Joan and Tom Steitz RNA Fellows Program

Eukaryotic translation initiation is the rate-limiting step for protein synthesis, determining the number of protein molecules synthesized from a single messenger RNA (mRNA). Canonical translation initiation relies on recognition of the 5ʹ N7-methylguanosine cap by eukaryotic initiation factors (eIFs), which subsequently recruit the ribosome to the mRNA. However, cellular and viral mRNAs use diverse, non-canonical mechanisms to initiate translation. Intriguingly, some well-structured RNA elements can recruit the translation initiation machinery directly, without using a 5ʹ cap. Such internal ribosome entry sites (IRESs) have been well characterized in certain viral untranslated regions (UTRs) including at atomic resolution; by contrast, the very existence of IRESs in human 5ʹ UTRs remains controversial. My project, identifying and dissecting mechanisms of internal translation initiation in human 5ʹ UTRs, will provide insight into fundamental biology by illuminating alternative mechanisms of ribosome recruitment that are likely to be important for regulating gene expression in human cells.

Since joining the Gilbert lab in May 2022, I have successfully adapted our novel high-throughput technique, Direct Analysis of Ribosome Targeting (DART), to investigate cap-independent translation initiation in human 5ʹ UTRs. In collaboration with the Thoreen lab, I have generated a pool of thousands of human 5ʹ UTRs embedded in circular RNAs. Without a free 5ʹ end, any ribosome recruitment to circular RNAs must proceed through internal initiation. Using DART, I aim to comprehensively identify putative IRESs in human UTRs and delineate the RNA elements required for IRES activity. I will then elucidate their mechanisms using low-throughput biochemistry. Importantly, my approach specifically examines the initiation stage of translation thereby overcoming the limitations of previous studies which could not disentangle effects on translation initiation from other cellular processes.

The significance of this project extends beyond the bench. Circular RNAs are appealing as a “next-generation” RNA therapeutic. Because these RNAs lack 5ʹ and 3ʹ ends, they evade host exonucleases, escaping many RNA degradation pathways and innate immune responses in cells. However, internal initiation is generally less efficient as compared to cap-dependent translation initiation. By identifying and characterizing novel sequences with high internal translation initiation in circular RNAs, I aim to engineer circular RNAs that sustain high levels of therapeutic protein production in patients.

The recent SARS-CoV2 outbreak demonstrates that viruses remain a threat to human health. Although vaccines can be developed retroactively, forecasting the next outbreak is challenging. Alternatively, we could aim to leverage the inherent cell-autonomous mechanisms of human cells to broadly boost our anti-viral resistance. This approach is crucial for stem cells, because, unlike differentiated cells, they lack the essential anti-viral interferons to co-ordinate the cell-based innate immune system.

I use the planarian Schmidtea mediterranea to investigate nucleic acid sensors and non-coding RNAs in anti-viral defense in stem cells. Planarians have an abundance of long-lived adult stem cells that are crucial for homeostasis. Absence of cell-based immune systems makes planarians a perfect model to study cell-autonomous immunity. My preliminary research demonstrates that dsRNA responders including RIG-I-like receptors (RLRs) and several components of RNAi-mediated silencing display increased expression upon viral challenges. Remarkably, I also identified a piRNA factor in this response, and knockdown of the identified RNAi and piRNA factors leads to increased viral load in the animals. This indicates the unexpected collaboration between these two small RNA pathways in stem cells for viral defense. I am using biochemical approaches to identify further factors in this pathway.

Currently, there is only one known planarian virus, so I developed an in-house method to isolate and identify viruses from S. mediterranea. Interestingly, most of the families identified were RNA viruses. We will study the differences in responses against dsDNA and RNA viruses as they threaten the cellular physiology differently. Moreover, we plan to leverage this information to develop a planarian transgenics system, a current limitation in the field. I have a longstanding interest in RNA biology. During my master’s degree, I developed a molecular diagnostic technique for detecting the endemic tropical disease (leptospirosis) outside of a lab setting, igniting my interest in research. This helped hospitals to diagnose this condition in remote areas. I currently develop a low-cost nucleic acid-based diagnosis kit for multiple zoonotic diseases. For my graduate work, I investigated how nuclear phosphatidylinositol-phosphate (PIP) lipid signaling controls mRNA 3'-end processing. We laid the groundwork for future RNA therapies by disclosing a novel role for alternative polyadenylation and PIP signaling in controlling translation in pathological states such as hypertrophy, and metastasis.

In summary, RNA has immense therapeutic potential, and I hope to eventually run an RNA research lab and continue to translate my working knowledge of RNA into low-cost diagnostics and treatments for current and future outbreaks.

Over the course of evolution, molecular adaptations within RNA structures allowed for functional versatility. Cis-acting RNA elements are secondary structures implicated in RNA metabolism, stability, translation, and processing. One cis-acting RNA element that is present in viruses and retrotransposons is the element for nuclear expression (ENE). First discovered in the PAN RNA of Kaposi’s sarcoma-associated herpesvirus, ENEs are elements that harbor U-rich internal loops flanked by short helices. Found in the 3’-proximal regions of transcripts, ENEs function by sequestering poly(A) tails into major-groove triple helices. This interaction protects poly(A) tails from rapid deadenylation-dependent decay, which is often the first step involved in eukaryotic RNA degradation. With that said, transcripts containing these ENEs have a stability advantage that is modulated through evolutionary pressures.

As part of the research I am doing in the lab of Dr. Joan Steitz, bioinformatic analyses of the genomes of vertebrates have revealed that some LINE retrotransposons are enriched in ENE sequences. To test whether these identified ENEs bind poly(A) tails, I first sought to determine if binding can occur in-trans using electrophoretic mobility shift assays (EMSAs). This was done by binding radio-labeled oligo(A12) to in-vitro transcribed ENE RNAs. Indeed, the ENEs identified computationally do bind oligo(A12) in-vitro as expected. To probe the role of highly conserved residues in the stability of the triple helix, mutagenesis was performed, followed by EMSA. Surprisingly, some mutations increased the binding affinity of the ENE for oligo(A12), suggesting a novel role for these ENEs. Upon analysis of the literature, we hypothesize that these ENEs are involved in stabilizing poly-uridylated transcripts in the cytoplasm, as cytoplasmic uridylation of poly(A) transcripts is often a marker for RNA degradation. This could have many implications for science and society, as ENEs are rarely studied in cytoplasmic environments. This could allow us to understand how retroelements and viruses passing through the cytoplasm avoid RNA degradation. As a result, one could theoretically target those elements using anti-morpholino oligos to destabilize the transcript and prevent viral progression. This research can also be utilized to develop better mRNA vaccines to fight pandemics, as therapeutic mRNAs can be stabilized by adding ENEs. This strategy should allow for the production of more robust immune responses upon vaccination. Thus, studying ENEs is one example of how basic advances in RNA biology can lead to improvements in therapeutics.

Not too long ago, most of the human genome was considered “junk” DNA – that is, DNA without protein-coding potential. We now know this to be false; instead, an intricate regulatory network exists within these noncoding regions. The center of this is noncoding RNA (ncRNA), whose novelty and ubiquitous appearance in numerous cellular pathways have garnered widespread attention. I am particularly interested in long ncRNA’s (lncRNAs) role in p53 tumor suppressive pathway and cancer development. My research can be categorized into two themes: mechanistic dissection of p53-regulated lncRNAs (PR-lncRNAs), and discovery of novel coding genes within the p53-regulatory network.

My fascination with lncRNAs started with Xist, a noncoding transcript that epigenetically silences chromatin for sex determination. Discovered in the 1990s, Xist is one of the most well-characterized lncRNAs. Yet, its mechanism of action largely remains a mystery. The diversity of lncRNAs has made it challenging to evaluate their roles in normal and diseased contexts, including cancer. My research focused on implementing gene editing technology to functionally characterize PR-lncRNAs. I developed a paired-guide RNA CRISPR/Cas9 deletion strategy that allows me to dissect elements within Pvt1, a PR-lncRNA that regulates the proto-oncogene Myc, and investigate their individual functions. This work unveiled key functional elements that regulate Myc and expanded our understanding of the Pvt1 locus and lncRNA activity in cancer. This work also validated the use of CRISPR/Cas9-mediated deletions in lncRNA studies, contributing to a growing list of tools for lncRNA biologists.

Recently, my interest with space exploration gave me the opportunity to investigate RNA from a unique perspective. Through the NASA CTSpace Consortium, I am currently studying novel coding genes within the ionizing radiation (IR) induced p53 response. Advancements in sequencing technology have revealed interesting coding potential within erroneously annotated lncRNAs. Therefore, my current work aims to discover, validate, and characterize novel proteins in the IR-response pathway that were once believed to be encoded by lncRNAs. My work will re-evaluate the functions of lncRNAs and further our knowledge of the p53 network in outer space. This intersection between RNA biology and space biology may bring about new solutions in improving human health.

My goal is to harness the potential that RNA has and utilize them to advance therapeutics for cancer and other diseases. Ultimately, I strive to pursue a career where I can apply my research in treating patients both on Earth and in space.

Greater than 75% of the human genome is pervasively transcribed, although only 1.5% encodes proteins. Non-coding RNAs (ncRNAs) are major functional and regulatory units of the non-coding part of the genome, yet remain under-explored. With increasing evidence for their perturbations in human disease pathogenesis, the remarkable variety of functions that ncRNAs can accomplish present particularly exciting areas for research.

Dissecting the mechanisms by which RNA molecules are translated into proteins in Dr. Cate’s lab at UC-Berkeley as an undergraduate student, I became fascinated by the versatile functions of RNA molecules fundamental to human health. I also wanted to integrate my research into understanding human disease and patient care. To this end, I exclusively applied to MD-PhD programs and chose Yale because of its strength in RNA biology and collaborative research community. Indeed, interdisciplinary collaboration and the supportive RNA community have been key aspects of my training at Yale.

My interest in RNA biology naturally led me to Dr. Steitz and a novel class of ncRNAs that her lab had just discovered. Relatively little is known about these RNA species, called Downstream-of-Gene (DoG) RNAs, other than the fact that they are expressed during various stress conditions such as viral infections and cancer. During my PhD, I wished to understand the function of these RNAs not only at a molecular but also at a systems level, especially in the context of human immune response. Systematically investigating their expression across human tissues, I discovered elevated levels of DoG RNAs in cerebellar neurons and described their potential role in suppressing the innate immune response by acting on retrotransposon transcripts. These findings suggest that DoG RNAs represent a strategy to prevent inflammation and cell death in non-replicating cell types and that their insufficient expression contributes to aberrant or hyperactive immune response.

The clinical and research training I have received at Yale continue to fuel my career goal as a physician-scientist in the fields of RNA biology, immunology, and medicine. I hope to synergize my clinical and research interests as an academic investigator dedicated to investigating mechanisms that drive autoimmunity, with a keen interest on the contributions of non-coding and retrotransposon-derived RNAs. Through my perseverance and commitment to uncovering RNA biology to better understand human disease, and the support of an exceptional community of RNA scholars and dedicated mentors, I will strive to become a physician-scientist who melds together her passions for medicine and research.

I have been fascinated by RNA since I first learned about the milieu of biomolecules essential for cell survival and proliferation. My research focuses on how ribosomes – the molecular machines that translate messenger RNA (mRNA) into protein – are made. Ribosomes have one small and one large subunit, each of which are comprised of ribosomal proteins and ribosomal RNA (rRNA). That these essential molecular machines translate one type of RNA while being comprised of another is a remarkable testament to the importance of RNA biology to all functions of living organisms.

In eukaryotic cells, ribosomes are made in the nucleolus, a membraneless organelle within the nucleus. During my graduate work, I studied chemotherapeutic platinum compounds that localize to the nucleolus and how they interfere with the creation of ribosomes. These anticancer drugs were discovered decades ago and are still widely used today, however they were only recently found to inhibit ribosome biogenesis. My research identified highly specific structural properties for platinum compounds to interfere in this process.

My current postdoctoral research in the lab of Dr. Susan Baserga seeks to address broad questions about the regulation of ribosome biogenesis. Aberrations in ribosome biogenesis and the nucleolus are strongly associated with disease. Elevated ribosome biogenesis is implicated in some cancers, and changes in nucleolar morphology have been associated with cancer for over a century. Ribosomopathies are genetic developmental diseases, often severe, driven by abnormal ribosome biogenesis. Despite the importance of ribosomes, there is much still to learn about how they are generated in the cell.

A critical line of inquiry is how changes in ribosome biogenesis regulation can lead to disease outcomes. Another open question driving my current research is how ribosome biogenesis is regulated by other important cellular processes, organelles, and pathways. I focus on proteins newly identified via screens to be regulators of ribosome biogenesis, especially those with disease relevancy and the potential to uncover previously undescribed relationships between ribosome biogenesis and other processes. For example, I am investigating how a protein primarily involved in transport from the Golgi apparatus to the endoplasmic reticulum seems to be regulating nucleolar ribosome biogenesis in human cells. Explaining these surprising connections will deepen our understanding of how ribosomes are made, with tremendous potential to provide insights into both basic RNA biology and human health.

Many aspects of RNA biology shape neural function: (i) The nervous system displays unmatched levels of transcriptional and post-transcriptional regulation, events crucial for neuronal identity and plasticity. (ii) Timing and localization of mRNA translation impact the dynamic of neuronal response. (iii) Numerous classes of RNA perform critical roles in neurons: non-coding (nc)RNAs, renowned for their multifaceted nature, are ideally suited for roles in modulation of neuronal activity and evolution. Several neurological disorders are associated with the dysregulation of these processes, highlighting the importance of mRNA tailoring and ncRNAs functions. Their investigation in the nervous system is a challenging frontier that requires exploration of uncharted territories.

In the Carlson laboratory, I examine such uncharted territory: neuronal RNA in Drosophila chemosensory systems. These well-characterized and highly adaptive neuronal systems are numerically simple yet drive multiple complex behaviors. With powerful genetic tools and low genetic redundancy, Drosophila offers an ideal opportunity for unparalleled investigation of mRNA tailoring and ncRNAs.

I began my studies by generating transcriptomes for two chemosensory organs, the antenna (olfaction) and the labellum (taste), and searched for non-canonical transcript variants of chemosensory genes and long ncRNAs. Such analyses divulged 3 layers of complexity to chemosensation:

1. Drosophila chemosensory genes encode more transcript variants than previously appreciated. RNA-Seq revealed evidence for alternative transcription start/termination sites and rare splicing events of chemoreceptor transcripts. Xueying Shang leads investigations into the role(s) of such events in neuronal adaptation.
2. Stop codon read-through prevails in chemoreceptor genes. Analysis of chemoreceptor transcripts revealed that a receptor for salt taste harbors an unexpected conserved premature termination codon (PTC). Genetic experiments showed that synthesis of a functional receptor requires PTC read-through.
3. Olfactory receptor neurons express unique lncRNAs. By combining bulk and single-nucleus RNA-Seq analyses, we generated the first database of antennal lncRNAs and an unprecedented lncRNA-to-neuron map. Intriguingly, a number of these lncRNAs are species-specific and expressed primarily in pheromone-sensitive neurons, inviting us to explore their role in species recognition.

My work provides the foundation for identifying mRNA tailoring and lncRNAs characteristic to the adaptation to new environments/internal states. I identified one of such RNAs: ANRUS, ANtennal RNA Upregulated by Starvation. ANRUS shows numerous ncRNA characteristics, yet it encodes 2 micropeptides. I am investigating their roles in modulating neuronal activity of fasted animals.

Overall, my research offers new biological insights into chemosensation and introduces a new platform to investigate the vast impact of RNA biology in neurons.