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. 2013 Apr 1;1(1):e24400.
doi: 10.4161/trla.24400. eCollection 2013.

Physical evidence supporting a ribosomal shunting mechanism of translation initiation for BACE1 mRNA

Dora C Koh  1 , Gerald M Edelman  1 , Vincent P Mauro  1
Affiliations

Physical evidence supporting a ribosomal shunting mechanism of translation initiation for BACE1 mRNA

Dora C Koh et al. Translation (Austin). .

Abstract

In Alzheimer disease, elevated levels of the BACE1 enzyme are correlated with increased production of amyloid peptides and disease pathology. The increase in BACE1 levels is post-transcriptional and may involve altered translation efficiency. Earlier studies have indicated that translation of BACE1 mRNA is cap-dependent. As ribosomal subunits move from the cap-structure to the initiation codon, they fail to recognize several AUG codons in the 5' leader. In this study, we looked for physical evidence of the mechanism underlying ribosomal scanning or shunting along the BACE1 5' leader by investigating structural stability in the 5' leaders of endogenous mRNAs in vivo. To perform this analysis, we probed RNAs using lead(II) acetate, a cell-permeable chemical that induces cleavage of unpaired nucleotides having conformational flexibility. The data revealed that the ≈440-nt 5' leader was generally resistant to cleavage except for a region upstream of the initiation codon. Cleavage continued into the coding region, consistent with destabilization of secondary structures by translating ribosomes. Evidence that a large segment of the BACE1 5' leader was not cleaved indicates that this region is structurally stable and suggests that it is not scanned. The data support a mechanism of translation initiation in which ribosomal subunits bypass (shunt) part of the BACE1 5' leader to reach the initiation codon. We suggest that a nucleotide bias in the 5' leader may predispose the initiation codon to be more accessible than other AUG codons in the 5' leader, leading to an increase in its relative utilization.

Keywords: BACE1; Initiation; RNase P; Ribosome; Shunt; Translation; mRNA.

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Figures

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Figure 1.Lead(II) acetate-induced cleavage of endogenous RNase P RNAs. (A) B104 cells were incubated with control buffer (0 mM Pb(II); first lane in each panel) or increasing concentrations of a lead(II) acetate solution (25, 50, or 100 mM; represented by the triangles). RNA was visualized on a denaturing agarose gel. Bands corresponding to rRNAs are indicated. (B) Schematic representation of primer extension assay. The black bar represents the RNase P RNA; the arrows show locations of DNA oligonucleotide primers P1-P4. The graphic below illustrates primer extension products as dashed blue lines that extend from the primer to either the 5′ end of the RNA or cleavage sites, indicated by red arrowheads. (C) Primer extension analysis of endogenous RNase P RNAs using primers P1 to P4. Cells were incubated with 0, 25, 50, or 100 mM lead(II) acetate; represented by the triangles. Primer extension products were resolved on denaturing polyacrylamide gels. Primer extension products appear as bands in the autoradiograms. The arrows indicate the position of full-length RNase P RNAs. Nucleotide positions of various cleavage sites in the RNase P RNA sequence are indicated. (D) Cleavage data mapped onto a previously predicted secondary structure of rat RNase P RNA. Individual nucleotides are indicated schematically as different colored circles (G = closed black, A = closed gray, U = open gray, and C = open black). G-C and A-U base pairs are indicated by black bars; G-U base pairs are indicated by 2 black dots. Major cleavage sites identified from different primers are indicated by red arrowheads. Nucleotides in CR 1-V, P10/11, and P12 are indicated by blue bars. A pseudoknot between complementary nucleotides in CR1 and CRV is indicated by the dashed blue line.
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Figure 2.Subcellular distribution and polysome analysis of BACE1 mRNAs. (A) Analysis of BACE1 mRNAs by RPA. RNA was isolated from nuclei or cytoplasm of rat B104 and PC12 cells. The top panel probes BACE1 mRNA, the bottom panel probes RNase P RNA. For both autoradiograms, lane 1 is undigested probe (u). Lanes 2–6 are RPAs performed using no cell lysate (lane 2), RNA from nuclear lysates (N, lanes 3 and 5) and RNA from cytoplasmic lysate (C, lanes 4 and 6). The RPA products indicated in lanes 4 and 6 (BACE1) are protected from digestion by the BACE1 probe. The RPA products indicated in lanes 3- 6 (RNase P) are protected from digestion by the RNase P probe (B) Polysome analysis of BACE1 mRNAs. Cell lysates were fractionated on a linear 10–50% sucrose gradient. The top of the gradient is on the left. The positions of the 40S and 60S ribosomal subunits, 80S monosomes, and polysomes are indicated. The A260 absorbance tracing is indicated by the red line. RNA from 14 fractions was analyzed by RPA using a BACE1 specific probe.
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Figure 3.Lead(II) acetate-induced cleavage of endogenous BACE1 mRNAs in cells. (A) PC12 cells were incubated with lead(II) acetate as indicated for 10 min and RNA cleavage analyzed as in Figure 1A. (B) Primer sites in BACE1 mRNA. The bar represents the BACE1 mRNA, with the 5′ leader as an open bar and the coding sequence (CDS) as a black bar. The locations of oligonucleotide primers are indicated below. (C) Primer extension analysis of BACE1 mRNAs with primers P5-P7. Cells were incubated with control buffer (0 mM Pb(II); lane 1 in each panel) or increasing concentrations of a lead(II) acetate solution (25, 50, or 100 mM; lanes 2, 3, and 4, respectively). Primer extension products were resolved on denaturing polyacrylamide gels. The arrows (5′) indicate the position of full-length BACE1 mRNAs. Nucleotide positions of uORFs1-3 and the BACE1 cistron are indicated; upstream AUG codons are highlighted with white bars. Sequencing reactions using the same primers are indicated to the left. The asterisk corresponds to the length of the reported cDNA sequence (NCBI accession #NM_019204).
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Figure 4.Comparison of Pb2+-induced cleavage sites in BACE1 mRNAs with predicted structural stabilities and nucleotide composition. (A) Linear representation of primer extension results. The BACE1 5′ leader and start of the coding region are indicated schematically. The range of the 5′ leader is indicated by an open bar, and uORF1, uORF2, and uORF3 are shown as gray boxes. Within uORF2 there is an AUG codon immediately upstream of the stop codon, which is indicated by a vertical line. The start of the coding sequence is indicated in black. Sites of Pb2+-induced cleavage are shown as red vertical bars and correspond to bands observed in primer extension reactions from at least two different primers in cells exposed to Pb2+ and not in control samples. (B) Minimum free energy predictions (MFEP) along BACE1 5′ leader and 5′ coding region. Each bar in the histogram is centered on the 15th nucleotide for each 30-nt window used for the RNAfold calculation. (C) Nucleotide composition of BACE1 5′ leader and 5′ coding region. Each nucleotide in the 5′ leader is indicated by a vertical bar: G is purple; C is black; A is green; and U is blue. The arrow is pointing to the start of the CDS (A1).
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