Nonsense-mediated messenger RNA decay (NMD) is triggered by premature translation termination, but the features distinguishing premature from normal termination are unknown. One model for NMD suggests that decay-inducing factors bound to mRNAs during early processing events are routinely removed by elongating ribosomes but remain associated with mRNAs when termination is premature, triggering rapid turnover. Recent experiments challenge this notion and suggest a model that posits that mRNA decay is activated by the intrinsically aberrant nature of premature termination. Here we use a primer extension inhibition (toeprinting) assay to delineate ribosome positioning and find that premature translation termination in yeast extracts is indeed aberrant. Ribosomes encountering premature UAA or UGA codons in the CAN1 mRNA fail to release and, instead, migrate to upstream AUGs. This anomaly depends on prior nonsense codon recognition and is eliminated in extracts derived from cells lacking the principal NMD factor, Upf1p, or by flanking the nonsense codon with a normal 3'-untranslated region (UTR). Tethered poly(A)-binding protein (Pab1p), used as a mimic of a normal 3'-UTR, recruits the termination factor Sup35p (eRF3) and stabilizes nonsense-containing mRNAs. These findings indicate that efficient termination and mRNA stability are dependent on a properly configured 3'-UTR.
Efficient translation initiation and optimal stability of most eukaryotic mRNAs depends on the formation of a closed loop structure and the resulting synergistic interplay between the 5′ m 7 G cap and the 3′ poly(A) tail 1,2 . Evidence of eIF4G and Pab1p interaction supports the notion of a closed loop mRNP 3 , but the mechanistic events that lead to its formation and maintenance are still unknown. Here we have used toeprinting and polysome profiling assays to delineate ribosome positioning at initiator AUG codons and ribosome:mRNA association, respectively, and find that two distinct stable (cap analog resistant) closed loop structures are formed during initiation in yeast cell-free extracts. The integrity of both forms requires the mRNA cap and poly(A) tail, as well as eIF4E, eIF4G, Pab1p, and eIF3, and is dependent on the length of both the mRNA and the poly(A) tail. Formation of the first structure requires the 48S ribosomal complex whereas the second requires an 80S ribosome and the termination factors eRF3/Sup35p and eRF1/Sup45p. Surprisingly, the involvement of the termination factors is independent of a termination event. Keywords Yeast; mRNA circularization; translation initiation; termination factorsIn vitro translation reactions utilized synthetic mRNAs derived from yeast transcripts, extracts that recapitulate cap/poly(A) tail synergy ( Supplementary Fig. 1a), competitive inhibition of translation initiation by m 7 GpppG (cap analog), and analyses of ribosome positioning or mRNA association by toeprinting and sucrose gradient sedimentation. Addition of the elongation inhibitor, cycloheximide (CHX), to translation reactions programmed by the 2135nt AAA and UAA mRNAs containing long or short ORFs, respectively, (Fig. 1a) allowed detection of CHX-dependent initiator AUG toeprints that reflect 80S ribosomes protecting 16-18 nt 3′ of the AUG 4-6 (Fig. 1b, top and middle panels, lanes 1 and 3). These toeprints were dependent on initiation codon recognition, the presence of yeast extract, and concurrent mRNA translation ( Supplementary Fig. 1b and c). Supporting the latter conclusion, toeprints were almost completely eliminated by 2.7mM cap analog, a concentration that distinguished bona fide toeprints from background bands (Fig. 1b, top and middle panels, lanes 2 and 4). Lower cap analog concentrations also inhibited AUG toeprint accumulation, with 70% and 96% sensitivity obtained at 0.05mM and 0.5mM, respectively (Fig. 1b, lower panel). A shorter mRNA (miniUAA1, 488nt, Fig. 1a) also yielded the AUG toeprint (Fig. 1c,upper panel, lane 1), but this band was resistant to 2.7mM cap analog (lane 2) and only manifested sensitivity at higher concentrations (Fig. 1c, lower panel). Thus, in wild-type extracts, the short capped (see Supplementary Fig. 1d) and polyadenylated miniUAA1 mRNA is ∼160-fold more resistant to cap analog than the longer AAA mRNA. The miniADE2 (485nt) and ADE2 (2070nt) mRNAs, whose respective sizes (but not sequences) are comparable to those of the miniUAA1 and AAA transcripts, also...
The inflammasome has emerged as an important molecular protein complex which initiates proteolytic processing of pro-IL-1β and IL-18 into mature inflammatory cytokines. In addition, inflammasomes initiate pyroptotic cell death that may be independent of those cytokines. Inflammasomes are central to elicit innate immune responses against many pathogens, and are key components in the induction of host defenses following bacterial infection. Here, we review recent discoveries related to NLRP1, NLRP3, NLRC4, NLRP6, NLRP7, NLRP12 and AIM2-mediated recognition of bacteria. Mechanisms for inflammasome activation and regulation are now suggested to involve kinases such as PKR and PKCδ, ligand binding proteins such as the NAIPs, and caspase-11 and caspase-8 in addition to caspase-1. Future research will determine how specific inflammasome components pair up in optimal responses to specific bacteria.
Background: Group B Streptococcus (GBS) activates the NLRP3 inflammasome. Results: GBS RNA escapes the phagolysosome and activates the NLRP3 inflammasome in a -hemolysin-dependent fashion. RNA-NLRP3 interaction and activation are enhanced by lysosomal leakage. Conclusion: RNA activates the NLRP3 inflammasome in synergy with phagolysosomal proteins. Significance: The NLRP3 inflammasome responds to bacterial invasion via RNA recognition subsequent to phagolysosomal degradation.
In addition to their well-documented roles in the promotion of nonsense-mediated mRNA decay (NMD), yeast Upf proteins (Upf1, Upf2/Nmd2, and Upf3) also manifest translational regulatory functions, at least in vitro, including roles in premature translation termination and subsequent reinitiation. Here, we find that all upfD strains also fail to reinitiate translation after encountering a premature termination codon (PTC) in vivo, a result that led us to seek a unifying mechanism for all of these translation phenomena. Comparisons of the in vitro translational activities of wild-type (WT) and upf1D extracts were utilized to test for a Upf1 role in post-termination ribosome reutilization. Relative to WT extracts, non-nucleased extracts lacking Upf1 had approximately twofold decreased activity for the translation of synthetic CAN1/LUC mRNA, a defect paralleled by fewer ribosomes per mRNA and reduced efficiency of the 60S joining step at initiation. These deficiencies could be complemented by purified FLAG-Upf1, or 60S subunits, and appeared to reflect diminished cycling of ribosomes from endogenous PTC-containing mRNAs to exogenously added synthetic mRNA in the same extracts. This hypothesis was tested, and supported, by experiments in which nucleased WT or upf1D extracts were first challenged with high concentrations of synthetic mRNAs that were templates for either normal or premature translation termination and then assayed for their capacity to translate a normal mRNA. Our results indicate that Upf1 plays a key role in a mechanism coupling termination and ribosome release at a PTC to subsequent ribosome reutilization for another round of translation initiation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.