Selective 40S footprinting reveals that scanning ribosomes remain cap-tethered in human cells
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ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE139391 | GEO | 2020/06/25
REPOSITORIES: GEO
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Project description:The recycling of ribosomal subunits after translation termination is critical for efficient gene expression. Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR) function as 40S recycling factors in vitro, but it is unknown whether they perform this function in vivo or serve as alternative initiation factors. Ribosome profiling of strains missing these factors revealed 80S ribosomes queued behind the stop codon, consistent with a block in 40S recycling. We found that unrecycled ribosomes could reinitiate translation at AUG codons in the 3’UTR, as evidenced by peaks in the footprint data and 3’UTR reporter analysis. In vitro translation experiments using reporter mRNAs containing upstream ORFs (uORFs) further established that reinitiation increased in the absence of these proteins. In some cases, 40S ribosomes appeared to rejoin with 60S subunits and undergo an alternative 80S reinitiation process in 3’UTRs. These results support a crucial role for Tma64, Tma20, and Tma22 in the recycling of 40S ribosomal subunits at stop codons and translation reinitiation.
2018-08-30 | GSE108942 | GEO
Project description:Most animal mRNAs contain upstream Open Reading Frames (uORFs). These uORFs represent an impediment to translation of the main ORF since ribosomes usually bind the mRNA cap at the 5’ end and then scan for ORFs in a 5’-to-3’ fashion. One way for ribosomes to bypass uORFs is via leaky scanning, whereby the ribosome disregards the uORF start codon. Hence leaky scanning is an important post-transcriptional mechanism affecting gene expression. Few molecular factors regulating or facilitating this process are known. Here we identify the PRRC2 proteins PRRC2A, PRRC2B and PRRC2C as translation initiation factors. We find that they bind other eukaryotic translation initiation factors and preinitiation complexes and are enriched on ribosomes translating mRNAs with uORFs. We find that PRRC2 proteins promote translation of mRNAs containing uORFs by facilitating leaky scanning past uORFs. Since PRRC2 proteins have been associated with cancer, this provides a mechanistic starting point for understanding their physiological and pathophysiological roles.
2023-02-11 | GSE211440 | GEO
Project description:Translation efficiency varies 1000-fold between different mRNAs, thereby strongly impacting protein expression. Translation of the master stress response gene ATF4 increases in response to stress, but the molecular mechanisms are not well understood. We discover here that translation initiation factors DENR, MCTS1 and eIF2D are absolutely required to induce ATF4 translation upon stress, by promoting translation reinitiation on the ATF4 5’UTR. Hence DENR and MCTS1 are important players in the cellular Integrated Stress Response. We find DENR and MCTS1 promote reinitiation after long uORFs with specific penultimate codons, due to the tRNA that remains attached to 40S ribosomes after translation termination. This provides a model for how DENR and MCTS1 promote translation reinitiation. Since cancer cells are exposed to many stresses, they require ATF4 for survival and proliferation. We find a strong correlation between DENR•MCTS1 expression and ATF4 activity across cancers. Additional oncogenes including a-Raf, c-Raf and Cdk4 have long uORFs and are translated in a DENR•MCTS1 dependent manner. This explains in part why DENR and MCTS1 are oncogenes.
2020-07-18 | GSE140084 | GEO
Project description:Among the transcript features that regulate translation rate, upstream open reading frames (uORFs) have emerged as a major class of inhibitory elements which limit how many scanning ribosomes reach the start codon of the main protein coding sequence (CDS). Two processes are implicated in ensuring that CDS translation of uORF-containing transcripts can take place nevertheless: first, leaky-scanning, which involves the bypassing of uORF start codons by the scanning 40S ribosome, and second, re-initiation (REI), which allows the 40S subunit of the ribosome that terminated on the uORF stop codon to resume scanning to reach a downstream start codon. REI has remained particularly enigmatic, as it is at odds with generally accepted principles of conventional translation initiation, termination and post-termination subunit recycling. MCTS1-DENR is the first re-initiation-specific factor that has been identified. Its homolog, EIF2D, is assumed to act in uORF REI as well, yet formal proof of EIF2D-mediated REI has remained scarce. In our study, we present our in vivo analyses of DENR and EIF2D in translation re-initiation in mammalian cells. At the transcriptome-wide scale, ribosome profiling clearly identifies DENR-dependent REI, yet disproves the assumption that EIF2D also functions as a REI-factor.
2024-10-30 | GSE263991 | GEO
Project description:mRNAs bound by ribosomes from yeast cells were analysed in order to determine the exact position of ribosomes in the presence or absence of Rio1p. Beside total Ribosome Protected Fragments (RPFs), RPFs from mRNAs protected by immature pre-40S pre-ribosomes was also analysed. The analysis showed that immature 40S ribosomes can carry out translation and their premature entry into translation is hindered by Rio1p.
2017-08-16 | GSE98333 | GEO
Project description:Terminal oligopyrimidine motif-containing (TOP) mRNAs encode all ribosomal proteins in mammals and are regulated to tune ribosome synthesis to cell state. Previous studies implicate LARP1 in 40S- or 80S-ribosome complexes that repress and stabilize TOP mRNAs. However, a mechanistic understanding of how LARP1 and TOP mRNAs interact with ribosomes to coordinate TOP mRNA outcomes is lacking. Here, we show that LARP1 senses the cellular supply of ribosomes by directly binding non-translating 80S ribosomes. Cryo-EM structures reveal a previously uncharacterized domain of LARP1 bound to and occluding the 40S mRNA channel and mutations at the LARP1-ribosome interface block formation of the 40S/80S-LARP1-TOP complexes. Free cytosolic ribosomes induce sequestration of TOP mRNAs in repressed 80S-LARP1-TOP complexes independent of alterations in mTOR signaling. Together, this work demonstrates a ribosome-sensing function of LARP1 that allows it to tune ribosome protein synthesis to the availability of free ribosomes.
2023-10-28 | GSE246077 | GEO
Project description:Upstream open reading frames (uORFs) initiate translation within mRNA 5’ leaders,and have the potential to altermain coding sequence(CDS) translationontranscripts in which they reside. Ribosome profiling(RP)studies suggest that translating ribosomes are pervasive within 5’ leadersacross model systems. However, the significance of this observationremains unclear. To explore a role for uORF usage in neuronal differentiation, we performed RP on undifferentiated and differentiated human neuroblastoma cells. Using a spectral coherence algorithm (SPECtre),we identify4,954uORFsacross31%of all neuroblastoma transcripts. These uORFspredominantly utilize non-AUG initiationcodonsand exhibit translational efficiencies(TE)comparable to annotated coding regions. Usage of both AUG initiated uORFs and aconservedandconsistently translated subset of non-AUG initiated uORFs correlateswith repressed CDS translation. Ribosomal protein transcripts are enriched in uORFs, and select uORFs on such transcripts were validated for expression. Withneuronal differentiation, we observedan overall positive correlation between translational shifts in uORF/CDSpairs. However,a subset of transcripts exhibit inverse shifts in translation of uORF/CDSpairs. TheseuORFsare enriched in AUG initiation sites, non-overlapping, and shorterin length. Cumulatively, CDSs downstream of uORFs characterized by persistent translation show smaller shifts in TE withneuronal differentiation relative to CDSs without a predicted uORF, suggesting that fluctuations in CDS translation are buffered by uORF translation. In sum, this work provides insights into the dynamic relationshipsand potential regulatory functionsof uORF/CDS pairs in a model of neuronal differentiation.
2018-09-07 | GSE119615 | GEO
Project description:eIF3 is a multi-subunit complex thought to execute numerous functions in canonical translation initiation, including mRNA recruitment to the 40S ribosome, scanning for the start codon, and inhibition of 60S subunit joining 1–3. eIF3 was also found to interact with 40S and 60S ribosomal proteins and translation elongation factors 4, but a direct involvement in translation elongation has never been demonstrated. Using selective ribosome profiling, we made the unexpected observation that eIF3 remains bound to post-initiation 80S ribosomes, followed by release after translation of ~50 codons. Furthermore, eIF3 deficiency reduces early ribosomal elongation speed, particularly on mRNAs encoding proteins associated with membrane-associated functions, resulting in defective synthesis of their encoded proteins and abnormal mitochondrial and lysosomal physiology. Accordingly, heterozygous eIF3e+/- knockout mice accumulate giant mitochondria in skeletal muscle and show a progressive decline in muscle strength with age. Hence, in addition to its canonical role in translation initiation, eIF3 interacts with 80S ribosomes to enhance, at the level of early elongation, the synthesis of proteins with membrane-associated functions, an activity that is critical for normal muscle health.
2020-06-01 | GSE131074 | GEO
Project description:eIF3 is a multi-subunit complex thought to execute numerous functions in canonical translation initiation, including mRNA recruitment to the 40S ribosome, scanning for the start codon, and inhibition of 60S subunit joining 1–3. eIF3 was also found to interact with 40S and 60S ribosomal proteins and translation elongation factors 4, but a direct involvement in translation elongation has never been demonstrated. Using selective ribosome profiling, we made the unexpected observation that eIF3 remains bound to post-initiation 80S ribosomes, followed by release after translation of ~50 codons. Furthermore, eIF3 deficiency reduces early ribosomal elongation speed, particularly on mRNAs encoding proteins associated with membrane-associated functions, resulting in defective synthesis of their encoded proteins and abnormal mitochondrial and lysosomal physiology. Accordingly, heterozygous eIF3e+/- knockout mice accumulate giant mitochondria in skeletal muscle and show a progressive decline in muscle strength with age. Hence, in addition to its canonical role in translation initiation, eIF3 interacts with 80S ribosomes to enhance, at the level of early elongation, the synthesis of proteins with membrane-associated functions, an activity that is critical for normal muscle health.
2020-06-15 | GSE149697 | GEO
Project description:Purpose: Ribosome profiling and RNA-Seq were used to map the location and abundance of translating ribosomes on mouse heart and skeletal muscle transcripts. Methods: Tissue was rapidly harvested and snap-frozen to minimize bias to the pool of translating ribosomes. RNA was prepared from a single homogenate for each tissue so that starting RNA populations for both libraries were closely matched. Homogenates were not clarified before RNase digestion to avoid loss of ribosomes associated with large molecular weight complexes, and RNA-Seq libraries were prepared after rRNA subtraction to avoid positional loss of 5’ reads. Trimmed reads from 50 cycles of Illumina single-end sequencing were mapped onto a non-redundant set of 18,499 mouse protein-coding RefSeq transcripts from the nuclear genome. Results: Mapped sequence reads to myosin, actin and the giant protein titin together account for ~20% of the total mRNA-derived ribosome protected fragments (RPFs). We observed large-scale uniformity in the distribution of RPFs on the >30,000 codon titin open reading frame, from which we inferred an in vivo ribosome elongation error rate of ≤10-5. Ribosome footprints on Ttn mRNA also uncovered a novel 5’ UTR within a phylogenetically conserved intronic element that would produce ~2.35 mDa titin isoform that corresponds to the titin 'T2' band frequently described as a proteolytic artifact. Local translation efficiency across several >10 kb muscle mRNAs was also uniform, while their global translation efficiencies varied by ~20-fold suggesting initiation rate plays a major role in the translation efficiency of large mRNAs. Evidence for RPFs on 5’ UTRs was widespread with particular enrichment for ribosomes positioned at CUG codons. Comparison of global translation efficiency in cardiac and skeletal muscle revealed novel examples of tissue-specific translational control including synthesis of the myogenic factor Mef2c, and the titin-binding stress response protein Ankrd23. Conclusions: Our study represents the first detailed analysis of translation in an adult mammalian tissue generated by ribosome profiling technology. Current limitations to using ribosomal profiling in tissues include unknown perturbations to the dynamic state of translation despite rapidly harvested and snap-frozen samples. The uniform 5’ to 3’ coverage observed on individual large mRNAs and the ability to observe footprints on the extremely small phospholamban coding sequence, suggests that initiation and elongation were halted on similar time scales. More detailed examination of the positional information within CDS region requires further understanding of the bias introduced during the library preparation steps for both RPF-and RNA-Seq, as well as local biases induced as translation is arrested. Despite these qualifications, this initial view of active translation in muscle tissue highlights the potential for ribosome profiling to monitor the dynamic translation response to exercise, injury or disease pathology in animal models at a level of resolution not easily attainable with other quantitative approaches. Heart and skeletal muscle ribosome-protected fragment and RNA-Seq profiles of 10-week old C57BL/6J male mice were generated by deep sequencing using the Illumina HiSeq 2000.
2015-09-01 | E-GEOD-41246 | biostudies-arrayexpress