Controlling protein translation: a message from the 3' end of mRNA

The control of mRNA translation is one of the many mechanisms utilized by cells to regulate gene expression. How well an mRNA is translated is often determined by ‘codes’ within the 3’ untranslated region (3’ UTR). Regulatory molecules, RNA and/or proteins, can bind these codes to influence when, where and how often mRNA is translated. One way in which the translational output of individual mRNA can be graded, is by regulated change in the poly(A)-tail length. A long poly(A)-tail is generally correlated with high translational efficiency whereas mRNA having a short-poly(A)-tail are poorly translated. Moreover, as a further level of control, recent work shows more than 50% of eukaryotic transcripts undergo condition dependant alternative 3’ end processing referred to as Alternative Polyadenylation (APA). This generates mRNA isoforms with differing 3’ UTR lengths and can result in the gain or loss of the regulatory elements. Variation of the position at which the pre-mRNA 3’ UTR is cleaved and polyadenylated can thus provide a mechanism by which the level of protein translation can be regulated. That is, a long 3’ UTR isoform might contain multiple regulatory elements that silence translation whereas a short isoform may be translationally deregulated. The translation of mRNA can be conveniently determined by its degree of association with ribosomes. Actively translating mRNA show better ribosomal association than silent or poorly translating mRNA. This study hypothesize that the deadenylation rate of mRNA poly(A)-tail is specified by proteins that bind to the 3’ UTR of specific transcripts and thereby regulate translational repression. However, it remains unresolved whether the poly(A)-tail shortening is a direct cause or indirect consequence of translational repression. In order to address this as well as to explore the functional consequence of APA, a ribosome affinity isolation assay has been established, which traps mRNA with translating ribosomes. The subsequent comparison between ribosome enriched RNA and the total RNA within the sample, serves as a surrogate read-out of mRNA translation at steady-state. In addition, to investigate if any adenylation-state modulators are responsible for differential translation, using reporters differing in their 3’ UTRs, a classic growth based assay was designed. This was based on modification of the HIS3 gene locus such that life on media lacking histidine depends on the stabilization of the HIS3 mRNA poly(A)-tail and thus increased His3p protein expression. However this study revealed an unappreciated complexity in the control of 3’ UTR dynamics in the endogenous genomic context that is not easily predicted or overcome by standard genetic engineering methodologies. Overall, this thesis will provide mechanistic insight into the 3’ end mediated control of mRNA translation.