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.

Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes

“…All 3′ FP intermediates show extended 5′ ends, some of which exceed the length of the typical 40S FP (∼30 nt; Figures 4 D, 4E, S6 H, S7 A, and S7B). We assume that the latter FPs originated from two queuing 40S species at the start codon region; i.e., the phenomenon that was demonstrated recently ( Shirokikh et al., 2019 ; Figure S3 A). With Sel-TCP-seq, an equal pull-down efficiency can be expected for early AUG recognition intermediates with a queuing 40S because both 40S species should still contain eIF2 and eIF3 ( Figure 4 B).…”

iRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation

“…Rapid rates of preinitiation complex loading and scanning relative to translation start would generate queues of potentially collided 43S preinitiation complexes within 5'UTRs. Evidence for such queueing has been demonstrated using in vitro translation systems and translation complex profile sequencing (TCP-seq) in yeast and human cells (Bohlen et al, 2020;Shirokikh et al, 2019;Sogorin et al, 2012;Wagner et al, 2020). Further, generating queues of preinitiation complexes using cycloheximide or insertion of an upstream open reading frame (uORF) resulted in alternative start codon utilization and translation recoding (Ivanov et al, 2018;Kearse et al, 2019).…”

Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes

“…In view of our earlier interpretation that the start codon FP with the longest 3' end represents a late PIC intermediate (Archer et al, 2016), these observations are consistent with the expectation that eIF2 leaves the initiating PIC before eIF3. The logic we apply to order unselected PIC intermediates represented by differing FP 3' ends into a time sequence is based on the extent of evidence for a queuing 40S upstream of the initiating 40S, which was recently demonstrated (Shirokikh et al, 2019) and which is inferred from the 5' extension of FPs, pronounced at a distance to fit a queuing 40S (around -30 nt) (see schematic in Figure S5A). These 5' extensions are seen most prominently for the FPs with the longest 3' end .…”