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TransCirc (https://www.biosino.org/transcirc/) is a specialized database that provide comprehensive evidences supporting the translation potential of circular RNAs (circRNAs). This database was generated by integrating various direct and indirect evidences to predict coding potential of each human circRNA and the putative translation products. Seven types of evidences for circRNA translation were included: (i) ribosome/polysome binding evidences supporting the occupancy of ribosomes onto circRNAs; (ii) experimentally mapped translation initiation sites on circRNAs; (iii) internal ribosome entry site on circRNAs; (iv) published N-6-methyladenosine modification data in circRNA that promote translation initiation; (v) lengths of the circRNA specific open reading frames; (vi) sequence composition scores from a machine learning prediction of all potential open reading frames; (vii) mass spectrometry data that directly support the circRNA encoded peptides across back-splice junctions. TransCirc provides a user-friendly searching/browsing interface and independent lines of evidences to predicte how likely a circRNA can be translated. In addition, several flexible tools have been developed to aid retrieval and analysis of the data. TransCirc can serve as an important resource for investigating the translation capacity of circRNAs and the potential circRNA-encoded peptides, and can be expanded to include new evidences or additional species in the future.

The generalized process of mRNA decay involves deadenylation followed by release from translating polysomes, decapping, and exonuclease decay of the mRNA body. In contrast the mRNA endonuclease PMR1 forms a selective complex with its translating substrate mRNA, where it initiates decay by cleaving within the mRNA body. In stressed cells the phosphorylation of the subunit of eukaryotic initiation factor 2 causes translating mRNAs to accumulate with stalled 48S subunits in large subcellular structures termed stress granules (SGs), wherein mRNAs undergo sorting for reinitiation, storage, or decay. Given the unique relationship between translation and PMR1-mediated mRNA decay, we examined the impact of stress-induced dissociation of polysomes on this process. Arsenite stress disrupts the polysome binding of PMR1 and its substrate mRNA but has no impact on the critical tyrosine phosphorylation of PMR1, its association with substrate mRNA, or its association with the functional 680-kDa mRNP complex in which it normally resides on polysomes. We show that arsenite stress drives PMR1 into an RNase-resistant complex with TIA-1, and we identify a distinct domain in the N terminus of PMR1 that facilitates its interaction with TIA-1. Finally, we show that arsenite promotes the delayed association of PMR1 with SGs under conditions which cause tristetraprolin and butyrate response factor 1, proteins that facilitate exonucleolytic mRNA, to exit SGs. Translation and mRNA decay are intimately linked processes, and the identification of cytoplasmic foci containing enzymes involved in mRNA decay in yeast (29) suggested that a dynamic process partitions these two key steps in posttranscriptional control into distinct physical complexes. These cytoplasmic foci, termed “processing bodies” (P-bodies) (PBs), were originally thought to function only as sites of mRNA

Epidermal growth factor receptor (EGFR) and tumour growth factor alpha (TGFalpha) are frequently overexpressed in renal cell carcinoma (RCC) yet responses to single-agent EGFR inhibitors are uncommon. Although von Hippel-Lindau (VHL) mutations are predominant, RCC also develops in individuals with tuberous sclerosis (TSC). Tuberous sclerosis mutations activate mammalian target of rapamycin (mTOR) and biochemically resemble VHL alterations. We found that RCC cell lines expressed EGFR mRNA in the near-absence of other ErbB family members. Combined EGFR and mTOR inhibition synergistically impaired growth in a VHL-dependent manner. Iressa blocked ERK1/2 phosphorylation specifically in wt-VHL cells, whereas rapamycin inhibited phospho-RPS6 and 4E-BP1 irrespective of VHL. In contrast, phospho-AKT was resistant to these agents and MYC translation initiation (polysome binding) was similarly unaffected unless AKT was inhibited. Primary RCCs vs cell lines contained similar amounts of phospho-ERK1/2, much higher levels of ErbB-3, less phospho-AKT, and no evidence of phospho-RPS6, suggesting that mTOR activity was reduced. A subset of tumours and cell lines expressed elevated eIF4E in the absence of upstream activation. Despite similar amounts of EGFR mRNA, cell lines (vs tumours) overexpressed EGFR protein. In the paired cell lines, PRC3 and WT8, EGFR protein was elevated post-transcriptionally in the VHL mutant and EGF-stimulated phosphorylation was prolonged. We propose that combined EGFR and mTOR inhibitors may be useful in the subset of RCCs with wt-VHL. However, apparent differences between primary tumours and cell lines require further investigation.

Prolonged translation arrest in post-ischemic hippocampal CA1 pyramidal neurons precludes translation of induced stress genes and directly correlates with cell death. We evaluated the regulation of mRNAs containing adenine- and uridine-rich elements (ARE) by assessing HuR protein and hsp70 mRNA nuclear translocation, HuR polysome binding, and translation state analysis of CA1 and CA3 at 8 h of reperfusion after 10 min of global cerebral ischemia. There was no difference between CA1 and CA3 at 8 h of reperfusion in nuclear or cytoplasmic HuR protein or hsp70 mRNA, or HuR polysome association, suggesting that neither mechanism contributed to post-ischemic outcome. Translation state analysis revealed that 28 and 58 % of unique mRNAs significantly different between 8hR and NIC, in CA3 and CA1, respectively, were not polysome-bound. There was significantly greater diversity of polysome-bound mRNAs in reperfused CA3 compared to CA1, and in both regions, ARE-containing mRNAs accounted for 4-5 % of the total. These data indicate that posttranscriptional ARE-containing mRNA regulation occurs in reperfused neurons and contributes to post-ischemic outcome. Understanding the differential responses of vulnerable and resistant neurons to ischemia will contribute to the development of effective neuroprotective therapies.

One of the principal functions of the actin cytoskeleton is to serve as a support for diverse physiological and biochemical processes, protein synthesis in particular. About 25 years ago, in experiments with cultured animal cells, it was discovered that most mRNAs, ribosomes, and protein synthesis factors were attached to the actin microfilaments (Lenk et al., 1977). Later, a similar phenomenon was demonstrated for plant tissues (Davis et al., 1991; Zak et al., 1995). The physiological basis for such an attachment is evident in some cases but obscure in others. Attachment of polysomes to the microfilaments might provide for the correct spatial arrangement of the translational machinery and protein synthesis activation. If the binding of polysomes to the cytoskeleton is considered an additional level of protein synthesis regulation, its physiological control is expected but is not shown, at least for plant cells. To evaluate the proportion of the cytoskeleton-bound polysomes (CBP) in the total polysome population, we used the differential sensitivity of membranes and cytoskeleton to nonionic detergents that destroy membranes but leave microfilaments intact. A difference in the polysome peak areas in sucrose density gradients of plant tissue homogenates treated and nontreated with Triton X-100 was a rough measure for the amount of CBP (stretched areas in Fig. 2). * Corresponding author. Tel.: +7-95-432-0233; fax: +7-95-977-8018.

The mycotoxin rubratoxin B binds to some component(s) of the microsomal fraction of mouse liver. The α-β unsaturated lactone ring was a structural requirement for binding. The rubratoxin B-microsomal adduct was not separated by Sephadex G-25 filtration. The binding occurred rapidly and persisted for at least 2 hr. Sulfhydryl reagents, dithiothreitol and cysteine partially protected from and/or reversed binding. Rubratoxin B inhibited the in vitro activity of an enzyme located in the endoplasmic reticulum associated with binding of the ribosomes to the membrane.

The putative role of membrane-bound sialic acids in the attachment of polysomes to the membranes of the rough endoplasmic reticulum (T. Scott-Burden and A. O. Hawtrey, 1973, Biochem. Biophys. Res. Commun. 54 , 1288–1295) was reinvestigated. In contrast with the previous report, it was found that although treatment of the stripped membranes with bacterial neuraminidase caused a release of 2.5 nmol of N -acetylneuraminic acid/mg of membrane protein, their capacity to reattach polysomes was not affected. Following incubation of the neuraminidase-treated membranes with labeled N -acetylneuraminic acid, nucleotide triphosphates, and an ATP-generating system, no incorporation of sialic acid into the membranes was observed. Thus, the increase in polysome binding reported by the above investigators following this treatment cannot be attributed to membrane resialylation. In all probability, the Mn 2+ present in the sialylating system was responsible for their finding. In fact, about threefold increases in the polysome-binding capacity of ribosomefree rough endoplasmic reticulum membranes were observed in the presence of 5 m m Mn 2+ . Phospholipase C and Ca 2+ , common contaminants of some commercial preparations of Clostridium perfringens neuraminidase, could conceivably be responsible for the effects observed by Scott-Burden and Hawtrey. This possible explanation is supported by the marked reduction of polysome-binding capacity observed after incubation of the membranes with phospholipase C from C. perfringens in the presence of Ca 2+ . These studies show that neuraminidase-susceptible membrane-bound sialic acids do not play a role in the attachment of polysomes to the membranes of the rough endoplasmic reticulum and provide novel insights into this complex phenomenon.

Translocation-competent microsomal membrane vesicles of dog pancreas were shown to selectively bind nascent, in vitro assembled polysomes synthesizing secretory protein (bovine prolactin) but not those synthesizing cytoplasmic protein (alpha and beta chain of rabbit globin). This selective polysome binding capacity was abolished when the microsomal vesicles were salt-extracted but was restored by an 11S protein (SRP, Signal Recognition Protein) previously purified from the salt-extract of microsomal vesicles (Walter and Blobel, 1980. Proc. Natl. Acad. Sci. U. S. A. 77:7112-7116). SRP-dependent polysome recognition and binding to the microsomal membrane was shown to be a prerequisite for chain translocation. Modification of SRP by N-ethyl maleimide abolished its ability to mediate nascent polysome binding to the microsomal vesicles. Likewise, polysome binding to the microsomal membrane was largely abolished when beta-hydroxy leucine, a Leu analogue, was incorporated into nascent secretory polypeptides. The data in this and the preceding paper provide conclusive experimental evidence that chain translocation across the endoplasmic reticulum membrane is a receptor-mediated event and thus rule out proposals that chain translocation occurs spontaneously and without the mediation by proteins. Moreover, our data here demonstrate conclusively that the initial events that lead to translocation and provide for its specificity are protein-protein (signal sequence plus ribosome with SRP) and not protein-lipid (signal sequence with lipid bilayer) interactions.

Summary Cytoplasmic extracts from female rat liver were incubated with 14 C-labeled free polysomes and the binding capacity of the membrane therein was assayed by CsC1 density gradient centrifugation after fixing the complex in formaldehyde. Phenobarbital treatment in vivo increased the binding capacity, while hepatectomy decreased it. Addition of ethanol in vitro into this system enhanced the binding capacity of the cytoplasmic extract from normal and phenobarbital treated rat liver but not from regenerating liver. The stimulation of binding by ethanol was inhibited by estradiol in this female system. Testosterone had no effect.

Growing yeast spheroplasts were shown to have, on the average, four times the number of cytoplasmic ribosomes in contact with the outer mitochondrial membrane compared to starved spheroplasts. Ribosomes in contact with mitochondria in the growing spheroplast preparation, like free cytoplasmic ribosomes, exist primarily as polysome structures. In the starved spheroplast preparation, both mitochondria-bound and free cytoplasmic ribosomes exist primarily as monosomes. Mitochondria isolated from growing spheroplasts in a medium containing lmM Mg++ have cytoplasmic ribosomes bound directly to the outer membrane. These ribosomes can be quantitatively removed by washing the mitochondria with 2 mM EDTA. Mitochondria from starved spheroplasts are capable of accepting either free cytoplasmic polysomes or cytoplasmic polysomes extracted from mitochondria. However, the extent of polysome binding to mitochondria was shown to be a direct function of the Mg++ concentration; a smaller percentage of the input polysomes bind as the Mg++ concentration is lowered. At 1 mM Mg++, neither free cytoplasmic nor mitochondria-bound polysomes bind to mitochondria. Nevertheless, when growing spheroplasts are broken and mitochondria isolated in medium containing 1 mM Mg++, the mitochondria are seen to have cytoplasmic ribosomes firmly attached to the outer membrane. This result, in addition to our earlier data (Kellems, R. E., and R. A. Butow. 1974. J. Biol. Chem. 249:3304-3310), support the view that cytoplasmic ribosomes attached to the outer membrane of purified mitochondria were attached in vivo. In preparations of mitochondria isolated from growing spheroplasts, ribosomes appear to be found to specific regions of the outer membrane, namely those regions which are in close association or in contact with the inner mitochondrial membrane. This is particularly evident with mitochondria in a condensed configuration. This finding suggests a mechanism whereby cytoplasmically synthesized mitochondrial protein could be transferred by a process of vectorial translation across both membranes of the organelle.

One method of destroying polysome binding sites which is reversible is mild-trypsin digestion. Unlike the original untreated sites, the trypsin altered sites when complexed with polysomes are unable to direct transfer of puromycin-discharged polypeptide to the membrane vesicle as can sites on native membranes.