Regulatory Role In Translation Research Articles

Visualizing the translation landscape in human cells at high resolution.

Obtaining comprehensive structural descriptions of macromolecules within their natural cellular context holds immense potential for understanding fundamental biology and improving health. Here, we present the landscape of protein synthesis inside human cells in unprecedented detail obtained using an approach which combines automated cryo-focused ion beam (FIB) milling and in situ single-particle cryo-electron microscopy (cryo-EM). With this in situ cryo-EM approach we resolved a 2.19 Å consensus structure of the human 80S ribosome and unveiled its 21 distinct functional states, nearly all higher than 3 Å resolution. In contrast to in vitro studies, we identified protein factors, including SERBP1, EDF1 and NAC/3, not enriched on purified ribosomes. Most strikingly, we observed that SERBP1 binds to the ribosome in almost all translating and non-translating states to bridge the 60S and 40S ribosomal subunits. These newly observed binding sites suggest that SERBP1 may serve an important regulatory role in translation. We also uncovered a detailed interface between adjacent translating ribosomes which can form the helical polysome structure. Finally, we resolved high-resolution structures from cells treated with homoharringtonine and cycloheximide, and identified numerous polyamines bound to the ribosome, including a spermidine that interacts with cycloheximide bound at the E site of the ribosome, underscoring the importance of high-resolution in situ studies in the complex native environment. Collectively, our work represents a significant advancement in detailed structural studies within cellular contexts.

Schlafen14 Impairs HIV-1 Expression in a Codon Usage-Dependent Manner.

Schlafen (SLFN) is a family of proteins upregulated by type I interferons with a regulatory role in translation. Intriguingly, SLFN14 associates with the ribosome and can degrade rRNA, tRNA, and mRNA in vitro, but a role in translation is still unknown. Ribosomes are important regulatory hubs during translation elongation of mRNAs rich in rare codons. Therefore, we evaluated the potential role of SLFN14 in the expression of mRNAs enriched in rare codons, using HIV-1 genes as a model. We found that, in a variety of cell types, including primary immune cells, SLFN14 regulates the expression of HIV-1 and non-viral genes based on their codon adaptation index, a measurement of the synonymous codon usage bias; consequently, SLFN14 inhibits the replication of HIV-1. The potent inhibitory effect of SLFN14 on the expression of the rare codon-rich transcript HIV-1 Gag was minimized by codon optimization. Mechanistically, we found that the endoribonuclease activity of SLFN14 is required, and that ribosomal RNA degradation is involved. Therefore, we propose that SLFN14 impairs the expression of HIV-1 transcripts rich in rare codons, in a catalytic-dependent manner.

Glucose-driven TOR-FIE-PRC2 signalling controls plant development.

Nutrients and energy have emerged as central modulators of developmental programmes in plants and animals1-3. The evolutionarily conserved target of rapamycin (TOR) kinase is a master integrator of nutrient and energy signalling that controls growth. Despite its key regulatory roles in translation, proliferation, metabolism and autophagy2-5, little is known about how TOR shapes developmental transitions and differentiation. Here we show that glucose-activated TOR kinase controls genome-wide histone H3 trimethylation at K27 (H3K27me3) in Arabidopsis thaliana, which regulates cell fate and development6-10. We identify FERTILIZATION-INDEPENDENT ENDOSPERM (FIE), an indispensable component of Polycomb repressive complex 2 (PRC2), which catalyses H3K27me3(refs.6-8,10-12), as a TOR target. Direct phosphorylation by TOR promotes the dynamic translocation of FIE from the cytoplasm to the nucleus. Mutation of the phosphorylation site on FIE abrogates the global H3K27me3 landscape, reprogrammes the transcriptome and disrupts organogenesis in plants. Moreover, glucose-TOR-FIE-PRC2 signalling modulates vernalization-induced floral transition. We propose that this signalling axis serves as a nutritional checkpoint leading to epigenetic silencing of key transcription factor genes that specify stem cell destiny in shoot and root meristems and control leaf, flower and silique patterning, branching and vegetative-to-reproduction transition. Our findings reveal a fundamental mechanism of nutrient signalling in direct epigenome reprogramming, with broad relevance for the developmental control of multicellular organisms.

Regulatory role of mitochondrial genes in the tenderisation of lamb meat during postmortem ageing

AbstractThe tenderness of mutton is one of the qualities of prime lamb meat. This study investigated the effects of postmortem time on different meat quality parameters and examined the transcriptome involved in the tenderisation of lamb meat during postmortem ageing. Results showed that the pH value and adenosine triphosphate (ATP) levels considerably decreased in the early stage (0–4 days) of postmortem ageing, whereas caspases‐3 activities increased (0–4 days). The pH value notably increased in the later stage (4–8 days), while ATP levels and caspases‐3 activities decreased (4–8 days). The transcriptional changes at the early stage of ageing (0–4 days) play important regulatory roles in translation and hydrogen transmembrane transport. Mitochondrial genes, ATP synthase F0 subunit 6, cytochrome c oxidase subunit I, cytochrome c oxidase subunit II, and cytochrome b were identified as the core genes associated with Tan lamb tenderness. In conclusion, mitochondrial genes most likely contribute to lamb meat tenderness as they affect the activation of caspase‐3.

Dynamic methylome of internal mRNA N7-methylguanosine and its regulatory role in translation.

Over 150 types of RNA modifications are identified in RNA molecules. Transcriptome profiling is one of the key steps in decoding the epitranscriptomic panorama of these chemical modifications and their potential functions. N7-methylguanosine (m7G) is one of the most abundant modifications present in tRNA, rRNA and mRNA 5'cap, and has critical roles in regulating RNA processing, metabolism and function. Besides its presence at the cap position in mRNAs, m7G is also identified in internal mRNA regions. However, its transcriptome-wide distribution and dynamic regulation within internal mRNA regions remain unknown. Here, we have established m7G individual-nucleotide-resolution cross-linking and immunoprecipitation with sequencing (m7G miCLIP-seq) to specifically detect internal mRNA m7G modification. Using this approach, we revealed that m7G is enriched at the 5'UTR region and AG-rich contexts, a feature that is well-conserved across different human/mouse cell lines and mouse tissues. Strikingly, the internal m7G modification is dynamically regulated under both H2O2 and heat shock treatments, with remarkable accumulations in the CDS and 3'UTR regions, and functions in promoting mRNA translation efficiency. Consistently, a PCNA 3'UTR minigene reporter harboring the native m7G modification site displays both enriched m7G modification and increased mRNA translation upon H2O2 treatment compared to the m7G site-mutated minigene reporter (G to A). Taken together, our findings unravel the dynamic profiles of internal mRNA m7G methylome and highlight m7G as a novel epitranscriptomic marker with regulatory roles in translation.

Roles of Long Noncoding RNAs and Circular RNAs in Translation.

Most of the eukaryotic genome is pervasively transcribed, yielding hundreds to thousands of long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs), some of which are well conserved during evolution. Functions have been described for a few lncRNAs and circRNAs but remain elusive for most. Both classes of RNAs play regulatory roles in translation by interacting with messenger RNAs (mRNAs), microRNAs (miRNAs), or mRNA-binding proteins (RBPs), thereby modulating translation in trans Moreover, although initially defined as noncoding, a number of lncRNAs and circRNAs have recently been reported to contain functional open reading frames (ORFs). Here, we review current understanding of the roles played by lncRNAs and circRNAs in protein synthesis and discuss challenges and open questions in the field.

Evolutionary analysis of polyproline motifs in Escherichia coli reveals their regulatory role in translation.

Translation of consecutive prolines causes ribosome stalling, which is alleviated but cannot be fully compensated by the elongation factor P. However, the presence of polyproline motifs in about one third of the E. coli proteins underlines their potential functional importance, which remains largely unexplored. We conducted an evolutionary analysis of polyproline motifs in the proteomes of 43 E. coli strains and found evidence of evolutionary selection against translational stalling, which is especially pronounced in proteins with high translational efficiency. Against the overall trend of polyproline motif loss in evolution, we observed their enrichment in the vicinity of translational start sites, in the inter-domain regions of multi-domain proteins, and downstream of transmembrane helices. Our analysis demonstrates that the time gain caused by ribosome pausing at polyproline motifs might be advantageous in protein regions bracketing domains and transmembrane helices. Polyproline motifs might therefore be crucial for co-translational folding and membrane insertion.

Unique localization of the plastid-specific ribosomal proteins in the chloroplast ribosome small subunit provides mechanistic insights into the chloroplastic translation

Chloroplastic translation is mediated by a bacterial-type 70S chloroplast ribosome. During the evolution, chloroplast ribosomes have acquired five plastid-specific ribosomal proteins or PSRPs (cS22, cS23, bTHXc, cL37 and cL38) which have been suggested to play important regulatory roles in translation. However, their exact locations on the chloroplast ribosome remain elusive due to lack of a high-resolution structure, hindering our progress to understand their possible roles. Here we present a cryo-EM structure of the 70S chloroplast ribosome from spinach resolved to 3.4 Å and focus our discussion mainly on the architecture of the 30S small subunit (SSU) which is resolved to 3.7 Å. cS22 localizes at the SSU foot where it seems to compensate for the deletions in 16S rRNA. The mRNA exit site is highly remodeled due to the presence of cS23 suggesting an alternative mode of translation initiation. bTHXc is positioned at the SSU head and appears to stabilize the intersubunit bridge B1b during thermal fluctuations. The translation factor plastid pY binds to the SSU on the intersubunit side and interacts with the conserved nucleotide bases involved in decoding. Most of the intersubunit bridges are conserved compared to the bacteria, except for a new bridge involving uL2c and bS6c.

YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs.

Besides translation, transfer RNAs (tRNAs) play many non-canonical roles in various biological pathways and exhibit highly variable expression profiles. To unravel the emerging complexities of tRNA biology and molecular mechanisms underlying them, an efficient tRNA sequencing method is required. However, the rigid structure of tRNA has been presenting a challenge to the development of such methods. We report the development of Y-shaped Adapter-ligated MAture TRNA sequencing (YAMAT-seq), an efficient and convenient method for high-throughput sequencing of mature tRNAs. YAMAT-seq circumvents the issue of inefficient adapter ligation, a characteristic of conventional RNA sequencing methods for mature tRNAs, by employing the efficient and specific ligation of Y-shaped adapter to mature tRNAs using T4 RNA Ligase 2. Subsequent cDNA amplification and next-generation sequencing successfully yield numerous mature tRNA sequences. YAMAT-seq has high specificity for mature tRNAs and high sensitivity to detect most isoacceptors from minute amount of total RNA. Moreover, YAMAT-seq shows quantitative capability to estimate expression levels of mature tRNAs, and has high reproducibility and broad applicability for various cell lines. YAMAT-seq thus provides high-throughput technique for identifying tRNA profiles and their regulations in various transcriptomes, which could play important regulatory roles in translation and other biological processes.

Nascent Proteins Interact with Key Regions of the Outer Surface of the Ribosome

The initial stages of a protein's life in the cell are crucial for the proper balance between folding, misfolding and aggregation. While an appreciable amount of information is known about protein folding/unfolding after biosynthesis, we lack a clear understanding of how ribosome bound nascent chains (RNCs) acquire any degree of structure and dynamics. Previous studies in the Cavagnero group showed that RNCs of the intrinsically disordered protein PIR (i.e., the phosphorylated insulin receptor) and its highly negatively charged mutant PIR 1.2.3 exhibit significant spatial bias towards the ribosomal surface. We now present findings that pinpoint the presence of well-defined interactions between RNCs of PIR, PIR 1.2.3, as well as the foldable proteins bacterial flavohemoglobin apoHmpH and sperm whale apomyoglobin, with specific ribosomal proteins and the co-translationally active chaperone trigger factor (TF). RNC-ribosome contacts were probed by chemical crosslinking followed by mass spectrometry analysis, and nascent protein local and global dynamics were assessed by time-resolved fluorescence depolarization. Initial results indicate that under physiologically relevant conditions RNCs of both PIR and apoHmpH crosslink to TF in a strategic manner. In addition to interactions with TF, multiple populations of apoHmpH RNCs were observed to transiently bind to specific proteins located on the outer surface of the ribosome, both near and far from the exit tunnel. We are currently exploring the implications of these specific interactions on the balance between protein folding and aggregation. Interestingly, these interactions are strongly Mg2+ concentration-dependent. The solubility profile of apoHmpH upon release from the ribosome is not Mg2+ concentration-dependent, suggesting that the above interactions may play a yet-unidentified regulatory role in translation.

Toward a non-coding RNA revolution in cancer society

microRNAs (miRNAs) are a large family of small regulatory RNA molecules found in all multicellular organisms. Since their discovery in 2001, there has been impressive progress in miRNA research, and a great deal is now known about the biosynthesis of miRNAs and their regulatory role in translation. It is becoming increasingly clear that miRNAs have fundamental roles to play in cellular responses to xenobiotic stress, the development of pathophysiological changes and other toxicological phenomenon such as susceptibility and resistance. Furthermore, the expression of miRNAs, like many of the genes important in toxicology, can be regulated by xenobiotics and DNA methylation. In this article we review the present understanding of the miRNA field with particular reference to toxicology. We also give an insight into our current projects within this exciting area and highlight some of the new challenges that now face miRNA research.

Structure of the catalytic fragment of translation initiation factor 2B and identification of a critically important catalytic residue.

Eukaryotic initiation factor (eIF) 2B catalyzes the nucleotide activation of eIF2 to its active GTP-bound state. The exchange activity has been mapped to the C terminus of the eIF2Bepsilon subunit. We have determined the crystal structure of residues 544-704 from yeast eIF2Bepsilon at 2.3-A resolution, and this fragment is an all-helical protein built around the conserved aromatic acidic (AA) boxes also found in eIF4G and eIF5. The eight helices are organized in a manner similar to HEAT repeats. The molecule is highly asymmetric with respect to surface charge and conservation. One area in the N terminus is proposed to be directly involved in catalysis. In agreement with this hypothesis, mutation of glutamate 569 is shown to be lethal. An acidic belt and a second area in the C terminus containing residues from the AA boxes are important for binding to eIF2. Two mutations causing the fatal human genetic disease leukoencephalopathy with vanishing white matter are buried and appear to disrupt the structural integrity of the catalytic domain rather than interfering directly with catalysis or binding of eIF2.

Classification and evolution of P-loop GTPases and related ATPases

Sequences and available structures were compared for all the widely distributed representatives of the P-loop GTPases and GTPase-related proteins with the aim of constructing an evolutionary classification for this superclass of proteins and reconstructing the principal events in their evolution. The GTPase superclass can be divided into two large classes, each of which has a unique set of sequence and structural signatures (synapomorphies). The first class, designated TRAFAC (after translation factors) includes enzymes involved in translation (initiation, elongation, and release factors), signal transduction (in particular, the extended Ras-like family), cell motility, and intracellular transport. The second class, designated SIMIBI (after signal recognition particle, MinD, and BioD), consists of signal recognition particle (SRP) GTPases, the assemblage of MinD-like ATPases, which are involved in protein localization, chromosome partitioning, and membrane transport, and a group of metabolic enzymes with kinase or related phosphate transferase activity. These two classes together contain over 20 distinct families that are further subdivided into 57 subfamilies (ancient lineages) on the basis of conserved sequence motifs, shared structural features, and domain architectures. Ten subfamilies show a universal phyletic distribution compatible with presence in the last universal common ancestor of the extant life forms (LUCA). These include four translation factors, two OBG-like GTPases, the YawG/YlqF-like GTPases (these two subfamilies also consist of predicted translation factors), the two signal-recognition-associated GTPases, and the MRP subfamily of MinD-like ATPases. The distribution of nucleotide specificity among the proteins of the GTPase superclass indicates that the common ancestor of the entire superclass was a GTPase and that a secondary switch to ATPase activity has occurred on several independent occasions during evolution. The functions of most GTPases that are traceable to LUCA are associated with translation. However, in contrast to other superclasses of P-loop NTPases (RecA-F1/F0, AAA+, helicases, ABC), GTPases do not participate in NTP-dependent nucleic acid unwinding and reorganizing activities. Hence, we hypothesize that the ancestral GTPase was an enzyme with a generic regulatory role in translation, with subsequent diversification resulting in acquisition of diverse functions in transport, protein trafficking, and signaling. In addition to the classification of previously known families of GTPases and related ATPases, we introduce several previously undetected families and describe new functional predictions.

Regulation by heme of the synthesis of cytochrome c oxidase subunits V and VII in yeast

The effects of heme on the synthesis of subunits V and VII of cytochrome c oxidase were examined in the heme-deficient yeast mutant, GL1 In vitro translation and immunodetection with subunit-specific antisera indicated a 50% decrease in both proteins, with RNA obtained from hemeless cells. Unsupplemented, pulse-labeled cells contained both V and VII polypeptides, but at extremely low levels as compared with those found in δ-aminolevulinic acid-supplemented cells. The data suggest that heme controls the formation of mRNAs for the two subunits, and may also have a regulatory role in translation and in the stability of the polypeptides.