Looking for CLUes and Motifes in mRNA Transcripts

Abstract

Mitochondrial damage and dysfunction are associated with the aging process, and can be attributed to mutations in both nuclear and mitochondrial genes involved in cellular respiration. Mitochondrial DNA is particularly prone to damage due to its proximity to reactive oxygen species in the mitochondrial matrix. With the current advances in gene therapy studies for both inherited and acquired diseases, mitochondrial dysfunction is an attractive therapeutic target. It could theoretically be corrected through allotropic expression of mitochondrial genes from the nucleus. Inherent to the success of this approach is identification of the way in which nuclear transcripts and proteins destined for the mitochondria are regulated. Recent evidence suggests the involvement of RNA-binding proteins in translation of mitochondrial mRNA. Several of these RNA-binding proteins have been shown to specifically interact with transcripts destined for the respiratory chain, including Pumilio family member Puf3p in yeast and the CLU protein in Drosophila, humans, and other eukaryotes. While numerous mRNA targets of Puf3p and CLU have been experimentally identified, the precise mechanisms of these interactions remain poorly understood. In this project, sets of mRNA transcripts that have been shown to interact with these RNA-binding proteins are screened for sequence motifs that may explain the specificity for mitochondrial transcripts and shed some light on the regulatory adaptations of nuclear-encoded mitochondrial proteins. A 7 nt long linear motif was identified, enriched in the coding region of CLUH target transcripts. Additionally, a motif recognized by human Pumilio proteins PUM1 and PUM2 was found to be enriched in the 3'-UTRs of a few nuclear-encoded mitochondrial transcripts.

Popular Abstract

CLUes and motifes in wayfaring mitochondrial mRNA

Generally speaking, eukaryotic organisms have mitochondria in each of their cells. Humans have them, fish have them, plants have them. Mitochondria are tiny organelles sporting two membranes, the inner one of which is the location of oxidative phosphorylation. In other words, this is where the oxygen we breathe and the food we eat is ultimately transformed into a molecule we call ATP. It is the fuel for most of the cell’s processes. Depending on cell type, there are between a hundred and a thousand mitochondria in every cell’s cytoplasm (the watery cavity bounded by the cell membrane and the nucleus).

Mitochondria are absolutely necessary for survival. The complexes of large proteins that form the Respiratory Chain Complex, the molecular machinery responsible for the oxidative phosphorylation, are in part encoded within rings of DNA found in the innermost space of the mitochondria themselves. If the mitochondria were lost somehow, there would be no way to recreate them from the much larger bank of DNA found in the cell nucleus.

This poses a problem because the environment of these mitochondrial DNA rings is highly reactive. The byproducts of oxidative phosphorylation, often referred to as free radicals, cause damage to the mitochondrial DNA at a relatively high rate. Over time, the DNA repair mechanisms fail, and mutations accumulate. Some mutations are harmless, but others cause serious dysfunction. Consequences of such dysfunction include diseases like Parkinson’s disease, several types of cancer, and diabetes.

One possible solution to this problem is to copy genes from the mitochondrial DNA into the nucleus’ DNA, where it would enjoy a safer environment. In order for this to work however, additional steps must be taken to ensure that these copied genes are expressed properly, i.e. that the genes’ proteins end up in the mitochondria as they normally do. This is a highly involved undertaking. Help may be sought by looking at how the nuclear-encoded mitochondrial genes are expressed. These are genes the proteins of which are located in the mitochondria, for example as parts of the Respiratory Chain Complex, but the genes themselves are written in the nucleus’ DNA.

It has been discovered that expression of these genes can be supported by RNA-binding proteins. These proteins bind to the genes’ mRNA transcripts – the single strands of RNA that are to be translated into proteins by ribosomes. One such RNA-binding protein in humans is CLUH. It binds to its (nuclear-encoded mitochondrial) transcripts and lets them go when they have reached the vicinity of mitochondria. It is currently not understood how CLUH recognizes the particular transcripts that it has been shown to transport. This thesis entails a search for a linear sequence motif amongst the mRNA transcripts bound by CLUH, in the hopes that such a motif could be what distinguishes these transcripts from other transcripts not bound by CLUH.

A program called MEME was used to perform this search. At first, only certain parts of the transcripts were searched. When this proved unfruitful, whole transcripts were searched, resulting in the discovery of a seven nucleotide long motif, GCTGCTG. The analyses performed implies that if this motif is part of the feature that causes CLUH to bind, it is not the whole story, but only part of a bigger picture. More work must be done in order to explain why CLUH is playing favourites, so that we can gain a complete understanding of how the nuclear-encoded mitochondrial genes are expressed.

Master’s Degree Project in Bioinformatics 30 credits 2018
Department of Biology, Lund University

Advisor: Björn Canbäck

• author: Wallenius, Joel
• supervisor: Amutha Boominathan; Matthew O´Connor; Björn Canbäck
• organization: Degree Projects in Bioinformatics
• course: BINP50 20181
• year: 2018
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 8950848
• date added to LUP: 2018-06-18 14:05:33
• date last changed: 2018-06-18 14:05:33