Most protein-coding sequences (CDSs) are predicted sequences based on criteria such as a size sufficient to encode a product of at least 100 amino acids and with translation starting at an AUG ...initiation codon. However, recent studies based on ribosome profiling and mass spectrometry have shown that several RNAs annotated as long as noncoding RNAs are actually translated to generate polypeptides of fewer than 100 amino acids and that many proteins are translated from near-cognate initiation codons such as CUG and GUG. Furthermore, studies of genetically engineered mouse models have revealed that such polypeptides and proteins contribute to diverse physiological processes. In this review, we describe the latest methods for the identification of unannotated CDSs and provide examples of their physiological functions.
Although several long noncoding RNAs (lncRNAs) have recently been shown to encode small polypeptides, those in testis remain largely uncharacterized. Here we identify two sperm-specific polypeptides, ...Kastor and Polluks, encoded by a single mouse locus (Gm9999) previously annotated as encoding a lncRNA. Both Kastor and Polluks are inserted in the outer mitochondrial membrane and directly interact with voltage-dependent anion channel (VDAC), despite their different amino acid sequences. Male VDAC3-deficient mice are infertile as a result of reduced sperm motility due to an abnormal mitochondrial sheath in spermatozoa, and deficiency of both Kastor and Polluks also severely impaired male fertility in association with formation of a similarly abnormal mitochondrial sheath. Spermatozoa lacking either Kastor or Polluks partially recapitulate the phenotype of those lacking both. Cooperative function of Kastor and Polluks in regulation of VDAC3 may thus be essential for mitochondrial sheath formation in spermatozoa and for male fertility.
Hepatic iron overload is a risk factor for progression of hepatocellular carcinoma (HCC), although the molecular mechanisms underlying this association have remained unclear. We now show that the ...iron-sensing ubiquitin ligase FBXL5 is a previously unrecognized oncosuppressor in liver carcinogenesis in mice. Hepatocellular iron overload elicited by FBXL5 ablation gave rise to oxidative stress, tissue damage, inflammation, and compensatory proliferation of hepatocytes and to consequent promotion of liver carcinogenesis induced by exposure to a chemical carcinogen. The tumor-promoting outcome of FBXL5 deficiency in the liver was also found to be effective in a model of virus-induced HCC. FBXL5-deficient mice thus constitute the first genetically engineered mouse model of liver carcinogenesis promoted by iron overload. In addition, dysregulation of FBXL5-mediated cellular iron homeostasis was found to be associated with poor prognosis in human HCC, suggesting that FBXL5 plays a key role in defense against hepatocarcinogenesis.
Abstract
Although ribosome-profiling and translation initiation sequencing (TI-seq) analyses have identified many noncanonical initiation codons, the precise detection of translation initiation sites ...(TISs) remains a challenge, mainly because of experimental artifacts of such analyses. Here, we describe a new method, TISCA (TIS detection by translation Complex Analysis), for the accurate identification of TISs. TISCA proved to be more reliable for TIS detection compared with existing tools, and it identified a substantial number of near-cognate codons in Kozak-like sequence contexts. Analysis of proteomics data revealed the presence of methionine at the NH2-terminus of most proteins derived from near-cognate initiation codons. Although eukaryotic initiation factor 2 (eIF2), eIF2A and eIF2D have previously been shown to contribute to translation initiation at near-cognate codons, we found that most noncanonical initiation events are most probably dependent on eIF2, consistent with the initial amino acid being methionine. Comprehensive identification of TISs by TISCA should facilitate characterization of the mechanism of noncanonical initiation.
Graphical Abstract
Graphical Abstract
TISCA reliably identifies translation initiation sites (TISs) and provides insight into the mechanism of translation initiation at near-cognate codons. TISCA efficiently reduces the background noise in GTI-seq analysis. The absence and nonrelease of eIF3-seq footprints are mostly eliminated as noise. Red and blue circles show the 5′ end of a GTI-seq peak and an eIF3 release point, respectively. TIS candidates are located at +11 to +13 nt from the GTI-seq peak and at −22 to −26 nt from the eIF3 release point (top). TISCA identifies a substantial number of near-cognate initiation codons, most of which are translated in a manner dependent on eIF2–Met-tRNAi (bottom).
Ribosomes polymerize nascent peptides through repeated inter-subunit rearrangements between the classic and hybrid states. The peptidyl-tRNA, the intermediate species during translation elongation, ...stabilizes the translating ribosome to ensure robust continuity of elongation. However, the translation of acidic residue-rich sequences destabilizes the ribosome, leading to a stochastic premature translation cessation termed intrinsic ribosome destabilization (IRD), which is still ill-defined. Here, we dissect the molecular mechanisms underlying IRD in Escherichia coli. Reconstitution of the IRD event reveals that (1) the prolonged ribosome stalling enhances IRD-mediated translation discontinuation, (2) IRD depends on temperature, (3) the destabilized 70S ribosome complex is not necessarily split, and (4) the destabilized ribosome is subjected to peptidyl-tRNA hydrolase-mediated hydrolysis of the peptidyl-tRNA without subunit splitting or recycling factors-mediated subunit splitting. Collectively, our data indicate that the translation of acidic-rich sequences alters the conformation of the 70S ribosome to an aberrant state that allows the noncanonical premature termination.
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•Temperature and elongation stalling affect the intrinsic destabilization of the ribosome•Intrinsically destabilized ribosomes are not necessarily split into subunits•Pth hydrolyzes the peptidyl-tRNA within the destabilized 70S without ribosome splitting•Ribosome recycling factors can split the destabilized 70S ribosome with peptidyl-tRNA
Chadani et al. characterize the molecular determinants of acidic-rich nascent chain-induced intrinsic ribosome destabilization (IRD) in Escherichia coli. IRD is affected by temperature, elongation stalling, and translation factors. IRD prematurely terminates translation through two distinct mechanisms: either Pth-mediated hydrolysis of peptidyl-tRNA within 70S complex or splitting facilitated by recycling factors.
Although several ribosomal protein paralogs are expressed in a tissue-specific manner, how these proteins affect translation and why they are required only in certain tissues have remained unclear. ...Here we show that RPL3L, a paralog of RPL3 specifically expressed in heart and skeletal muscle, influences translation elongation dynamics. Deficiency of RPL3L-containing ribosomes in RPL3L knockout male mice resulted in impaired cardiac contractility. Ribosome occupancy at mRNA codons was found to be altered in the RPL3L-deficient heart, and the changes were negatively correlated with those observed in myoblasts overexpressing RPL3L. RPL3L-containing ribosomes were less prone to collisions compared with RPL3-containing canonical ribosomes. Although the loss of RPL3L-containing ribosomes altered translation elongation dynamics for the entire transcriptome, its effects were most pronounced for transcripts related to cardiac muscle contraction and dilated cardiomyopathy, with the abundance of the encoded proteins being correspondingly decreased. Our results provide further insight into the mechanisms and physiological relevance of tissue-specific translational regulation.
Although long noncoding RNAs (lncRNAs) are transcripts that do not encode proteins by definition, some lncRNAs actually contain small open reading frames that are translated. TINCR (terminal ...differentiation–induced ncRNA) has been recognized as a lncRNA that contributes to keratinocyte differentiation. However, we here show that TINCR encodes a ubiquitin-like protein that is well conserved among species and whose expression was confirmed by the generation of mice harboring a FLAG epitope tag sequence in the endogenous open reading frame as well as by targeted proteomics. Forced expression of this protein promoted cell cycle progression in normal human epidermal keratinocytes, and mice lacking this protein manifested a delay in skin wound healing associated with attenuated cell cycle progression in keratinocytes. We termed this protein TINCR-encoded ubiquitin-like protein (TUBL), and our results reveal a role for TINCR in the regulation of keratinocyte proliferation and skin regeneration that is dependent on TUBL.
The CHD (chromodomain helicase DNA binding protein) family consists of nine chromatin remodeling factors that alter chromatin structure in an ATP‐dependent manner. CHD4 contributes to the regulation ...of various cellular activities and processes including development through interaction with multiple proteins including formation of the NuRD (nucleosome remodeling and deacetylase activity) complex. Functions of CHD4 that appear not to be mediated by the NuRD complex or other known interactors have also been identified, however, suggesting the existence of unrecognized proteins that also associate with CHD4. We here generated HeLa‐S3 and HEK293T cells with a knock‐in allele for FLAG epitope‐tagged CHD4 and used these cells to identify proteins that bind to CHD4 with the use of immunoprecipitation followed by liquid chromatography and tandem mass spectrometry. LCORL (ligand‐dependent nuclear receptor corepressor like) and NOL4L (nucleolar protein 4 like) were reproducibly identified as novel CHD4 interactors. Furthermore, RNA‐sequencing analysis of HEK293T cells depleted of CHD4, LCORL, or NOL4L revealed consistent up‐regulation of genes related to the Notch signaling pathway. Our results thus suggest that both LCORL and NOL4L may cooperate with CHD4 to suppress the Notch pathway in mammalian cells.
A proteomics approach identified new CHD4 interactors, LCORL (ligand‐dependent nuclear receptor corepressor like) and NOL4L (nucleolar protein 4 like). Both LCORL and NOL4L may cooperate with CHD4 to suppress the Notch pathway in mammalian cells.
Translation initiates when the eIF4F complex binds the 5′ mRNA cap, followed by 5′ untranslated region scanning for the start codon by scanning ribosomes. Here, we demonstrate that the ASC‐1 complex ...(ASCC), which was previously shown to promote the dissociation of colliding 80S ribosomes, associates with scanning ribosomes to regulate translation initiation. Selective translation complex profiling (TCP‐seq) analysis revealed that ASCC3, a helicase domain‐containing subunit of ASCC, localizes predominantly to the 5′ untranslated region of mRNAs. Ribo‐seq, TCP‐seq, and luciferase reporter analyses showed that ASCC3 knockdown impairs 43S preinitiation complex loading and scanning dynamics, thereby reducing translation efficiency. Whereas eIF4A, an RNA helicase in the eIF4F complex, is important for global translation, ASCC was found to regulate the scanning process for a specific subset of transcripts. Our results have thus revealed that ASCC is required not only for dissociation of colliding 80S ribosomes but also for efficient translation initiation by scanning ribosomes at a subset of transcripts.
Synopsis
The ASC‐1 complex (ASCC) was shown to dissociate colliding 80S ribosomes. This study shows that ASCC also associates with scanning ribosomes, which is required for efficient translation initiation by scanning ribosomes at a subset of transcripts.
ASCC associates with the scanning ribosomes.
ASCC localizes predominantly to the 5′ untranslated region of mRNAs.
Loss of ASCC3 impairs preinitiation complex loading and scanning dynamics and thereby reduces translation efficiency.
Whereas eIF4A is important for global translation, ASCC regulates the scanning process for a subset of transcripts.
Different from RNA helicase eIF4A that globally controls translation initiation, the ASC‐1 complex regulates start codon scanning for a specific subset of transcripts.
Background
Recently, two molecular subtypes of pancreatic ductal adenocarcinoma (PDAC) have been proposed: the “Classical” and “Basal-like” subtypes, with the former showing better clinical outcomes ...than the latter. However, the “molecular” classification has not been applied in real-world clinical practice. This study aimed to establish patient-derived organoids (PDOs) for PDAC and evaluate their application in subtype classification and clinical outcome prediction.
Methods
We utilized tumor samples acquired through endoscopic ultrasound-guided fine-needle biopsy and established a PDO library for subsequent use in morphological assessments, RNA-seq analyses, and in vitro drug response assays. We also conducted a prospective clinical study to evaluate whether analysis using PDOs can predict treatment response and prognosis.
Results
PDOs of PDAC were established at a high efficiency (> 70%) with at least 100,000 live cells. Morphologically, PDOs were classified as gland-like structures (GL type) and densely proliferating inside (DP type) less than 2 weeks after tissue sampling. RNA-seq analysis revealed that the “morphological” subtype (GL vs. DP) corresponded to the “molecular” subtype (“Classical” vs. “Basal-like”). The “morphological” classification predicted the clinical treatment response and prognosis; the median overall survival of patients with GL type was significantly longer than that with DP type (
P
< 0.005). The GL type showed a better response to gemcitabine than the DP type in vitro, whereas the drug response of the DP type was improved by the combination of ERK inhibitor and chloroquine.
Conclusions
PDAC PDOs help in subtype determination and clinical outcome prediction, thereby facilitating the bench-to-bedside precision medicine for PDAC.
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