Biochemistry and Cell Biology - Doctoral Theses
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Item Cancer proteogenomics – identifying coding, long non-coding RNAs relevant in cancer(University College Cork, 2024) Zaheed, Oza; Dean, Kellie; Baranov, Pavel V.; Mészáros, Bálint; Science Foundation IrelandLong non-coding RNAs (lncRNAs) are RNA molecules exceeding 500 nucleotides that were traditionally considered non-functional and not translated (Mattick et al., 2023). However, recent advancements in next-generation sequencing have unveiled pivotal roles for lncRNAs in cellular processes, especially in disease contexts such as cancer. My work proposes that the non-coding transcriptome significantly impacts cancer cell biology, influencing aggressiveness and offering potential applications in patient diagnosis, disease stratification, and treatment. Cancer cells, characterised by altered metabolism and adaptations to cellular stress, may tip the balance between non-coding and coding functions. This shift could potentially result in the production of peptides from RNAs previously annotated as non-coding. Emerging evidence suggests that some of these 'non-coding' RNAs particularly lncRNAs may, in fact, encode proteins, often originating from small open reading frames (sORFs). Examples of these peptides generated from sORFs within non-coding transcripts span across species, from plants to humans, and many exhibit critical biological functions. My research focuses on identifying bifunctional RNAs that exhibit dual non-coding and coding functions in cancer. Leveraging RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) datasets, I aim to challenge the conventional classification of 'noncoding' RNAs. My hypothesis posits that a single RNA molecule may serve both noncoding and coding roles, influenced by contextual cues and cellular conditions. To explore this hypothesis, I propose a multi-omic approach, aiming to identify potential peptides with pivotal roles in oncogenesis and cancer progression. This approach uncovered a total of 400 lncRNAs that were differentially expressed in malignant breast cancer cell lines. From this list of differentially expressed long non-coding RNAs, 64 candidate translated open reading frames were identified. By employing sequence analysis in combination with proteomic evidence and translated open reading frame curation, a scoring system was created to rank these candidates on the likelihood of producing a detectable microprotein. A total of nine parameters were assessed and fit into evidencebased categories including, translation, sequence information, and proteomics. This scoring system identified ten candidates most likely to produce a stable detectable microprotein with the three best scoring candidates arising from the lncRNAs ENSG00000253477, LINC02163, and MRPS30-DT. This integrated methodology holds promise for uncovering hidden coding potential within lncRNAs, offering novel insights into therapeutic targets and biomarkers. By examining the interplay between non-coding and coding functions in cancer, this research seeks to reshape our understanding of lncRNAs and their applications in cancer diagnosis and treatment.Item Structural and biochemical investigation of viral translational recoding events(University College Cork, 2024) O'Connor, Kate; Atkins, John F.; Loughran, Gary; Irish Research CouncilIn standard translation elongation, sequential non-overlapping codons encode amino acids, with 61 sense codons and 3 stop codons (UGA, UAG, and UAA). Translational recoding involves dynamic changes to this standard readout. One type of recoding, called readthrough, involves stop codons being read as sense codons. Other recoding events involve temporary suspension of the linear, non-overlapping reading of codons. This thesis focuses on these such recoding cases characterised by dissociation of peptidyl-tRNA from the mRNA within the ribosome, ribosome slippage, and tRNA repairing with the mRNA. Frameshifting refers to instances where the tRNA repairs with the mRNA at an overlapping codon, thereby allowing translation to continue downstream in a different reading frame. Bypassing relates to cases where repairing occurs at a non-overlapping codon, which may be in the same or a different reading frame as the upstream translated region. All documented natural instances of bypassing involve repairing at a codon 3’ of the take-off codon. In chapter 2, the first results chapter, the mechanism of -1 programmed ribosomal frameshifting (-1PRF) in SARS-CoV-2 is investigated. Frameshifting is vital in coronaviral decoding to allow translating ribosome access open reading frame (ORF) 1b which encodes several essential proteins for virus replication. Site-directed mutagenesis is used in this chapter to examine the key features involved in stimulating frameshifting, including of the frameshift stimulatory pseudoknot and the nascent chain. Furthermore, the importance of a zero frame stop codon found 5 codons downstream of the slippery site is investigated. It is found that the stop codon is important for frameshifting when there is a high ribosome load. We hypothesise that it allows ribosomes that do not successfully frameshift to quickly terminate and disassemble from the mRNA, allowing rapid pseudoknot refolding before trailing ribosomes reach the slippery site. Finally, various potential modulators of SARS-CoV-2 frameshifting are assessed, with Merafloxacin emerging as a promising candidate for further investigation. In Chapter 3, experimental investigations of several bioinformatically predicted novel gene 60 bypassing cases are presented and discussed. The temperature dependency of these cases is compared to that of the well-studied example of phage T4. Among them, two cases stand out. MGS12, which was sequenced from a sample taken from a 50C hot spring, was found to have a higher optimal temperature for bypassing higher than that of the E. coli phage, T4. MGS13 was found to lack temperature dependence between 12C and 37C. This is unlike T4 and the other bypassing cases tested within this temperature range, which exhibited a decrease in bypass efficiency at reduced temperatures. The temperature insensitivity of MGS13 vs T4 can be attributed primarily to the nascent chain sequence. Furthermore, the role of the A-site stem-loop (SL) of T4 is further investigated with a cryo-EM study, revealing two distinct SL structures. Intriguingly, one of these SL conformations potentially functions as a tRNA mimic, aiding the recruitment of EF-G to the ribosome. Chapter 4 focuses on an intriguing novel gene 60 bypassing case, R13, that lacks the potential to form a tetraloop-capped SL in the ribosomal A-site at take-off. A combination of a cryo-EM structure of the E. coli ribosome stalled at the take-off site and mutational assays measuring bypass efficiency, demonstrate the lack of an A-site mRNA secondary structure. Instead, the UAG codon in the ribosomal A-site at take-off adopts an unusual conformation, with substitutions of UGA or UAA proving detrimental for bypassing. Further differences between T4 and R13 bypassing are identified, including stacking of R13 mRNA bases within the ribosomal mRNA entrance channel. Unlike T4, R13 has higher bypass efficiency under high ribosome load compared to low ribosome load. However, parallels between the two cases also emerge. The nascent chain encoded 5’ of the take-off site, is also crucial for bypassing in R13, with efficient bypassing occurring upon replacement with the nascent chain of T4. A Shine-Dalgarno-like sequence within the coding gap aids in promoting landing at the correct site in the case of R13.Item The development of a platform for novel sweet taste receptor ligand discovery & an optimisation method for heterologous protein secretion in Saccharomyces cerevisiae(University College Cork, 2024) O'Riordan, Nicola; Young, Paul; Irish Research Council; Milis Bio LtdExcess sugar and artificial sweetener consumption is associated with many modern-day diseases which impose a major burden on global healthcare systems (Malik et al., 2019). Sweet-tasting proteins represent a promising alternative and could help control the incidence of diabetes, obesity, hyperlipemia, and many other metabolic-related diseases (Zhao et al., 2021). Unfortunately, the cultivation of sweet proteins from their natural sources is technically challenging and commercially unfeasible (Bilal et al., 2022). Sweet taste is initiated by the interaction of an appropriate ligand with the sweet taste receptor which functions as an obligate heterodimer, composed of two subunits; TAS1R2 and TAS1R3 (taste receptor type 1 member 2 and 3) (Ahmad and Dalziel, 2020). Widespread issues with heterologous protein expression of the sweet taste receptor and its ligand binding domains (LBDs) have been reported (Smith et al., 2021, Nango et al., 2016). Therefore, expression of the sweet taste receptors LBDs across insect, bacterial, and mammalian cells was compared, and protein expression was optimised to facilitate screening for a novel sweet protein. A stable cell line expressing the full-length versions of the receptors was characterised to assess the interaction of the anticipated novel sweet protein candidates with the sweet taste receptor. An affibody-A phage display library and a nanobody yeast display library were screened and binding to TAS1R2 and TAS1R3 was assessed. Unfortunately, no suitable ligand was identified therefore, a novel method to optimise protein secretion in Saccharomyces cerevisiae (S. cerevisiae) was established to facilitate simplified downstream processing of commercially significant proteins. The expansion involved modification of the yeast modular cloning (MoClo) toolkit (Lee et al., 2015) to include a panel of secretion-promoting sequences and translational fusion partners (TFPs) selected from the literature. The utility of this expansion was validated using 5 unique proteins of commercial interest including the naturally occurring sweet proteins, brazzein and monellin. This work represents the first reliable documentation of brazzein secretion in S. cerevisiae close to its expected molecular weight but its utility extends to the production of many heterologous proteins of commercial interest. It is anticipated that this will help to overcome the barriers associated with sweet protein production and presents a viable commercial alternative to sugar and artificial sweeteners.Item Representing translation: concepts, methods, and resources for protein coding loci representation and ribosome profiling data analysis(University College Cork, 2024) Tierney, Jack; Baranov, Pavel V.; Science Foundation IrelandThe advent of the ribosome profiling (Ribo-Seq) sequencing technique has led to the genome-wide characterisation of translatomic complexity across the tree of life. The understanding of the extent to which translation occurs outside of annotated protein coding regions has been greatly expanded, as has the understanding of the role of translation dynamics in gene expression. As more and more Ribo-Seq data is made available and consequently more translated regions identified, the need for resources and methods for annotating, representing and analysing this translation grows. Current representation schemas all but ignore translational complexity and where it is accounted for, the reality of the gene's expression is often further obscured. The Ribo-Seq data used to uncover this translational complexity is often generated for independent investigations prior to its deposition in public depositories. The re-use of this data is essential for the annotation of translational complexity yet the need for elaborate data processing and lack of standardised metadata hinder this reuse at scale. In this thesis, we introduce a set of interoperable frameworks and resources for the representation of eukaryotic translational complexity, and the increased accessibility of high-quality Ribo-Seq data in order to facilitate further investigations into translational complexity. First, introduce the concept of a Ribosome Decision Graph (RDG) for the accurate representation of translatomic complexity. RDGs address the shortcomings of current annotation frameworks by representing translation events as branching points in a directed acyclic graph enabling the representation of the set of potential paths a ribosome may take as it traverses the mRNA. In order to facilitate the adoption of this concept, we proceed to introduce a comprehensive Python package that enables researchers to construct, visualise and analyse these graphs. To address the barriers to large-scale reanalysis of public Ribo-Seq data, we introduce the RiboSeq Data Portal. This resource makes 14,840 Ribo-Seq samples from 969 studies across 96 different species/strains. This resource serves as a database of standardised metadata for each of these samples and also offers pre-processed data in various formats. This portal’s integration with the existing resources maintained by RiboSeq.Org enhances the exploration of Ribo-Seq data within the browser. We also introduce RiboMetric a comprehensive command line application for the assessment of Ribo-Seq dataset properties. RiboMetric generates detailed sample reports as well as a set of metrics that quantify important Ribo-Seq data properties including, periodicity, uniformity and read length distribution. In combination with the data available in the RiboSeq Data Portal, these metrics enable large-scale comparisons of datasets enhancing the reproducibility of translatomic research. Collectively, these advances provide robust foundations for future research into translatomic complexity in particular through the reanalysis of Ribo-Seq data. RDGs provide new perspectives on how translatomic can be represented and analysed while the RiboSeq Data Portal and RiboMetric make large-scale reanalysis of Ribo-Seq datasets with desired properties more accessible to the entire translatomic research community. This work collectively lays the groundwork for more comprehensive and reproducible investigations into translatomic complexity, potentially revolutionising our understanding of translation dynamics in gene regulation.Item Development of new photoluminescent sensing materials, systems and applications for cell analysis(University College Cork, 2024) Li, Liang; Papkovsky, Dmitri B.; Science Foundation Ireland; Agilent TechnologiesCell analysis using optochemical sensors represents large and important niche in life and biomedical science. Sensor calibration and detection platforms are critical during sensor fabrication, characterization and application. However, existing calibration setups, platforms, and systems have limitations with respect to their robustness, analytical performance and capabilities. In this project, several new photoluminescent pH and oxygen (O2) sensing materials, platforms and systems were developed, evaluated comparatively and then applied for bio-analysis particularly for samples containing cells. The first experimental section describes a new barometric calibration setup which provides accurate, convenient and versatile O2 calibration. Using this setup, a detailed characterization of several sensor types based on PtBP dye (sensor stickers, membrane inserts, liquid probe NanO2-IR) was conducted in phosphorescence lifetime (PLT) mode by handheld sensor readers, and their key optional parameters (τ0, Ksv, T-coefficients, etc) and calibration equations were determined and cross-compared. The second experimental section evaluates comparatively the PLT measurements of several Pt-porphyrin based oxygen sensors on three different platforms: the time-revolved fluorescence reader Victor2, luminescence spectrometer Cary Eclipse, and confocal laser-scanning FLIM/PLIM-TCSPC microscope. The merits and limitations of each platform, including instrumental factors, analytical performance, lifetime calculation methods, potential sources of error were assessed. The third experimental section presents a new photoluminescent materials for cell analysis based on the substituted phosphorescent Pt(II)- or Pd(II)-porphyrin indicator dyes bearing dual O2 and pH sensing functionality (MePor-SB). The study includes screening of different host matrices for the sensor, synthesis and evaluation of new MePor-SB derivatives with altered protonation behaviour, development of ratiometric version of the pH sensor, photoluminescent signal enhancement options, deposition of sensor coatings on common cell analysis substrates and their demonstration in OCR/ECAR measurements with relevant cell models (PC 12 cells). The fourth experimental section, describes novel solid-state pH sensors for cell analysis based on hydrophobic protonable metal-free porphyrins, octaethylporphine (OEP), and octaethylporphine-ketone (OEPK), as fluorescent pH indicators. The internally referenced ratiometric intensity and nanosecond lifetime-based versions of these pH sensors were developed and also multiplexed with the O2 sensors based on phosphorescent PtOEP dye. The key parameters of the pH sensor were optimized, including the type and concentration of proton transfer agent, working pH range and pKa, dye concentrations and cross-talk with the O2 sensor (Pt-OEP). The optimised two-analyte sensors with stable internally referenced calibrations, convenient spectral characteristics and low cytotoxicity were coated on microplates and demonstrated with cultured cells and 3D spheroids made of HCT 116 cells, measuring their ECAR/OCR and responses to stimulation. Finally, the planar solid-state pH sensors on polyester films (Mylar) were used to measure extracellular pH in grafted tumours produced by CT26 cells and freshly excised from animals on a portable FLIM imager. The new pH sensors and dual pH/O2 sensors showed high stability (>1 year of storage), fast response and stable calibration, being well-suited for detailed metabolic studies of biological samples on widely available laboratory equipment.