Transcript leaders : annotation and insight into functions in translation

Abstract

For a eukaryotic mRNA to be properly expressed, it undergoes a series of several steps, including transcription, modification, splicing, packaging, export, localization, translation, and decay. Of these steps transcription is the most extensively studied, though the remaining steps are also indispensible for proper protein production. While we understand many of these steps in biochemical detail in vitro, we have a much poorer knowledge of how they occur and are regulated for a given gene in vivo. Posttranscriptional regulation is carried out primarily through the noncoding portions of the mRNA: the Transcript Leader (TL or 5'UTR) upstream of the Open Reading Frame (ORF), and the 3'Untranslated Region (3'UTR) downstream. To understand how these regions affect post-transcriptional gene expression, it is critical to have precise annotations of the mRNA(s) produced from a gene. In Chapter 2 I describe the development of Transcript Leader Sequencing (TL-seq), a technique to annotate TLs, and demonstrate its utility in yeast. TL-seq annotations reveal interesting TL-dependent regulation, including transcription within ORFs and short TLs that are associated with translation initiation at the second AUG of the ORE. To further study the roles of TLs in translation, I develop Translation-Associated Transcript Leader Sequencing (TATL-seq). TATL-seq works by applying TL-seq across fractions of a polysome gradient, generating TL-specific translational measurements. This approach demonstrates a widespread inhibitory function for upstream AUGs (uAUGs), and that ~6% of yeast genes express multiple TL species with distinct translational activities. This demonstrates that alternative TLs are prevalent and functional even in a relatively simple eukaryote like yeast. My interest in alternative TLs prompted me to explore TL variation in mammals, where many thousands of genes are known to have alternative TLs. In Chapter 3 I enumerate the contributions of alternative mRNA processing events to alternative TLs in mice. I observe alternative TLs produced by alternative Transcription Start Sites (TSSs), and also demonstrate that alternative splicing events, such as skipped exons and alternative splice sites, contribute substantially to functional TL diversity. To facilitate the future study of alternative TLs in mammals, in Appendix I I modify TL-seq to sequence longer TL fragments and optimize TL-seq's enzymatic steps to reduce input RNA requirements. This thesis is concerned with understanding post-transcriptional mRNA expression both globally and gene-specifically. In particular, I seek to understand the role the Transcript Leader has in affecting translation and degradation of its transcript. The findings detailed here define and analyze discernable features of TLs that relate to translational properties of the downstream message. Furthermore, the techniques developed enable analyses of TLs and translation that could not be carried out with previous technologies.