Scientific Report 2007

Neurobiology

Translational Regulation of Gene Expression

V.P. Mauro, S.A. Chappell, W. Zhou, J. Dresios, D.C.Y. Koh, P. Panopoulos, D. Maar, G.M. Edelman

In eukaryotes, translation of mRNA into protein begins with recruitment by the mRNA of the translation machinery, which consists of the 40S ribosomal subunit, the initiator methionine-tRNA, and various other factors. This recruitment can occur via the cap structure, which is found at the 5′ ends of mRNAs, or at internal sequences contained within some mRNAs. For most mRNAs, the recruitment site is distant from the nucleotides encoding the protein; thus, before protein synthesis can commence, the translation machinery must reach the initiation codon. These early events are essential for protein synthesis and are key sites of regulation, yet they remain poorly understood. We focus on understanding the mechanisms that underlie these essential initiation events in translation.

Ribosomal Recruitment

Our earlier studies provided the first direct evidence for a mechanism of ribosomal recruitment in eukaryotes that involved base pairing between complementary nucleotides in mRNA and 18S rRNA, which is the RNA component of the 40S ribosomal subunit. The mRNA element in these earlier studies was isolated from the Gtx homeodomain mRNA. We showed that this element base pairs with 18S rRNA in the platform of the 40S ribosomal subunit and facilitates initiation of translation. Currently, we are investigating the extent to which this particular base-pairing interaction affects the translation of other mRNAs; we are blocking the interaction in various ways and assessing the effects on the proteome. Preliminary results indicate that this binding site specifically affects the expression of a subset of proteins. In addition to the Gtx-binding site, we have identified other putative mRNA-binding sites in the 18S rRNA and are now examining these binding sites and their physiologic relevance.

Reaching the Initiation Codon

A generally held model of how ribosomal subunits reach the initiation codon is that they scan from the recruitment site to the initiation codon. Ribosomal scanning is suggested to be linear, that is, each nucleotide is inspected until the initiation codon is encountered, at which point the initiator tRNA base pairs to the initiation codon and scanning stops. However, this model cannot explain various observations reported in the literature and our own findings, a situation that prompted us to suggest alternative mechanisms of translation initiation.

These alternative mechanisms involve tethering or clustering of ribosomal complexes. The notion of tethering suggests that the ribosomal subunits reach the initiation codon while attached to a fixed point in the mRNA, which may be the cap structure or internal mRNA sequences. The tethered ribosomal subunit effectively bypasses sequences located between the ribosomal recruitment site and the initiation codon. In contrast, clustering is a dynamic process in which ribosomal subunits bind to and detach from various sites in the mRNA. This reversible binding at various sites is postulated to increase the local concentration of ribosomal subunits, increasing the probability that the initiator tRNA will base pair to an initiation codon in the vicinity.

We tested the feasibility of these ideas in studies with model mRNAs. The results indicated that translation efficiency varied with the distance between the ribosomal recruitment site and the initiation codon. In addition, we found that translation could initiate efficiently at AUG codons located upstream of an internal recruitment site. These results are consistent with the notion of ribosomal tethering at the cap structure and clustering at internal sites.

An important prediction of the ribosomal tethering/clustering models is that the accessibility of the initiation codon is an important factor determining the use of the codon. To test this notion, we are studying the BACE1 mRNA, which encodes the enzyme β-secretase. This enzyme is overexpressed in Alzheimer’s disease without a corresponding increase in BACE1 mRNA levels, suggesting that the translation efficiency of this mRNA is increased in the disease. Our earlier studies indicated that the translation of this mRNA is affected by factors that alter the use of the BACE1 initiation codon. To assess whether the altered use of this initiation codon is correlated with the accessibility of this codon, we are probing the accessibility of nucleotides within this mRNA in living cells. We have adapted a lead acetate cleavage method that was first used in bacteria to probe short, highly structured RNAs. Our preliminary data indicate that we can detect the endogenous BACE1 mRNA. Although many of the nucleotides preceding the initiation codon are highly inaccessible to lead cleavage, the initiation codon itself is highly accessible. In ongoing studies, we will experimentally test our hypotheses about accessibility and use of the initiation codon.