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Proper regulation of gene expression is critical for organismal life and is dictated by numerous complex transcriptional and post-transcriptional regulatory pathways. Profiling the transcriptome by sequencing to assess gene expression is a powerful approach that facilitates understanding these modes of regulation. This dissertation explores gene expression and post- transcriptional regulation in numerous contexts.Chapter 2 summarizes my in-depth analysis of RNA expression and regulation in aging C. elegans, where we leveraged the Oxford Nanopore Technologies platform to perform full- length direct RNA sequencing in tandem with short-read sequencing. I investigated changes to gene expression that occur during aging and identified 814 novel transcript isoforms. I report an age-dependent increase in poly(A) tail lengths genome-wide and a decrease in splice site fidelity. I also leveraged these methods to examine pseudouridine and inosine modified RNA nucleotides and found that there are more pseudouridine sites in old animals compared to young. Together, these results forge a deeper understanding of age-dependent transcriptomic changes that may contribute to degenerative aging phenotypes.In Chapter 3, I summarize work where I improved tools to conditionally deplete microRNA Argonuate proteins, ALG-1 and ALG-2, which are important post-transcriptional regulators that downregulate target RNA expression. Unexpectedly, depletion of these proteins does not elicit upregulation of target RNAs, which I report and explore. In Chapter 4 I report limitations of the original auxin inducible degron (AID) system, which I improved upon in Chapter 3. In its original iteration, we observed basal degradation of AID-tagged proteins, which limited its applications. Chapter 5 is an examination of poly(A) tail lengths in Influenza A virus (IAV), which uses a non-canonical mode of polyadenylation. We observed that poly(A) tail lengths of IAV are longer than predicted and are subject to shortening over the course of viral infection.

The auxin inducible degron (AID) system was developed to enable conditional depletion of proteins of interest. In this system, a plant-derived F-box protein, TIR1, is expressed and complexes with endogenous Skp1 and Cullin proteins to form an E3 ubiquitin ligase complex. Proteins of interest can be tagged with a plant-derived degron, which is recognized by the TIR1-ubiquitin ligase complex in the presence of the plant hormone auxin (indole-3-acetic acid) (Nishimura et al. 2009). This system was recently adapted for Caenorhabditis elegans, wherein TIR1 constitutive expression enables rapid depletion of degron-tagged proteins of interest when animals are transferred to NGM plates supplemented with auxin. In C. elegans, the AID system has allowed for fine-tuned spatiotemporal control of protein expression and has been used to deplete many essential proteins, which are difficult to study using classical genetic approaches (Zhang et al. 2015). Here, we highlight a limitation of this system by demonstrating that TIR1 expression can cause depletion of degron-tagged proteins without auxin addition. These results corroborate observations from human cell lines, where TIR1 has been shown to act independently of auxin to chronically deplete degron-tagged proteins (Sathyan et al. 2019). In an attempt to study the effects of depletion of the essential protein ERFA-3, we used CRISPR-Cas9 to insert a degron sequence at its N-terminus. The resulting 3xflag::degron::erfa-3 strain (WJA2126), was backcrossed three times to N2 and then outcrossed to CA1200, a publicly available pan-somatic TIR1::mRuby strain (ieSi57 [eft-3p::TIR1::mRuby::unc-54(3’UTR) + Cbr-unc-119(+)] II), to make a new strain, PQ631. Expression of TIR1 in the 3xflag::degron::erfa-3 strain led to gross morphological defects that are not seen in the 3xflag::degron::erfa-3 or TIR1-expressing strains (Zhang et al. 2015) (Figure 1A). The 3xflag::degron::erfa-3; ieSi57 [Peft-3::TIR1::mRuby::unc-54 3’UTR, cb-unc-119(+)] II strain exhibited bagging and dumpy phenotypes as well as fertility defects that manifested as an observed slower growth rate of the overall population in comparison to its parental strains. Individuals of this strain tended to lay fewer viable eggs than control strains. It is yet unclear whether this is due to defects in meiosis, embryogenesis, or in any of the numerous steps in-between. To understand the cause of these phenotypic abnormalities, we performed a quantitative Western blot against ERFA-3 using an anti-FLAG antibody (Figure 1B). Here, it is evident that expression of TIR1 alone, despite no addition of auxin to the media, leads to depletion of 3xFLAG::degron::ERFA-3 to approximately 30% of the levels observed in the absence of TIR1. We observed a similar phenomenon with the alg-1 locus. We analyzed QK155 (4xflag::degron::alg-1; ieSi57 [eft-3p::TIR1::mRuby::unc-54(3’UTR) + Cbr-unc-119(+)] II). This strain expresses degron-tagged ALG-1 and pan-somatic TIR1. We performed a quantitative Western blot to compare ALG-1 expression in our 4xFLAG::degron::ALG-1; TIR1 strain to N2 using an antibody against endogenous ALG-1. As with ERFA-3, degron-tagged ALG-1 was chronically depleted when TIR1 was expressed pan-somatically. The ALG-1 protein level in the 4xFLAG::degron::ALG-1;TIR1 strain was approximately 25% that of endogenous protein expression (Figure 1C). Taken together, these results indicate that TIR1 can act without the addition of auxin to the media, ultimately resulting in chronic depletion of degron-tagged proteins. There are at least two, non-mutually exclusive possibilities to explain our observations: (1) Auxin may exist in one or more components of the C. elegans media used. Seaweed is a common source of agar and does contain some auxin, though it is unclear if the concentration would be sufficient to activate TIR1. (2) There may be auxin-independent recognition of the degron by TIR1. Regardless of the mechanism of our observation, the effects were observed in at least two separate labs over the span of several months, indicating it is a potentially widespread caveat of the approach. The phenotypes we observed for the 3xflag::degron::erfa-3 strain in the presence of TIR1 are consistent with published phenotypes associated with RNAi knockdown of erfa-3, which include sterility, slow development, and a high rate of embryonic lethality (Kamath et al. 2003). Yet, when the AID system was adapted for C. elegans, various phenotypic assays were performed to demonstrate that TIR1 expression, with multiple different degron-tagged essential proteins, resulted in no observable abnormalities (Zhang et al. 2015). This suggests that not all degron-tagged proteins may be subject to auxin-independent degradation by TIR1 or that chronically lower levels of some essential proteins can be tolerated. In conclusion, our results highlight the need to carefully analyze expression levels of degron-tagged proteins of interest as they may be subject to TIR1-mediated degradation prior to auxin exposure.