Ribosomal shunting mediated by a translational enhancer element that base pairs to 18S rRNA - PubMed (original) (raw)

Ribosomal shunting mediated by a translational enhancer element that base pairs to 18S rRNA

Stephen A Chappell et al. Proc Natl Acad Sci U S A. 2006.

Abstract

In eukaryotes, 40S ribosomal subunits move from their recruitment site on the mRNA to the initiation codon by an as yet poorly understood process. One postulated mechanism involves ribosomal shunting, in which ribosomal subunits completely bypass regions of the 5' leader. For some mRNAs, shunting has been shown to require various mRNA elements, some of which are thought to base pair to 18S rRNA; however, the role of base pairing has not yet been tested directly. In earlier studies, we demonstrated that a short mRNA element in the 5' leader of the Gtx homeodomain mRNA functioned as a ribosomal recruitment site by base pairing to the 18S rRNA. Using a model system to assess translation in transfected cells, we now show that this intermolecular interaction also facilitates ribosomal shunting across two types of obstacles: an upstream AUG codon in excellent context or a stable hairpin structure. Highly efficient shunting occurred when multiple Gtx elements were present upstream of the obstacles, and a single Gtx element was present downstream. Shunting was less efficient, however, when the multiple Gtx elements were present only upstream of the obstacles. In addition, control experiments with mRNAs lacking the upstream elements showed that these results could not be attributed to recruitment by the single downstream element. Experiments in yeast in which the mRNA elements and 18S rRNA sequences were both mutated indicated that shunting required an intact complementary match. The data obtained by this model system provide direct evidence that ribosomal shunting can be mediated by mRNA-rRNA base pairing, a finding that may have general implications for mechanisms of ribosome movement.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Shunting across an uAUG can be mediated by _Gtx_-translational enhancer elements. (A) Photinus luciferase reporter mRNAs are indicated schematically. In the 5′ leaders, the Gtx elements are indicated by dark gray boxes, SI spacer sequences are indicated by black boxes, and poly(A) spacer sequences are indicated by a thin black line. The white arrow indicates the Photinus luciferase initiation codon (AUGi). The gray arrow indicates an uAUG, the resulting ORF overlaps the luciferase cistron in a different reading frame. The black arrow indicates an uAUG, the resulting ORF overlaps the luciferase cistron in the same reading frame. In the histogram, translation efficiencies (see Materials and Methods) in mouse N2a cells are expressed as raw light units (rlu) per unit mRNA, in the absence or presence of an uAUG, respectively. Error bars indicate SEM. The extent to which the uAUGs block translation is indicated as percent inhibition. In these experiments, the calculated translation efficiencies reflected the raw luciferase enzyme activities. (B) Western blot analysis for Photinus luciferase of lysates from N2a cells transfected with the indicated constructs.

Fig. 2.

Shunting across a hairpin structure can be mediated by _Gtx_-translational enhancer elements. (A) Photinus luciferase reporter mRNAs are indicated schematically. Gray bars and lines are as in Fig. 1. The histogram represents translation efficiencies in mouse N2a cells. Error bars indicate SEM. In these experiments, the calculated translation efficiencies reflected the raw luciferase enzyme activities. (B) Ribonuclease protection assays. Riboprobes complementary to the 3′ stem of the hairpin (hp probe) and to the Photinus luciferase coding sequence (CDS probe) are indicated schematically as gray bars. Below is an autoradiograph of ribonuclease protection assays performed on in vitro transcribed RNAs containing the full hairpin (full hp) or 3′ stem of the hairpin (half hp) and on total RNAs isolated from N2a cells transfected with the constructs indicated. Full-length probes are indicated in the sample lacking RNase (−), and protected probes are indicated in the samples treated with RNase (+).

Fig. 3.

The ability of Gtx elements to mediate shunting requires base pairing to 18S rRNA. Schematic representations of the luciferase constructs are indicated to the left of the histogram. The 8-nt Gtx elements are indicated as dark gray boxes, and the mutated Gtx elements (8nt-m1) are indicated as white boxes. Constructs were tested in yeast strain NOY908 transformed with constructs expressing the 18S rRNAs indicated. Yeast rRNA sequences are indicated by the white bar and mouse sequences by the black bar. The histogram represents translation efficiencies in the indicated yeast cells expressed as raw light units per unit of mRNA. Error bars indicate SEM. (A) Sequence matches between the rRNA sequences (nucleotides 1132–1124 in the mouse), and the 8-nt Gtx element are indicated. Complementary nucleotides are indicated by vertical bars, and a black dot represents a potential G-U base pair. (B) Sequence matches between the rRNA sequences and the 8nt-m1 element are indicated as above. In both A and B, the calculated translation efficiencies reflected the raw luciferase enzyme activities.

Fig. 4.

A model of ribosome shunting. 5′ leader sequences are represented schematically as bold black lines, the coding regions as a hatched bar, the cap as gray circles, and the key mRNA elements that can affect shunting by interacting with ribosomal subunits directly or indirectly as gray boxes. The “X” in the 5′ leaders represents an obstacle, for example, hairpin or uAUG. The 40S ribosomal subunits and associated factors (ribosomal complexes) are indicated as black circles. Ribosomal subunits are recruited to the mRNA via the cap structure and mRNA elements (indicated by the dashed arrows). For clarity, we indicate individual ribosomal complexes only. Likewise, we indicate one mRNA element upstream of the obstacle in A and two mRNA elements flanking the obstacle in B. These recruitment events increase the local concentration of the translation machinery and the likelihood of ribosomes interacting with other accessible sites in the mRNA (indicated by the solid arrows). The presence of one or more mRNA elements downstream of the obstacle in B increases the efficiency of shunting. One assumption of the model is that movement of the translational machinery between mRNA elements need not necessarily be unidirectional.

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Ribosomal shunting mediated by a translational enhancer element that base pairs to 18S rRNA - PubMed (original) (raw)