Physiological Significance of Stop Codon Readthrough

Abstract

Regulation of translation, the process by which ribosomes synthesize proteins from mRNAs, is critical to maintain cellular proteostasis. Dysregulation of translation is implicated in several neurodegenerative, metabolic, and genetic diseases. Ribosomes initiate translation at the start codon (canonically AUG) and continue translation till they encounter one of the three stop codons (UAA, UAG, UGA). However, in certain instances, ribosomes continue translation beyond the canonical stop codon till the next in-frame stop codon in the 3′-UTR, generating a C-terminally extended protein that can have different functions, localization, and activities. This process is known as stop codon readthrough (SCR). In mammals, SCR has been demonstrated in several genes including HBB, VEGFA, AGO1, LDHB, MDH1, MTCH2. However, the physiological significance of SCR is not known for most of these genes. This thesis focuses on the in vivo significance of stop codon readthrough in MTCH2. The second part of the thesis describes a method to study the post-transcriptional gene regulation in vivo.

The thesis is divided into two broad chapters:

Chapter 1: Investigation of physiological significance of stop codon readthrough of MTCH2 using a mouse model

MTCH2 or mitochondrial carrier homolog 2 has previously been identified in our lab to undergo double stop codon readthrough, generating two SCR isoforms: MTCH2x and MTCH2xx. Ablation of SCR in MTCH2 in HEK293 cells lead to a decrease in the mitochondrial membrane potential and ATP levels. In this study, we employed a mouse model to investigate the in vivo physiological significance of SCR in MTCH2. Our experiments demonstrate that SCR in MTCH2 plays a critical role in regulating MTCH2 protein levels. We have shown that optimal MTCH2 protein level is necessary to regulate mitochondrial OXPHOS and ATP levels in the skeletal muscle of these mice. In the absence of SCR in MTCH2, the mice show decreased musculoskeletal activity. We have also demonstrated that SCR of MTCH2 protects the mice from diet-induced obesity, implicating its potential significance in the function of adipocytes.

Chapter 1.1: Introduction

This chapter introduces the mammalian translation process and the phenomenon of stop codon readthrough (SCR). It then presents an in-depth review of the regulatory mechanisms governing SCR and its biological significance, illustrated with examples of various mRNAs that have been known to undergo SCR. This chapter also introduces our protein of interest, MTCH2, discussing its roles in cellular and in vivo functions in maintaining mitochondrial metabolism. It also describes the significance of SCR in MTCH2 in HEK293 cells.

Chapter 1.2: Materials and methods

This chapter demonstrates various molecular and physiological assays used to study the in vivo significance of SCR in MTCH2. First, it provides a detailed description of the process of generating the mouse model to study SCR of MTCH2 in mice. It also includes methodological details of histological analysis of the mouse tissues and molecular assays like western blotting, qRT-PCR. It also describes the rotarod assay used in this study to assess the musculoskeletal activity of mice.

Chapter 1.3: Generation of MTCH2 SCR-deficient (∆RTMTCH2) mice

This chapter demonstrates the establishment of a MTCH2 SCR-deficient mouse line by CRISPR-Cas9-mediated deletion of the proximal 3′-UTR region of MTCH2 genomic locus. These mice, denoted as ∆RTMTCH2 mice, can generate canonical MTCH2 protein but not the SCR isoform MTCH2x. This chapter also explains the general physiological characterization, such as body weight and fertility of the mice.

Chapter 1.4: Investigation of skeletal muscle in ∆RTMTCH2 mice

This chapter discusses the physiological effects of MTCH2 SCR in skeletal muscles. In the histological analysis of the skeletal muscle, a decreased muscle fibre cross- sectional area was observed in ∆RTMTCH2 mice compared to wild-type (WT) mice. Subsequently, molecular changes in the skeletal muscle of these mice were examined. Increased expression of the canonical MTCH2 protein, along with reduced ATP levels, was detected in the ∆RTMTCH2 mice. In agreement with decreased ATP levels, oxygen consumption by the mitochondrial oxidative phosphorylation (OXPHOS) complex was also found to be reduced. A decreased expression of specific OXPHOS complex proteins was also observed in the ∆RTMTCH2 mice. These findings suggest that ATP production is impaired due to diminished OXPHOS activity in skeletal muscles. Furthermore, mitochondrial translation was shown to be reduced as a consequence of decreased ATP levels. This potentially creates a negative feedback loop that further reduces the OXPHOS protein expression, because some key proteins of the OXPHOS complex are translated in the mitochondria. As a cumulative outcome of these alterations, reduced musculoskeletal strength was observed in the rotarod assay in ∆RTMTCH2 mice.

Chapter 1.5: Investigation of other organ systems in the ∆RTMTCH2 mice

This chapter demonstrates the role of MTCH2 SCR in other organ systems. Consistent with increased MTCH2 level, the ∆RTMTCH2 mice also showed increased weight gain with a high-fat diet. This indicates a potential role of MTCH2 SCR in adipocyte maturation and metabolism. In the liver of ∆RTMTCH2 mice, elevated MTCH2 protein levels were detected alongside a concomitant reduction in ATP levels. Although no apparent alterations were observed in the liver histology and in the liver function tests, it is possible that these molecular imbalances may contribute to phenotypic changes under certain stress conditions, which need further investigation. Additionally, no significant differences were observed in the hematological parameters between ∆RTMTCH2 and WT mice.

Chapter 2: IVISc-L: A quick and simple in vivo assay to study post transcriptional gene regulation

Temporal and spatial regulation of gene expression is essential for cells to maintain homeostasis. Gene expression changes are often associated with differences in cellular, immunological, metabolic, and stress response functions. Though a number of methods are available to study gene expression regulation in vitro, there are only a few methods that can be applied in vivo. Most of them are cumbersome and time-consuming, often requiring the sacrifice of the mice. To fill this gap, a minimally invasive and rapid in vivo bioluminescence assay was developed to study post-transcriptional gene regulation in live mice, without the need for sacrificing the animals.

Chapter 2.1: Introduction

This chapter briefly introduces different modes of gene expression regulation in mammalian cells and different techniques to study the regulation of gene expression both in vitro and in vivo, discussing their advantages and disadvantages.

Chapter 2.2: Materials and methods

This chapter describes the techniques involved in developing this assay protocol and all the plasmid constructs used in this chapter.

Chapter 2.3: Development of a simple in vivo gene expression system

In this chapter, a simple and minimally invasive subcutaneous injection in the tail region to study post-transcriptional gene regulation in vivo is described. Using a bioluminescent reporter system, this method enables visualization of the emitted light in live mice within 24 hours without requiring tissue collection or animal sacrifice. The system was further characterized by evaluating its dose dependency and temporal kinetics. This new technique was named as IVISc-L (In Vivo Imaging of Subcutaneous Luminescence).

Chapter 2.4: Detection of microRNA-mediated regulation of gene expression in vivo

In this section, the capability of IVISc-L to investigate post-transcriptional gene regulation by microRNAs is described. To achieve this, miRNA-mediated regulation of a firefly luciferase reporter was validated using the 3′-UTR of PDCD4, a well-characterized target of multiple microRNAs. Additionally, miRNA-mediated gene silencing was recapitulated by tethering AGO2—a key component of the miRNA-induced silencing complex—to the 3′-UTR of the reporter through BOXB-N-peptide-mediated interaction. These experiments collectively establish the potency of IVISc-L in studying microRNA- driven regulatory mechanisms in vivo.

Chapter 2.5: Detection of translational regulation in vivo

In this section, IVISc-L was used to investigate translational regulation of gene expression. First, stop codon readthrough in MTCH2 was described using a luciferase encoding construct bearing the partial CDS of MTCH2 either in the presence or absence of its proximal 3′-UTR. Rare codon-mediated modulation of translational elongation with IVISc-L is also described in this chapter. A significant decrease in the luminescence occurred when there was a stretch of rare codons present upstream of luciferase.

Chapter 2.6: Detection of promoter-mediated regulation of gene expression in vivo

Finally, this chapter describes how IVISc-L can be applied to study promoter-mediated transcriptional gene regulation, as evidenced by a reduction in luminescence signal in the absence of an upstream promoter and enhancer.