. Despite this observation, CERES promotes translation in vivo interacts with eIF4A and with eIF3 in vivo and cosediments with translation initiation complexes in sucrose gradients. Moreover, ceres mutants display a sharp increase of the 80S peak and a reduction of polysome content at specific periods of the diel cycle. Super-resolution ribosome profiling demonstrates that these mutants show a change of translation efficiency of mRNAs related to light response and glucose management. Consistently, these mutants show a hypersensitive response to glucose. These data show that CERES is a “non canonical” translation initiation factor that, through the formation of alternative translation initiation complexes, modulates translation during the light cycle in plants.
2019-12-04 | GSE124290 | GEO
Project description:Phosphorylation 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. 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 and the functional consequence on translation.
2021-12-22 | GSE168977 | GEO