Translatome profiling analysis during chloroplast biogenesis

Abstract

Gene regulation is critical to cellular homeostasis. Many of the most fundamental processes for energy production take place in semi-autonomous organelles. Almost all eukaryotes contain mitochondria which house the oxidative phosphorylation complex. Furthermore, photosynthetic organisms additionally contain photosystems for turning light and CO2 into carbohydrates. Intriguingly, the genetic instructions encoding the components of these energy-producing organelles are split between a private organellar genome and the shared nuclear genome. These complexes have come to be known as dual-origin complexes and pose an exciting regulatory puzzle because the gene expression machinery is specific for each compartment. In this thesis, I aim to create a greater understanding of how dual-origin complexes can be regulated by utilizing methods I developed in Chlamydomonas reinhardtii, a unicellular photosynthetic algae. In Chapter 1, I established polysome profiling for all ribosome populations (cytoplasmic, chloroplastic, and mitochondrial) as well as ribosome profiling for the chloroplast. These methods allowed for the cataloging of the nascent translatome and uncovered unique features of translation in the chloroplast. To understand dynamic gene expression changes, I utilized a classical system characterized by a lack of chloroplast in the dark and rapid chloroplast biogenesis when shifted to light. In Chapter 2, I used methods developed in Chapter 1 to characterize how the cell modulates expression during chloroplast biogenesis. I found that nuclear expression of mitochondrial energy production pathways were down-regulated upon shift to light, coinciding with an increased expression of chloroplast-destined proteins. To understand how dual-origin complexes are regulated, I examined the 5 large dual-origin complexes(minimum of three components from each genetic loci) in Chlamydomonas reinhardtii (chloroplast ribosome, Photosystem II, Cytochrome b6f, Photosystem I, and ATP synthase). The nuclear-encoded components of dual-origin complexes showed more balanced expression relative to their chloroplast-encoded counterparts. Finally, I found that dual-origin complexes were co-regulated quite differently, the chloroplast ribosome was highly co-expressed across compartments while the chloroplast-encoded components tended to increase in expression prior to the nuclear components of the other complexes. This builds upon the diversity of regulation already identified in yeast and metazoans, hopefully leading to a deeper understanding of this challenging regulatory problem.