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Browsing by Author "Stasevich, Timothy, advisor"

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    Elucidating translation control of Argonaute in live cells by developing a tetherable biosensor with single-mRNA resolution
    (Colorado State University. Libraries, 2021) Cialek, Charlotte A., author; Stasevich, Timothy, advisor; Montgomery, Taiowa, advisor; Hoerndli, Fred, committee member; Nishimura, Erin, committee member; Ross, Eric, committee member
    Translation is an essential step for all living beings. It stands as the final hurdle of converting our genetic code into functional protein products. A culmination of specific factors heavily regulates what, when, and how much of a given peptide product is translated (Chapter 1). Though many technological advancements have expanded our understanding of translational control, they have often opened many new questions due to the high complexity of this process. Recently, a technique called Nascent Chain Tracking (NCT) was able to image translation with single molecule resolution in living cells (Chapter 2). NCT-based technologies have overcome some of the limitations of conventional in vivo and in vitro approaches to study translation. Though these NCT-based technologies accomplished detection of heterogeneity of translation dynamics, they were not capable of studying how specific factors mediate translational control. The process of translation can be controlled by small RNA silencing pathways that restrict or completely block protein production. Of these, microRNAs (miRNAs) direct translational repression and mRNA decay by guiding the enzyme Argonaute and its associated proteins to partially complementary sequences on target mRNAs (Chapter 1). The dynamics of miRNA-mediated gene silencing, and in particular the role of Argonaute on translation, remain difficult to interpret due to pathway's complexity, long (minutes-to-hours) timescale, and conflicting results from different studies. To address these problems, we developed technology to directly visualize and quantify the impact of human Argonaute2 (Ago2) on translation and subcellular localization of individual reporter mRNAs in living cells (Chapter 3). Translation and Tethering (TnT) is a tethering-based single molecule reporter that simultaneously monitors translation and Ago2-tethering status in live human cells. Since this technique is microscopy-based, its readout includes valuable subcellular localization and intensity information over timeframes ranging from seconds to hours, which describe when, where, and how much translation and tethering is occurring per single-mRNA. Finally, to simplify using this multi-construct, multi-probe system, we adapted a cell loading technique, called bead loading, to introduce TnT plasmids and proteins simultaneously into adherent cells (Chapter 4). Our TnT system reflects endogenous miRNA-mediated gene silencing when we compared it to natural miRNA-target site recruitment. Using the TnT system, we find that Ago2 association leads to progressive repression of translation at individual mRNA. The timescale of silencing was similar to that of translation, consistent with a role for Ago2 in blocking translation initiation and subsequent runoff of the ribosomes already engaged in translation elongation. At early timepoints, we observed occasional brief bursts of translational activity at Ago2-tethered mRNAs undergoing silencing, suggesting that translational repression may initially be reversible. At late timepoints, Ago2-tethered mRNA were redirected into P-bodies where they remained translationally silenced for 10+ hours, which was the duration of the experiment. These results provide a framework for exploring miRNA-mediated gene regulation in live cells at the single molecule level (Chapter 5). Furthermore, due to the adaptability of the TnT system, it will likely have wide-ranging application in studying RNA-protein interactions more generally.
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    Modulating translation dynamics with tunable optogenetic protein recruitment
    (Colorado State University. Libraries, 2024) Fixen, Gretchen M., author; Stasevich, Timothy, advisor; Nishimura, Erin, committee member; Chung, Jean, committee member
    Genes encoded in our DNA are fundamental to human health and well-being. Their imperative role requires tight regulation throughout their journey to becoming functional proteins. These regulations, when disrupted, have been linked to many neurodegenerative disorders and cancers, stressing the importance of deconvolving their components. Translation is one of the final steps in this journey that has been extensively explored, resulting in a recent technique developed known as nascent chain tracking (NCT) coupled with MS2 stem loop tagging. Using this technique, we are able to track translation dynamics in real-time and in live cells. Despite this, there are still limitations in spatially and temporally tracking the recruitment of translation effectors to translation sites and accurately measuring these dynamics. With the incorporation of optogenetic blue-light-sensitive proteins, we can generate inducible biomolecular condensates that recruit green fluorescent protein (GFP)-tagged proteins and our reporter mRNAs. Using this controlled test-tube-like environment, we can discover the direct effects ribosomal quality control proteins have on translation dynamics. A main quality control pathway involves ZNF598, GIGYF2, and 4EHP proteins that mediate translation control during ribosome stalling. We discovered that both GIGYF2 and 4EHP can be recruited to these clusters and co-localize with our active translation sites in live cells. Further exploration found that 4EHP alone cannot fully cause translation inhibition with our system. Despite this, we do see translation initiation occurring over time due to complex formation with HIF-2∝. However, GIGYF2 has distinct effects on these kinetics that are variable. This tool, when optimized, will be able to describe different proteins' effects on translation kinetics in an isolated environment in live cells.
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    Multi-color visualization and quantification of single RNA translation and HIV-1 programmed ribosomal frameshifting in living cells
    (Colorado State University. Libraries, 2019) Lyon, Kenneth Ray, Jr., author; Stasevich, Timothy, advisor; Munsky, Brian, committee member; Cohen, Bob, committee member; DeLuca, Jake, committee member
    Sixty years ago, Francis Crick first stated the central dogma of molecular biology: DNA makes RNA, RNA makes protein. At that time the central dogma could only be imagined, but over the past two decades revolutionary advances in fluorescence microscopy have now made it possible to directly image transcription and translation in living cells and organisms. A key breakthrough was the discovery and development of GFP, which can be genetically fused to other proteins to selectively light them up and track their expression in vivo. While this powerful technology can illuminate mature protein products in live cells, processes like translation remain in the dark. This is because fluorescent fusion tags take too long to mature and light up. By the time the fluorescence becomes visible, translation of a nascent protein is over and has long since separated from its parental RNA strand. This fundamental challenge has made it difficult to visualize, quantify, and study translational gene regulatory mechanisms in living cells and organisms with fluorescence microscopy. To achieve this, nascent chain tracking (NCT) was developed, a technique that uses multi-epitope tags and antibody-based fluorescent probes to visualize and quantify protein synthesis dynamics at the single-RNA level. NCT revealed an elongation rate of ~10 amino acids per second, with initiation occurring stochastically every ~30 seconds. Polysomes contain ~1 ribosome every 200 to 900 nucleotides. By employing a multi-color probe strategy, NCT shows that a small fraction (~5%) form multi-RNA sites in which two distinct RNAs are translated simultaneously while spatially overlapping. NCT was then applied to further understand the dynamics of translating RNAs interactions with ribonucleoprotein (RNP) granules during cellular stress. NCT was used to image real-time single RNAs, their translational output, and RNA-granule interactions during stress. Although translating mRNAs only interact with RNP granules dynamically, non-translating mRNAs can form stable, and sometimes rigid, associations with RNP granules. These stable associations increase with both mRNA length and granule size. Live and fixed-cell imaging demonstrated that mRNAs can extend beyond the protein surface of a stress granule, which may facilitate interactions between RNP granules. Thus, the recruitment of mRNPs to RNP granules involves dynamic, stable and extended interactions affected by translation status, mRNA length and granule size that collectively regulate RNP granule dynamics. Finally, NCT was utilized to quantify the dynamics of multiple open reading frames at the individual RNA level in a living system. An NCT multi-color imaging modality was used to investigate ribosomal frameshifting during translation. Frameshifts occur through a ribosomal +1(-2) or -1 (+2) nucleotide(s) "slip" into another frame and are implicated in both human disease and viral infections. While previous work has uncovered many mechanistic details about single-RNA frameshifting kinetics in vitro, very little is known about how single RNAs frameshift in living systems. Applying multi-frame NCT technology to RNA encoding the -1 programmed ribosomal frameshift (-1PRF) sequence of HIV-1 revealed that a small subset (~8%) of translating RNAs frameshift in living cells. This multi-color NCT method also revealed that frameshifting RNA are preferentially in multi-RNA sites, are translated at about the same rate as non-frameshifting RNA, and can continuously frameshift for more than four rounds of translation. Interestingly, fits to a bursty model of frameshifting constrain frameshifting kinetic rates and demonstrate how ribosomal traffic jams contribute to the persistence of the frameshifting state. These data provide fresh insight into retroviral frameshifting and could lead to alternative strategies to perturb the process in living cells.
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    Strange translation - investigating ires-mediated and codon non-optimal translation dynamics at the single mRNA level in living cells
    (Colorado State University. Libraries, 2021) Koch, Amanda Lynn, author; Stasevich, Timothy, advisor; Munsky, Brian, committee member; Markus, Steven, committee member; Peersen, Olve, committee member; Wilusz, Jeffrey, committee member
    With the advent of Nascent Chain Tracking (NCT), a technique used to visualize single-molecule events of translation in living cells, answering detailed questions about how, when, and where translation is occurring in living cells is possible. Since its publishing debut in 2016, NCT has provided a wealth of information about translation initiation and elongation dynamics, subcellular localization, translation site structure, and reaction to stress for both canonical and non-canonical translation in living cells. Here, we slightly modify the NCT assay to quantify translation dynamics when a ribosome is recruited to an mRNA in a non-canonical fashion and when a ribosome encounters codon non-optimal stretches on a transcript. The first step of translation requires a primed ribosome to be recruited to a readied mRNA. Canonically, this recruitment takes place on the 5' cap of an mRNA and is termed cap-dependent initiation. However, some eukaryotic messages and many viral RNAs use an internal ribosome entry site (IRES) to recruit ribosomes and initiate translation in a cap-independent manner. Specifically, viruses use IRES elements to hijack host ribosomes to translate viral proteins and properly propagate in host cells. While well- studied in bulk, the dynamics of IRES-mediated translation remain unexplored at the single-molecule level. Here, we developed a bicistronic biosensor encoding distinct repeat epitopes in two open reading frames (ORFs), one translated from the 5'-cap, the other from the Encephalomyocarditis Virus IRES. When combined with a pair of complementary probes that bind the epitopes co-translationally, the biosensor lights up in different colors depending on which ORF is translated. Using the sensor together with single-molecule tracking and computational modeling, we measured the kinetics of cap- dependent versus IRES-mediated translation in living human cells. We show that bursts of IRES translation are shorter and rarer than bursts of cap translation, although the situation reverses upon stress. Collectively our data support a model for translational regulation primarily driven by transitions between translationally active and inactive RNA states. Once the ribosome has been recruited to the mRNA and a start codon located, the ribosome will begin decoding the mRNA in nucleotide triplets or codons to ultimately create a protein. In some cases, the ribosome encounters a codon that it cannot decode efficiently. The relationship between codons and ribosome efficiency is termed codon optimality. It has been shown that codon non-optimal mRNA are less stable in cells. However, little is known about the effects of codon non-optimality on translation kinetics and overall translation regulation. In an ongoing collaboration with the Rissland group, we use bulk assays and NCT to address unanswered questions about how codon non- optimality leads to translation regulation along with mRNA instability. Thus far, we have evidence to support that translation repression is occurring in codon non-optimal conditions through inhibition of ribosome initiation and slower elongation. Further investigations of exact translation repression mechanisms are ongoing.