Fluorescent toeprinting to study the dynamics of ribosomal complexes

Егорова, Т. В.; Sokolova, Elizaveta; Shuvalova, Ekaterina; Matrosova, Vera A.; Shuvalov, Alexey; Alkalaeva, Elena

doi:10.1016/j.ymeth.2019.06.010

Cited by 15 publications

(19 citation statements)

References 29 publications

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“…The poly(A) 5′UTR mRNA is not dependent on active scanning, allowing us to observe inhibitory effects other than caused by, e.g., complementarity-based structural impediments. SSU:mRNA complex formation was assessed based on inhibition of reverse transcription (toeprinting) [42,74,93,94]; the resultant cDNA fragments were detected by fluorescent labelling and capillary electrophoresis [16,74]. We introduced several modifications to channel initiation through cap-dependent scanning (see Figure 3a for a schematic of the experiment; more details in Materials and Methods).…”

Section: Resultsmentioning

confidence: 99%

Migration of Small Ribosomal Subunits on the 5′ Untranslated Regions of Capped Messenger RNA

Shirokikh

Dutikova

Staroverova

et al. 2019

IJMS

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Several control mechanisms of eukaryotic gene expression target the initiation step of mRNA translation. The canonical translation initiation pathway begins with cap-dependent attachment of the small ribosomal subunit (SSU) to the messenger ribonucleic acid (mRNA) followed by an energy-dependent, sequential ‘scanning’ of the 5′ untranslated regions (UTRs). Scanning through the 5′UTR requires the adenosine triphosphate (ATP)-dependent RNA helicase eukaryotic initiation factor (eIF) 4A and its efficiency contributes to the specific rate of protein synthesis. Thus, understanding the molecular details of the scanning mechanism remains a priority task for the field. Here, we studied the effects of inhibiting ATP-dependent translation and eIF4A in cell-free translation and reconstituted initiation reactions programmed with capped mRNAs featuring different 5′UTRs. An aptamer that blocks eIF4A in an inactive state away from mRNA inhibited translation of capped mRNA with the moderately structured β-globin sequences in the 5′UTR but not that of an mRNA with a poly(A) sequence as the 5′UTR. By contrast, the nonhydrolysable ATP analogue β,γ-imidoadenosine 5′-triphosphate (AMP-PNP) inhibited translation irrespective of the 5′UTR sequence, suggesting that complexes that contain ATP-binding proteins in their ATP-bound form can obstruct and/or actively block progression of ribosome recruitment and/or scanning on mRNA. Further, using primer extension inhibition to locate SSUs on mRNA (‘toeprinting’), we identify an SSU complex which inhibits primer extension approximately eight nucleotides upstream from the usual toeprinting stop generated by SSUs positioned over the start codon. This ‘−8 nt toeprint’ was seen with mRNA 5′UTRs of different length, sequence and structure potential. Importantly, the ‘−8 nt toeprint’ was strongly stimulated by the presence of the cap on the mRNA, as well as the presence of eIFs 4F, 4A/4B and ATP, implying active scanning. We assembled cell-free translation reactions with capped mRNA featuring an extended 5′UTR and used cycloheximide to arrest elongating ribosomes at the start codon. Impeding scanning through the 5′UTR in this system with elevated magnesium and AMP-PNP (similar to the toeprinting conditions), we visualised assemblies consisting of several SSUs together with one full ribosome by electron microscopy, suggesting direct detection of scanning intermediates. Collectively, our data provide additional biochemical, molecular and physical evidence to underpin the scanning model of translation initiation in eukaryotes.

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Section: Resultsmentioning

confidence: 99%

Migration of Small Ribosomal Subunits on the 5′ Untranslated Regions of Capped Messenger RNA

Shirokikh

Dutikova

Staroverova

et al. 2019

IJMS

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“…These complexes were then used to test the effect of Nsp1 on the formation of termination complexes (TCs) by fluorescent toe-print analysis, which shows the position of ribosomal complexes on mRNA. When stop codon is recognized by eRF1, the ribosome changes conformation of the +4 nucleotide following the stop codon, which is then detected as a nucleotide shift of the toe-print signal (Alkalaeva et al, 2006; Egorova et al, 2019). It should be noted that in each experiment we compared the relative amounts of cDNAs as areas of the preTC and the TC peaks according to the following formula TC formation efficiency = TC / (TC + preTC), which is presented on the histograms (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Samples were analysed using the primer extension protocol. The toe-printing analysis was performed with AMV reverse transcriptase and 5’-carboxyfluorescein-labelled primers complementary to 3’-UTR sequences, as described previously (Egorova et al, 2019). cDNAs were separated via electrophoresis using standard GeneScan® conditions on an ABI Prism® Genetic Analyser 3100 (Applera).…”

Section: Methodsmentioning

confidence: 99%

Nsp1 of SARS-CoV-2 Stimulates Host Translation Termination

Shuvalov

Shuvalova

Biziaev

et al. 2020

Preprint

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SUMMARYThe Nsp1 protein of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by binding to the entry channel of the 40S ribosome subunit. The structural study of SARS-CoV-2 Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1 and the eRF1 and ABCE1 proteins. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex simultaneously with eRF1, we hypothesized that Nsp1 may be involved in translation termination. We show the direct influence of Nsp1 on translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we reveal that Nsp1 stimulates translation termination in the stop codon recognition stage. We identify that activity of Nsp1 in translation termination is localized in its N-terminal domain. The data obtained will enable an investigation of new classes of potential therapeutic agents from coronavirus infection competing with Nsp1 for binding with the termination complex.

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“…Understanding the molecular basis and mechanisms of translation termination is required for the development of targeted therapy of more than 10% of inherited human disorders caused by nonsense mutations [40]. Despite the availability of biochemical approaches for investigating this process in reconstituted systems [10,82,108], the methods used in vivo or in complete cell extracts are limited to the estimation of read-through efficiency, based on the stop codon containing reporters [67,68,107]. The latter techniques, however, said little about the kinetics and molecular Our results indicated that the VEEV read-through promoting context caused marked problems at the termination stage, visualized as a delay in FLUC product appearance, similar to that observed in the zero-length 3 UTR construct (although less severe than in the case of the nonstop mRNA).…”

Section: Discussionmentioning

confidence: 99%

“…However, it is also hardly used for analysis of translation dynamics. A wide arsenal of biochemical approaches has been applied to study translation termination in reconstituted systems, including toe-printing [10,82] and radiolabeled Met-tRNA hydrolysis or peptide release [10]. Although much important information has been obtained with these methods, purified ribosomes and translation factors poorly recapitulate situation in complete system or in living cells.…”

Section: Introductionmentioning

confidence: 99%

CTELS: A Cell-Free System for the Analysis of Translation Termination Rate

et al. 2020

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Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3′ UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a “leaky” stop codon context, which likely defines the basis of nonsense suppression.

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Fluorescent toeprinting to study the dynamics of ribosomal complexes

Cited by 15 publications

References 29 publications

Migration of Small Ribosomal Subunits on the 5′ Untranslated Regions of Capped Messenger RNA

Migration of Small Ribosomal Subunits on the 5′ Untranslated Regions of Capped Messenger RNA

Nsp1 of SARS-CoV-2 Stimulates Host Translation Termination

CTELS: A Cell-Free System for the Analysis of Translation Termination Rate

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