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

Abstract

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.

Keywords: 40S ribosomal subunit; 5′ UTR; 5′UTR; SSU; cap-dependent initiation; eIF4A; eIF4F; eukaryotes; gene expression control; mRNA translation; ribosomal scanning; translation initiation.

Figure 1

In situ luminescence catalysed by firefly luciferase accumulating during translation of capped mRNAs in a cell-free translation system based on mouse Krebs-2 cells lysate, in the absence or presence of different concentrations of nonhydrolysable ATP analogue (AMP-PNP). (a,c) Translation of capped 5′poly(A)-Luc-3′TMV mRNA at 0.15 µM (a) or 0.45 µM (b). (b,d) Translation of capped 5′β-globin-Luc-3′TMV mRNA. AMP-PNP was added to 1.25, 2.5 or 5 mM (lines colour-coded by shades of red) or omitted (black line).

Figure 2

In situ luminescence catalysed by firefly luciferase accumulating during translation of capped mRNAs in a cell-free translation system based on mouse Krebs-2 cells lysate, either without or in the presence of different concentrations of eIF4A-blocking RNA aptamer (a4Aa20). (a,c) Translation of capped 5′poly(A)-Luc-3′TMV mRNA at 0.15 µM. (b,d) Translation of capped 5′β-globin-Luc-3′TMV mRNA at 0.15 µM. (a,b) Addition of eIF4A-blocking RNA aptamer a4Aa20. (c,d) Addition of Escherichia coli 5S ribosomal RNA (rRNA) used as negative control. The short RNAs were added at 0.08, 0.3 or 1.2 µM (lines colour-coded by shades of red) or omitted (black line).

Figure 3

Summary of the analysis of stepwise, cap-guided assembly of ribosome:poly(A)-Luc (capped) mRNA complexes, in the presence or absence of ATP or its nonhydrolysable analogue (AMP-PNP), and different sets of group 4 initiation factors (eIFs). (a) Schematic representing steps taken to channel translation initiation predominantly through cap-dependent attachment to mRNA on a cap- and powered scanning-independent mRNA with poly(A) 5′UTR. (b) Overview of the results for (a) with the sets of eIFs indicated on left (omitted eIFs shown in red with the minus ‘−’ sign). Numbers in boxes indicate percent of the complex assembly by the fluorescence in the start codon-corresponding toeprint stop relative to the total of the 5′UTR (see Materials and Methods and reference [74] for more detail) and are colour-coded by the complex yield (blue, more; red, less). Numbers between boxes represent mean of complex yield fold change between ATP (left) or no nucleotides (‘none’, right) and AMP-PNP (centre) conditions (using repeat shown in Supplementary Figure S2d), ± double standard deviation.

Figure 4

Relative fluorescence of cDNA fragments generated in a reverse transcription reaction with ribosome:poly(A)-Luc (capped) mRNA complexes after electrophoretic separation (toeprint assay). Percent values indicate amounts of fluorescence (area under the curve) corresponding to all signals related to the cognate start codon of this mRNA (+16, +17, +18 nt peaks), relative to the total signal in the 5′UTR. +16 nt denotes the toeprinting peak located 16 nt downstream of the first nucleotide of the start codon, which is considered as the first nucleotide located in the P-site of the SSU:mRNA complex upon start codon recognition. ‘−8 nt’ denotes position of a more 5′-proximal polymerase stop located 8 nt upstream of the +16 nt peak. Capped poly(A)-Luc mRNA at 40 nM was preincubated with mixtures containing different sets of eIFs (i–viii; as described to the right of the plots) and Escherichia coli Met-tRNA^fMet for 5 min at 37 °C. Subsequently, ATP to 2 mM (a), AMP-PNP to 2 mM (b) or water (c), as well as SSUs (all) were added and toeprinting performed as previously described, see Methods for further details and Figure 3 for the experiment schematic and summary of the results. Where ATP or AMP-PNP were added, we assumed 1:1 magnesium ion binding to the solubilized compounds [95] and supplemented the nucleotide triphosphates together with an equimolar amount of magnesium ions using freshly prepared equimolar premixes with magnesium acetate. See Supplementary Figure S2d for replicate assays corresponding to data shown here in rows i and iv. For the complete unprocessed fluorescence cDNA traces, see Supplementary Figure S3a–c; note the cDNA signal will appear flipped by the horizontal axis in the unprocessed plots.

Figure 5

(a) Sequences of the synthetic capped mRNAs used in the reconstituted translation initiation system assembly. (b) Toeprint assay of ribosome:mRNA complexes assembled on synthetic and natural capped mRNAs. (top two plots), poly(A,U)-Luc mRNA; (middle two plots), poly(U)-Luc mRNA; (bottom two plots), β-globin mRNA. Reactions were assembled as described in Materials and Methods and in Figure 4 legend; reaction mixtures included 40 nM capped mRNAs and were supplemented with either 2 mM ATP or water instead (indicated in the legend on right).

Figure 6

Electron micrographs of, and their possible interpretation for, mRNA:ribosomal complexes assembled in Krebs cells lysate using capped mRNA with LL1 5′UTR. The capped mRNA was preincubated with micrococcal nuclease-treated 50% Krebs-2 cells lysate in the presence of cycloheximide at 30 °C for 30 min and supplemented with 5 mM AMP-PNP and 10 mM magnesium acetate premix. The resultant reaction mixtures were gel-filtered using Illustra MicroSpin S300 columns, contrasted with uranyl acetate and imaged (see more details in Materials and Methods). (a) A representative electron microscopy field with particles of ribosome and SSU size and appearance. (b) Outlines of rows of SSU-appearing particles (smaller) located close to the singular full ribosome-appearing particles (larger). (c) Overlay of (b) over (a). (d, left) Cut-outs from several representative electron microscopy fields with SSUs appearing in rows located close to the singular full ribosomes. (d, middle) Outlines of SSUs and ribosomes are overlaid over the electron micrographs. (d, right) Schematic with possible interpretation of the (d, left) panels; note that the location of mRNA (represented by the dotted line) cannot be predicted from the electron microscopy imaging used in panels (a,c,d) and is used for illustrative purposes only.

Figure 7

Schematic explaining the detection of ‘open’ entry channel or queuing (stacked) SSUs at the capped mRNAs via toeprinting approach. (left) SSU position over mRNA in the beginning and end of a limiting (a,b) or excessive (c) initiation reaction. (right) Results of the toeprinting reaction for each of the initiation states shown in the left panel. (b) The start codon SSU complex in the ‘open’ entry channel configuration stops the reverse transcriptase (RT) at a distance from the usual start codon toeprint signal (right; b). (c) The stacked SSU scanning complex, together with the reverse transcriptase (RT), displace the start codon SSU complex while adopting the ‘closed’ entry channel configuration, resulting in RT stop at a distance from the usual start codon toeprint signal (right; c).